M Mertens

S S Essbauer

A Rang

J Schröder

W D Splettstoesser

C Kretzschmar

D H Krüger

M H Groschup

K Mätz-Rensing

R G Ulrich

Non-human primates in outdoor enclosures: Risk for infection with rodent-borne hantaviruses

Keywords: Non-human primates, haemorrhagic fever with renal syndrome, hantavirus, rodent, bank vole, common vole, field vole

Introduction

In natural infections hantaviruses are transmitted from persistently infected reservoir hosts such as rodents or insectivores to humans (and other non-reservoir species) by inhalation of virus-contaminated aerosols (for review see [START_REF] Schönrich | Hantavirus-induced immunity in rodent reservoirs and humans[END_REF]. Human infections with viruses indigenous in Europe and Asia can result in haemorrhagic fevers with renal syndrome (HFRS) of different severity levels and case fatality rates (for review see [START_REF] Schönrich | Hantavirus-induced immunity in rodent reservoirs and humans[END_REF]. As hantaviruses can cause life-threatening diseases in humans, non-human primates have been employed to establish suitable disease models. In a first attempt, three cynomolgous monkeys (Macaca fascicularis) and a chimpanzee (Pan troglodytes) were intravenously inoculated with Prospect Hill virus (PHV), a hantavirus believed to be non pathogenic to humans. Surprisingly it caused acute nephropathy with mild, transient proteinuria and azotemia (Yanagihara et al., 1988). Later, an experimental intratracheal infection of cynomolgus macaques with cell culture-adapted Puumala virus A c c e p t e d M a n u s c r i p t 3 (PUUV) resulted in signs of lethargy followed by mild proteinuria and microhematuria and histopathological abnormalities in hantavirus RNA-and antigen-positive kidneys [START_REF] Groen | A macaque model for hantavirus infection[END_REF]. Infection of cynomolgus macaques with a PUUV strain, that was exclusively replicated in its natural host, the bank vole (Myodes glareolus), induced some clinical symptoms such as loss of appetite, apathetic behaviour, fever, proteinuria, biochemical markers and immunological characteristics of HFRS typically observed in human patients (Klingström et al., 2002a;[START_REF] Sironen | Pathology of Puumala hantavirus infection in macaques[END_REF]. A first indication for a naturally acquired hantavirus infection of non-human primates was reported for Macaca mulatta in China (see [START_REF] Clement | ed) Virus infections of Rodents and lagomorphs, 5th Volume in a series (Osterhaus, ed) "Virus infections in Vertebrates[END_REF].

Two Arvicolinae-associated hantaviruses, i.e. PUUV and Microtus-associated Tula virus (TULV), have a broad geographical distribution in Germany (Ulrich et al., 2004;[START_REF] Schmidt-Chanasit | Extensive host sharing of Central European Tula virus[END_REF]. Large numbers and clusters of human PUUV infections have been recorded during the outbreaks in 2005 and 2007, mainly affecting the federal states Baden-Wuerttemberg, Bavaria, Lower Saxony and North Rhine Westphalia [START_REF] Essbauer | A new Puumala hantavirus subtype in rodents associated with an outbreak of Nephropathia epidemica in South-East Germany in 2004[END_REF][START_REF] Essbauer | Nephropathia epidemica outbreak in a metropolitan area, Germany[END_REF][START_REF] Hofmann | Molecular epidemiology of a large hantavirus outbreak in Germany[END_REF]. Whereas PUUV is causing the majority of human HFRS cases in Germany, little is known about the relevance of TULV as a human pathogen. Besides a single description of a HFRS case in north-eastern Germany [START_REF] Klempa | Occurrence of renal and pulmonary syndrome in a region of northeast Germany where Tula virus circulates[END_REF], TULV-specific antibodies were detected in human samples from a few seroprevalence studies (Ulrich et al., 2004;Mertens et al., unpublished data). A recent longitudinal study revealed a sympatric occurence of TULV in common (Microtus arvalis) and field voles (M. agrestis) from Sennickerode, district Göttingen, federal state of Lower Saxony [START_REF] Schmidt-Chanasit | Extensive host sharing of Central European Tula virus[END_REF].

As this site is in close vicinity of the German Primate Center (see Fig. 1) we wanted to prove if non-human primates in outdoor enclosures are at risk to get infected by hantaviruses circulating in the vole populations close to the husbandry. For this purpose we investigated the prevalence of hantavirus-reactive antibodies in three different simian

Materials and methods

Breeding colonies of non-human primates

The German Primate Center (GPC) is housing and breeding the Old World monkey species Macaca mulatta, M. fascicularis and Papio anubis in in-and outdoor units. Each unit is composed of an indoor area, a heated and/or roofed outdoor room and a large outdoor exhibition bordered by fences. Contacts to any other animal species except wild birds or small mammals are efficiently prevented. About one half of the investigated animals were born at the GPC, whereas the other animals originated from other husbandries in Germany, France or the USA. All animals are kept in accordance with the guidelines of the European Union for the accommodation and care of animals used for experimental and other scientific purposes (2007/526/EG, D-AFF 008-EWG). The primate husbandry is controlled by local and regional veterinary authorities in accordance with the German Animal Protection Law. All procedures are supervised by an animal welfare officer and the ethical committee for experiments using animals in the federal state of Lower Saxony. During the yearly routine health check 295 serum samples were collected in 1999 -2007 from 254 monkeys including 211 rhesus macaques (M. mulatta), 26 cynomolgus monkeys (M. fascicularis) and 17 olive baboons (P. anubis). This panel contains also serum samples from two M. mulatta and one M. fascicularis, that died in 2004 or 2005 due to tularemia. The blood collection was carried out in Ketamin-anesthesia (Ketavet®, Pfizer, Karlsruhe, Germany) in a dosage of 10 mg/kg. The serological investigations of the samples for hantavirus-specific antibodies were performed following the recently described scheme for seroepidemiological investigations [START_REF] Mertens | Seroepidemiological study in a Puumala virus outbreak area in South-East Germany[END_REF]. Briefly, the sera were initially investigated in parallel by PUUV-and TULV-based screening IgG-ELISAs. For confirmation, ELISA-positive sera were tested in the corresponding PUUV-and TULV-IgG immunoblot tests [START_REF] Mertens | Seroepidemiological study in a Puumala virus outbreak area in South-East Germany[END_REF][START_REF] Schmidt-Chanasit | Extensive host sharing of Central European Tula virus[END_REF], but using a horseradish peroxidase-labeled rabbit anti-monkey IgG (Heavy and Light chain, H+L) (Nordic Immunology, Offenbach, Germany). Finally, selected ELISA-reactive, immunoblot-confirmed sera were investigated by chemiluminescence focus reduction neutralization test (cFRNT) using PUUV, strain Sotkamo, TULV, strain Moravia, and Apodemus flavicollis-borne Dobrava-Belgrade virus (DOBV), strain Slovenia, as a control [START_REF] Heider | A chemiluminescence detection method of hantaviral antigens in neutralisation assays and inhibitor studies[END_REF].

Serological analysis of monkey serum samples

Rodent trapping and hantavirus analysis

The trapping of rodents in Sennickerode and at an area of approximately 30,000 m 2 around the GPC of Göttingen, both in district Göttingen (see Fig. 1), as well as the TULV investigations of Microtus arvalis and M. agrestis from Sennickerode have already been described in detail [START_REF] Kaysser | Re-emergence of tularemia in Germany: presence of Francisella tularensis in different rodent species in endemic areas[END_REF][START_REF] Schmidt-Chanasit | Extensive host sharing of Central European Tula virus[END_REF]. Hantavirus-Ssegment-specific reverse transcriptase-PCR (RT-PCR) of animals trapped around the GPC and of bank voles from Sennickerode followed previously described detailed protocols [START_REF] Essbauer | A new Puumala hantavirus subtype in rodents associated with an outbreak of Nephropathia epidemica in South-East Germany in 2004[END_REF][START_REF] Schmidt-Chanasit | Extensive host sharing of Central European Tula virus[END_REF]. Retrieved novel partial PUUV and TULV S-segment sequences were submitted to GenBank with accession numbers GU300138-GU300143 and GU300135-GU300137, respectively.

Results

Using recombinant PUUV-and TULV-antigen-based IgG screening ELISA and confirmation immunoblot assays, 24 of the 251 (9.6 %) investigated animal serum samples 1). Out of these 24 samples 17 exclusively reacted with PUUV antigen, three with TULV antigen and four with both antigens to the same extent. A total of 16 of the 209 (7.7 %) sera from 13 female and three male rhesus macaques (M. mulatta)

were seropositive with isolated reaction to PUUV antigen for 13 samples, to TULV antigen for two samples and a cross-reactivity to both antigens for one sample. In the olive baboons (P. anubis) serum panel 6 of 17 (35.3 %) were reactive with at least one of the antigens. Out of these 6 samples, with five of six originating from female animals, three reacted with the PUUV, one with TULV and two with both antigens. The level of seroreactivity in the cynomolgus monkey (M. fascicularis) panel was 8.0 % (2/25) with one serum reacting only with PUUV antigen and one with the two antigens. The two seroreactive sera originated from female individuals (Table 1). Subsequent analysis of five selected ELISA-positive, immunoblot-confirmed sera from M. mulatta by PUUV, TULV and DOBV cFRNT revealed one PUUV/TULV cross-reactive, but DOBV non-reactive serum originating from a six year-old female animal (#2116) born at the GPC. The endpoint titer to PUUV (640) was slightly higher than that to TULV (320).

Next, follow-up sera of 12 animals including 11 M. mulatta and one M. fascicularis, which were in the initially investigated serum samples from 2006 positive, were tested in the same way as described above (Table 1). From four M. mulatta (#4986, #9290, # 9796 and #9821) all serum samples were found to be positive, with at least one antigen. Four developed symptoms typical for a hantavirus infection like apathy, vomiting, diarrhea or hematuria. At the time of blood sample collection a complete health check was done. All animals were clinically healthy and kidney and liver enzymes were within the normal range. Six of the seropositive animals died during the investigation period. There was no correlation between the cause of death and the former hantavirus infection. Two animals (#2039, #2242) had to be humanly euthanasized because of severe injuries after heavy ranking fights. One animal died after severe aspiration pneumonia (#9292). One of the baboons (#13315) died because of several changes due to old age. Systemic echinococcosis was the cause of death in the last two animals (#9858, #9870). The histological samples of the deceased monkeys were screened for signs of a hantavirus infection but none of the animals showed kidney alterations in form of interstitial nephritis.

In addition, serum samples from two M. mulatta and one M. fascicularis, that died due to tularemia, tested in the PUUV and TULV ELISA revealed for all investigated samples of one M. mulatta PUUV-reactive antibodies (Table 1; #9411). The other two animals did not show any reactivity in the PUUV and TULV IgG screening ELISAs (data not shown).

Interestingly, in a recent study TULV infections have been described in M. arvalis and M. agrestis from Sennickerode, district Göttingen, about 20 km apart from the GPC [START_REF] Schmidt-Chanasit | Extensive host sharing of Central European Tula virus[END_REF]; see Fig. 1). In addition, herein we investigated lung tissue samples from 60 bank voles (M. glareolus) from the same trapping site. Novel partial PUUV S-segment-specific fragments were detected by RT-PCR in 6 of the lung samples (10%). These sequences are closely related to each other, but can be differentiated at the nucleotide level from PUUV sequences originating from other regions in Germany, e.g. Baden-Wuerttemberg, northern and southern Bavaria and Cologne (see supplementary 2).

Discussion

Spillover infections of non-reservoir rodent species are believed to be a rare event (Klingström et al., 2002b), but represent a pre-requisite for a subsequent establishment of a transmission cycle in a novel reservoir host as well as genetic reassortment processes between different hantaviruses. In line, detection of multiple spillover infections of M. agrestis with M. arvalis-associated TULV has been discussed as the beginning establishment of a novel reservoir host transmission cycle [START_REF] Schmidt-Chanasit | Extensive host sharing of Central European Tula virus[END_REF].

Similarly, multiple spillover infections of A. agrarius-borne DOBV to A. flavicollis have been observed in northern Germany [START_REF] Schlegel | Dobrava-Belgrade virus spillover infections, Germany[END_REF]. Detection of hantavirus-reactive antibodies in carnivores, like foxes, cats and dogs, and in other wildlife species, such as wild moose, demonstrated the infectivity of the pathogen for other species (for review see Zeier et al. 2005).

Here we demonstrated a stable hantavirus seropositivity over a period of up to seven years in all investigated longitudinal samples of six animals including five M. mulatta (#4986, #9290, #9411, #9796, #9821) and one M. fascicularis (#9858). These data suggest a long-term immunity induced by naturally acquired hantavirus infection in these primates. Interestingly, for two rhesus monkeys (#9291 and #12083) an obvious PHV [START_REF] Chu | Cross-neutralization of hantaviruses with immune sera from experimentally infected animals and from hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome patients[END_REF], might have occurred at their breeding origin at other places in Germany, France or the USA, this, nevertheless, again confirms the susceptibility of nonhuman primates, i.e. M. mulatta, M. fascicularis and P. anubis for natural hantavirus infection.

Experimental hantavirus infection of non-human primates resulted in disease symptoms which resemble those observed in humans (Yanagihara et al., 1988;[START_REF] Groen | A macaque model for hantavirus infection[END_REF]Klingström et al., 2002a). A retrospective analysis of the clinical records of all animals investigated here did not show hints for clinical symptoms after hantavirus exposure. In addition, for three animals that died in 2004/2005 due to tularemia no signs of a co-morbidity of hantavirus and Francisella infections could be found. Additional studies are needed to prove whether naturally acquired hantavirus infections in different nonhuman primate species may result in an asymptomatic or mild course of disease with unspecific symptoms.

The herein reported detection of hantavirus-reactive antibodies, and a seroconversion in particular, in non-human primates in outdoor enclosures and the parallel demonstration of PUUV-and TULV-infected reservoir hosts in the surroundings suggest A c c e p t e d M a n u s c r i p t 10 that infection events occurred in the past. The exposure of monkeys in the GPC to wild rodents from the surroundings was previously already suggested due to the death of a group of cynomolgus monkeys (M. fascicularis) and the parallel detection of Francisella tularensis in rodents from the surroundings [START_REF] Mätz-Rensing | Epizootic of tularemia in an outdoor housed group of cynomolgus monkeys (Macaca fascicularis)[END_REF][START_REF] Kaysser | Re-emergence of tularemia in Germany: presence of Francisella tularensis in different rodent species in endemic areas[END_REF].

In conclusion, this is, to our knowledge, the first report of natural infections of nonhuman primates in outdoor enclosures in Europe raising important questions on future hantavirus surveillance in such husbandries. In addition, the results of the study accentuate the need of an efficient rodent combat and management in primate husbandries to protect susceptible primate species from hantavirus infection.

  the findings with the results from molecular hantavirus investigations in the local rodent populations.

  A c c e p t e d M a n u s c r i p t 6 collected during 2006 were positive for hantavirus-specific IgG antibodies with at least one of the used antigens (Table

  additional M. mulatta (#9291, #9292, #12083, #12103) demonstrated an initially negative or equivocal sample with all corresponding following samples being positive. The remaining three M. mulatta showed an oscillation of positive and negative reactivity (#2148, #13032) or vice versa (#11660). All three follow-up samples from M. fascicularis #9858 reacted with both antigens. Finally, the medical records were retrospectively checked for potential indications of a hantavirus infection. None of the seropositive animals

  2005 a total of 46 rodents, including 8 M. agrestis, 11 M. arvalis and 13 M. glareolus, have been trapped in the immediate vicinity and surrounding coppice of the GPC (Kaysser et al., 2008). RT-PCR analysis of lung tissue samples from M. arvalis, M. agrestis and M. glareolus revealed 3/11 (27.3 %) TULV-positive M. arvalis, but no indication for hantavirus infections in field and bank voles (data not shown). A pairwise comparison of the derived TULV sequences from Göttingen revealed a close relationship to the TULV sequences recently described from Sennickerode (see supplementary Table

  was observed between 2003/2005 and 2005/2006 suggesting an exposure to PUUV at the GPC during 2003-2006. Due to the hantavirus outbreak in Germany in 2005 with a total of 447 HFRS cases and an increased number of cases (n=11) in the district Göttingen (Robert Koch-Institut: SurvStat, www.rki.de; data as of February 10, 2009), one may speculate on a high infection burden in the bank vole populations resulting in increased numbers of human cases and the potential exposure to some primates in the outdoor enclosures. The serological findings of TULV-reactive antibodies in M. mulatta animal #9292 may also suggest a seroconversion with an exposure in 2003 or before. Although for some seroreactive monkeys in this study the exposure and infection with PUUV, TULV or PUUV/TULV-cross-reactive hantaviruses, i.e.

  Fig. 1. Maps of Germany showing the federal state Lower Saxony (marked in black; A) and

  reactivity of non-human primate sera from the German Primate Center (GPC) in Puumala virus (PUUV) and Tula virus (TULV) IgG-ELISA and -immunoblot assay (IB).

Table 1 )

 1 .

Table

	Species	Animal no.	Origin	Age a (years)/ sex/ year of death	Year of collection blood	PUUV	TULV
	Macaca mulatta	2039	GPC	8/f/2008	2006	-	+
	Macaca mulatta	2116	GPC	6/f	2006	+	+
				1/f	2002	E	+
	Macaca mulatta	2148	GPC	2/f 4/f	2003 2005	-+	--
				5/f	2006	(+)	-
	Macaca mulatta	2242	GPC	3/f/2008	2006	+	-
	Macaca mulatta	2305	GPC	2/m	2006	+	-
	Macaca mulatta	2323	GPC	1/f	2006	+	-
	Macaca mulatta	4986	USA, since 1998 in GPC	13/m 14/m 15/m	2005 2006 2007	+ + +	+ + +
	Macaca mulatta	9290	Strasbourg, since 1998 in GPC	6/f 8/f 9/f	2003 2005 2006	+ + +	+ E -
				7/f	2003	-	-
	Macaca mulatta	9291	Strasbourg, since 1998 in GPC	9/f 10/f	2005 2006	+ +	--
				11/f	2007	+	E
				11/f/2008	2003	-	E
	Macaca mulatta	9292	Strasbourg, since 1998 in GPC	13/f/2008 14/f/2008	2005 2006	--	+ +
				15/f/2008	2007	-	+

Acknowledgments

The excellent technical assistance of Brigitte Pohl, Angelika Lander (Berlin), Dörte Kaufmann (Greifswald-Insel Riems), Kerstin Weiß, Aileene Lorber and Astrid Thomas (Munich) is kindly acknowledged.

The serological investigations and the subsequent result evaluations were performed following the recently described scheme for seroepidemiological investigations using PUUV and TULV IgG-ELISA as screening tests and the corresponding IgG-IB for confirmation [START_REF] Mertens | Seroepidemiological study in a Puumala virus outbreak area in South-East Germany[END_REF]


Ruiqing Li

· Timo

M Takala

Mingqiang Qiao

Haijin Xu

· Per

E J Saris
email: per.saris@helsinki.fi

P E J Saris

T M Takala

· Mingqiang

Microbial and Enzyme Technology

Keywords: food-grade plasmid, pediocin, nisin selection

A nisin-resistant Lactococcus lactis strain TML01 was isolated from crude milk. A gene with 99% homology to the nisin-resistance gene, nsr, was identified. The food-grade secretion plasmid, pLEB690 (3746 bp), was constructed based on this novel nsr gene enabling primary selection with up to 5 µg nisin/ml. The functionality of pLEB690 as a secretion vector was shown by expressing and secreting the pediocin AcH gene papA in L. lactis. pLEB690 is therefore a functional food-grade secretion vector potentially useful for the food industry.

Introduction

Lactococcus lactis is widely used in dairy fermentations. A set of genetic tools has been developed to improve the natural properties of L. lactis [START_REF] Mierau | 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis[END_REF]. Potential tools for genetic engineering of food bacteria are the food-grade plasmid vectors (Reviriego et al., 2007a;[START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF]. The DNA of these vectors is derived from strains, which are generally regarded as safe (GRAS). In addition, these vectors do not rely on antibiotics for their selection. Some vectors endow the host strains with new properties, such as nisin-cadmium-or copper-resistance [START_REF] Liu | Development of food-grade cloning and expression vectors for Lactococcus lactis[END_REF][START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF]. Some vectors adopt sugar utilization or auxotrophic supplement for selection [START_REF] Platteeuw | Food-grade cloning and expression system for Lactococcus lactis[END_REF]Sørensen et al., 2000). These complementation vectors require host strains with special genotypes, thus limiting their usability. An ideal food-grade vector would be small, stable, allow strong selection, and contain elements for intra-or extracellular protein production.

Nisin is the only bacteriocin used as a food preservative and is therefore acceptable as a selective agent upon transformation of food-grade plasmids. The nisin immunity gene, nisI, from a nisin-producing L. lactis strain has been exploited as a selection marker in a food-grade vector [START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF]. Some non-nisin-producing L. lactis strains carry the nisin-resistance gene nsr [START_REF] Von Wright | Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin-resistance determinant[END_REF], which encodes a protease that digests nisin at the MeLan 28 site, resulting in 100-fold reduction of nisin activity [START_REF] Sun | Novel mechanism for nisin-resistance via proteolytic degradation of nisin by the nisin-resistance protein NSR[END_REF]. The nsr gene has also been used as a selection marker in food-grade vectors [START_REF] Froseth | Molecular characterization of the nisin-resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3[END_REF][START_REF] Liu | Development of food-grade cloning and expression vectors for Lactococcus lactis[END_REF][START_REF] Von Wright | Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin-resistance determinant[END_REF]. Even though the nisI and nsr vectors make cells resistant against ≥ 5 µg nisin/ml, efficient primary selection of plasmid transformants after electroporation has only been possible with lower nisin concentrations [START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF][START_REF] Von Wright | Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin-resistance determinant[END_REF]. Other issues limiting the applicability of nisin-selectable vectors have been the lack of inducible promoters and secretion signals for induced and extracellular production of proteins.

In this work, a novel nisin-resistance gene nsr was identified and cloned from the nisin resistant L. lactis strain TML01. The nsr gene was used to construct food-grade vectors for constitutive and inducible gene expression, as well as for secretion and intracellular protein production. The new nsr-vectors presented here mediated higher nisin-resistance than previously reported food-grade vectors.

Materials and methods

Bacteria, plasmids and culture media

Lactococcus lactis NZ9000 [START_REF] Kuipers | Controlled overproduction of proteins by lactic acid bacteria[END_REF], L. lactis N8 [START_REF] Graeffe | A natural nisin variant[END_REF], and pediocin-sensitive strain Listeria monocytogenes WSLC 1018 (gift from Prof Martin Loessner, ETH Zürich, Switzerland) were cultivated at 30°C in M17GS medium (M17 broth supplemented with 0.5% w/v glucose and 0.55% w/v sucrose). Pediocin-producer Lactobacillus plantarum WHE 92 [START_REF] Ennahar | Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese[END_REF] was cultivated at 30°C in MRS broth. M17GST medium (M17GS with 1% v/v Tween80) supplemented with nisin was used for isolation of nisin-resistant strains and detection of pediocin in agar diffusion plates. The plasmid pLEB590 [START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF] was used as a template in construction of the food-grade vectors.

DNA manipulations

PCR primers used in this study are listed in the Supplementary Table 1. PCR products were purified with the EZNA Cycle-pure kit (Omega Bio-tek) and sequenced (Institute of Biotechnology, Helsinki, Finland). Plasmids were isolated with the Plasmid Mini Kit (Omega Bio-tek) with following modifications: lysozyme was used at 1 mg/ml in solution I followed by incubation at 37°C for 1 h. Electrotransformation was performed as described previously [START_REF] Wu | Mu transposition complex mutagenesis in Lactococcus lactis--identification of genes affecting nisin production[END_REF].

Strain isolation, identification, and nisin-resistance gene amplification

Raw bovine milk from a local farm was serially 10-fold diluted. Aliquots were spread on M17GST agar plates containing 5 µg nisin/ml and 1% (v/v) Bromocresol Purple as pH indicator. The plates were incubated overnight at 30°C. The white round colonies which changed the medium from purple to yellow were transferred to new M17GST agar plates supplemented with 12.5 µg nisin/ml. The 16S rRNA gene of the isolated strain was amplified and sequenced using the primers 16S1 and 16S2.

Primers nsr1 and nsr2 were used to amplify the nisin-resistance gene nsr using the isolated strain as template. PCR was performed using the Dynazyme DNA polymerase (Finnzymes) under the conditions of: 95°C for 2 min; 30 cycles of 94 °C for 20 s, 40 °C for 15 s, and 72 °C for 1 min; and finally 72 °C for 10 min. PCR products were analysed in 1% agarose gel with 1 kb DNA ladder (Fermentas) as the size marker.

Construction of food-grade vectors

The gene, nsr, was amplified by PCR using Phusion DNA polymerase (Finnzymes) with primers nsr3 and nsr4, digested with HindIII and EcoRV, and ligated with a HindIII-EcoRV -fragment from pLEB590 containing the replicon and P45 promoter. The formed food-grade vector, named pLEB688, was digested with HindIII and BglII, and ligated with the nisZ promoter amplified from L. lactis N8 with primers PnisZ1 and PnisZ2 (digested with HindIII and BglII).

The constructed nisin-inducible, food-grade vector pLEB689 was used as a template for construction of the food-grade secretion vector pLEB690 by overlap PCR. In the first step PCR, primers usp1 and usp2 were used to amplify usp45 signal sequence from NZ9000, while nsr5 and nsr6 were used to amplify pLEB689. The first step products were used as templates in the second step PCR with the primers nsr5 and usp2. Finally, the second step product was restricted with BglII and self-ligated.

Analysis of nisin-resistance level and plasmid stability L. lactis NZ9000 was electrotransformed with the constructed plasmids. Nisin-resistance was determined with a plate reader at 30°C for 20 h with wide band filter of 420-580 nm, by inoculating 3 μl overnight bacterial culture into 300 μl M17GS medium supplemented with nisin (0 to 12.5 µg/ml) in 100-well plates. Of each sample, three parallels were made. Plasmid stability was determined essentially as described before [START_REF] Platteeuw | Food-grade cloning and expression system for Lactococcus lactis[END_REF].

Pediocin secretion vector and pediocin bioassay

The pediocin gene papA without secretion signal was amplified by PCR with primers ped1 and ped2 using Lb. plantarum WHE 92 cells as template. The PCR product was digested with BglII and linked to the NaeI and BglII digested pLEB690 plasmid. The formed pediocin secretion vector pLEB691 (Fig. 1d) was introduced into L. lactis NZ9000.

The antimicrobial activity of the pediocin produced by the NZ9000/pLEB691 strain was tested by agar diffusion bioassay (Reviriego et al., 2007a) with L. monocytogenes WSLC 1018 as the indicator organism. L. lactis N8 and Lb. plantarum WHE 92 were used as positive controls for antimicrobial activity. NZ9000 harboring plasmid pLEB690 (no papA gene) was used as a negative control. All tested strains were cultured overnight at 30°C. For the induced gene expression, 0.5 µg nisin/ml was added after 4 h. Next day, the OD 600 of each culture was measured and the cultures were diluted with fresh medium to equal densities. The diluted cultures were then centrifuged and sterilized through a 0.2 µm filter. Each supernatant, 200 µl, was added into the well (diam. 9 mm) in M17GST agar with a lawn of the indicator L. monocytogenes strain. The plate was incubated overnight at 30°C.

Results

Isolation of a nisin-resistant strain and nsr identification Nisin resistant strains were screened from raw milk on M17GST agar plates with 5 µg nisin/ml. Eight colonies were picked onto a new plate containing 12.5 µg nisin/ml. One isolate, TML01, survived and was identified as Lactococcus lactis, according to 99% homology to 16S rRNA sequences of L. lactis strains (Supplementary Table 2). Genes for nisin production were not detected from L. lactis TML01 (data not shown).

Many nisin-resistant but non-producing strains express the NSR, a protease cleaving nisin between MeLan 28 and Ser 29 [START_REF] Sun | Novel mechanism for nisin-resistance via proteolytic degradation of nisin by the nisin-resistance protein NSR[END_REF]. To determine if the nisin-resistance of the strain TML01 was also caused by NSR, the nsr gene was sought by PCR with nsr-specific primers. The PCR showed that L. lactis TML01 contains an nsr gene. The gene was sequenced (accession number 1384934 in NCBI) and the derived amino acid sequence showed 99% homology to NSR nisin-resistance proteins, as shown in Supplementary Figure 1.

Construction of the food-grade vectors

The food-grade vector pLEB688 was constructed by replacing the nisI gene of pLEB590 with the novel nsr gene. New multiple cloning site were introduced by PCR using nsr3 and nsr4 primers (Fig. 1a). A transcription terminator from Bifidobacterium longum DJO10A putA2 operon [START_REF] Lee | Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth[END_REF] was added downstream from nsr with the primer nsr4.

For inducible gene expression, another food-grade vector pLEB689 was constructed by inserting the nisZ promoter with its own ribosome binding site and the initiation codon ATG into the HindIII/BglII sites of pLEB688 (Fig. 1b). Two nucleotides, AA, before the initiation codon ATG of nisZ were changed to CC, creating an NcoI restriction site for in-frame gene cloning. Lastly, the food-grade secretion vector was constructed by cloning the usp45 signal sequence into pLEB689 by overlap PCR. Simultaneously, the NcoI was replaced with the BspHI site between the P nisZ and the SSusp45 (Fig. 1c). The last codon, GCT encoding Ala 27 in the usp45 signal sequence, was changed to another alanine codon GCC. Hence, a NaeI site was created, enabling blunt-end fusion with the SSusp45 and a cloned gene with no extra N-terminal amino acids in the mature secreted protein (Fig. 1c).

Vector stability and nisin-resistance of L. lactis strains The stabilities of the constructed food-grade vectors were examined with serial subcultures in non-selective M17GS media. All food-grade vectors were stably maintained for more than 100 generations. Only 2-4% of the cells carrying the food-grade vectors lost their nisin-resistance. Single colonies obtained from the M17GST+nisin agar plates were found to contain plasmids with the expected size and restriction pattern (data not shown).

Nisin-resistance levels of the food-grade vector strains were determined by culturing the strains with different nisin concentrations. Nisin-resistances of the NZ9000 strain harboring the different vectors are shown in Fig. 2. NZ9000 is a nisin-sensitive strain which could not grow even in the lowest nisin concentration tested, 1.25 µg/ml. All the other strains grew in all tested nisin concentrations but variations in growth were observed. The nisI strain was visibly suffering in 2.5 µg nisin/ml, and its growth was strongly retarded with 5 µg nisin/ml. Also, with 5 µg nisin/ml the strain carrying the secretion vector pLEB690 grew slower than the other nsr vector strains. Nevertheless, the novel food-grade vectors were shown to be stable and to provide higher nisin-resistance than the previously constructed nisin-selectable plasmids.

Intra-and extra-cellular protein production from the nsr food-grade vectors Intracellular protein production from the expression vectors pLEB688 and pLEB689 was examined by the expression of the Lactobacillus helveticus pepI gene similarly as described by [START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF] (data not shown). To determine the applicability of the novel secretion vector pLEB690, a pediocin gene was fused with SSusp45 in the vector. The Lb. plantarum pediocin gene papA without leader sequence was amplified by PCR with primers ped1 and ped2. The PCR product was digested by BglII and cloned into NaeI/BglII -cut pLEB690, resulting in the papA secretion plasmid pLEB691 (Fig. 1d). Plasmid pLEB691 was transferred into the non-pediocin-producer L. lactis NZ9000.

Anti-listerial potential of the putative pediocin-secretion strain was tested by agar diffusion assay. L. lactis NZ9000 harboring the empty vector pLEB690 showed no anti-Listeria activity. L. lactis NZ9000 with the pediocin secretion plasmid pLEB691 exhibited antilisterial activity both in 0.5 µg nisin/ml -induced and non-induced conditions, indicating that the strain expressed and secreted pediocin from the plasmid (Fig 3). Nisin-induced pediocin production was higher than that without induction. The native pediocin producer Lb. plantarum WHE 92 was still better Listeria-killer than the NZ9000/pLEB691 or the nisin producer L. lactis N8. However, the successful secretion of pediocin concluded that the food-grade vector pLEB690 can be regarded as a secretion vector for L. lactis.

Discussion

A nisin-resistance gene nsr was found in L. lactis TML01 isolated from raw milk. This new NSR showed 99% homology to previously reported nisin-resistance proteins derived from different L. lactis strains. The nsr gene is a good candidate as a food-grade selection marker due to its nisin inactivation capacity [START_REF] Sun | Novel mechanism for nisin-resistance via proteolytic degradation of nisin by the nisin-resistance protein NSR[END_REF]. Three food-grade vector variants, pLEB688, pLEB689 and pLEB690, were constructed with this new nsr gene as the selection marker (Fig. 1). All these vectors are small (< 3.8 kb) and stable (only 2 to 4% loss in 100 generations) for gene cloning.

The functionality of the nsr food-grade vectors for intracellular protein production was verified by cloning the pepI gene into the plasmids pLEB688 and pLEB689 followed by analysis of PepI activity (results not shown) similarly as described by [START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF]. The potential for protein secretion of the vector pLEB690 was tested by cloning the lactobacillar pediocin gene papA. Pediocin is a broad-spectrum bacteriocin that shows particularly strong anti-Listeria activity [START_REF] Dabour | In vivo study on the effectiveness of pediocin PA-1 and Pediococcus acidilactici UL5 at inhibiting Listeria monocytogenes[END_REF]. Several studies have demonstrated that L. lactis is a good host for pediocin expression [START_REF] Arques | Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains[END_REF]Reviriego et al., 2007a;[START_REF] Lee | Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth[END_REF]. Nisin-inducible expression of pediocin has also been successfully applied (Renye & Somkuti, 2009;Reviriego et al., 2007a). However, use of the usp45 signal sequence has not been previously reported for pediocin secretion, and the secretion of pediocin from a food-grade vector with nisin selection is also novel. L. lactis NZ9000 carrying the pediocin plasmid, pLEB691, provided higher anti-listerial activity in presence of nisin than without nisin, showing that the nisZ promoter and nisin induction are functional in the pLEB690 vector. NSR is a protease reducing nisin activity and induction sensitivity [START_REF] Liang | Adverse effect of nisin-resistance protein on nisin-induced expression system in Lactococcus lactis[END_REF][START_REF] Sun | Novel mechanism for nisin-resistance via proteolytic degradation of nisin by the nisin-resistance protein NSR[END_REF], which could lead to inhibition of induction. However, the concentration of nisin needed for promoter induction is very low, compared to e.g. the concentrations used in food or for plasmid selection pressure. The recommended concentration for nisin induction is 0.1-5 ng/ml [START_REF] Mierau | 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis[END_REF]. Here, the expression of the pediocin gene was induced with 500 ng nisin/ml, about 100-1000 times more than what is sufficient. It is unlikely that NSR would degrade nisin from 500 ng/ml to below the inducible concentrations before nisin induction would occur. The higher pediocin production with nisin showed that nisin induction occurred fast enough before NSR would degrade the nisin concentration below an inducible level.

Even though L. lactis is naturally resistant to pediocin, co-expression of the pediocin immunity gene with the structural gene has been shown to increase the pediocin production up to the wild-type level [START_REF] Arques | Immunity gene pedB enhances production of pediocin PA-1 in naturally resistant Lactococcus lactis strains[END_REF]. Thus, in the wild-type, pediocin producer, Lb. plantarum WHE 92, the expression of the native immunity gene may explain why it was still clearly better pediocin producer than the heterologous host L. lactis with no immunity gene. However, in this work, the aim was not to produce large amounts of pediocin, but to show the applicability of pLEB690 by secreting biologically active pediocin. Moreover, L. lactis NZ9000 harboring pLEB691 could also produce pediocin without nisin induction due to the constitutive P45 promoter. The promoter P45 broadens the applicability of the vector, as it can be used in L. lactis strains lacking the nisin regulatory genes. To sum up, the food-grade secretion vector pLEB690 enables nisin selection in transformation, is small and stable, and it can be used both for constitutive and inducible secretion of polypeptides in L. lactis.

The nsr gene used in this study made cells more resistant to nisin than the gene nisI, which was used as a selection marker in previous food-grade vector pLEB590 (Fig. 2). In the previous study, plasmid pLEB590 enabled higher nisin concentration in selection than the nsr vector pVS40 [START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF][START_REF] Von Wright | Isolation of a replication region of a large lactococcal plasmid and use in cloning of a nisin-resistance determinant[END_REF]. This was probably because pVS40 (7.8 kb) is larger than pLEB590 (3.1 kb), which may have reduced the nisin-resistance capacity due to a copy number effect. The nsr plasmids in this study are smaller than 4 kb, and the expression of nsr is directed not only from its own promoter but also from P45 and P nisZ promoters. These differences may lead to a stronger expression of nsr, explaining the obtained higher nisin-resistance than with previously constructed vectors.

However, the secretion vector pLEB690 grew slower than the other nsr vectors in higher nisin concentrations. This could be explained by the secretion of a 7-aa nonsense peptide, caused by the expression of a fusion with SSusp45 and the 21-bp open frame in MCS after the SSusp45 (Fig. 1). In addition to the waste of energy, secretion of this useless peptide causes higher load for the sec-dependent secretion system, which may decelerate the growth. In the other nsr vector pLEB689, there is an in-frame stop codon 3 bp after the start ATG, thus saving energy from production of unwanted useless peptides.

With the nisI vector pLEB590, transformant colonies had to be selected by pour plate method [START_REF] Takala | A food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI[END_REF]. In this study, the nsr vectors could be selected by conventional surface spread plate method, when 1% v/v Tween 80 was added into the selection medium (results not shown). Tween 80 is a surfactant of which increases nisin solubility and reduces its adsorption onto surfaces [START_REF] Mocquot | A simple procedure to detect nisin in cheese[END_REF]. However, the function of Tween 80 in nisin selection is not fully clear. After transformation, the cells spread onto the agar form a layer. By adsorbing nisin molecules, the cell layer may hinder nisin from the medium to reach the cells on the top of the layer. Tween 80 could reduce nisin adsorption onto the dead cells, allowing nisin to diffuse through the cell layer to the live cells on the top. This would inhibit the growth of background colonies and allow transformant selection on the agar surface.

In conclusion, three food-grade vectors were constructed, all of which enabled higher nisin selection pressure than earlier published nisin selectable food-grade vectors. The secretion vector pLEB690 contains a novel combination of the usp45 secretion signal, dual expression promoters (constitutive P45 and nisin-inducible P nisZ ), and food-grade nisin-resistance gene nsr selection marker. In particular, this vector could be useful for secreting bioactive peptides in the intestine or in food applications. Fig. 2 Nisin-resistance analysis of L. lactis N8 (black square), L. lactis NZ9000 (empty diamond) and L. lactis NZ9000 strains containing pLEB688 (empty square), pLEB689 (black triangle), pLEB690 (empty triangle) and pLEB590 (black diamond). a, 0 µg nisin/ml; b, 1.25 µg nisin/ml; c, 2.5 µg nisin/ml; d, 5 µg nisin/ml. Fig. 3 Agar diffusion bioassay with Listeria monocytogenes WSLC 1018 as indicator. A, 0.5 µg nisin/ml induced NZ9000/pLEB691; B, non-induced NZ9000/pLEB691; C, pediocin producer Lb. plantarum WHE 92; D, 0.5 µg nisin/ml induced NZ9000/pLEB690; E, non-induced NZ9000/pLEB690; F, nisin producer L. lactis N8.

Fig. 1

 1 Fig. 1 Vectors constructed in this work. P45: lactococcal constitutive promoter; P nisZ : nisin-inducible promoter of nisZ; SSusp45: signal sequence of lactococcal gene usp45; papA: gene encoding mature pediocin; nsr: gene encoding the nisin-resistance protease; Ter: transcription terminator. Changed nucleotides are marked by gray shade. The functional ATG initiation codon of pLEB689 is bolded.

  Fig. 1

Fig. 3

 3 Fig. 3

Table 2

 2 Homology of the 16S rRNA gene sequence of Lactococcus lactis TML01 with L. lactis Lactococcus lactis subsp. lactis bv. diacetylactis gene for 16S rRNA, partial

	Accession	Description

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 30970095), China Scholarship Council and the University of Helsinki, Finland.

Supplementary Table 1 Primers used in this study

Name


Minna M Pulkki
email: minna.pulkki@helsinki.fi

Samu Myllymaa

Arja Pasternack

Stanley Lun

Helen Ludlow

Ahmed Al-Qahtani

Olexandr Korchynskyi

Nigel Groome

Jennifer L Juengel

Nisse Kalkkinen

Mika Laitinen

Olli Ritvos

Ahmed Al- Qahtani

David G Mottershead

The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: characterization of the purified untagged processed mature region

Keywords: BMP15, bone morphogenetic protein, granulosa cell, human _______________________________________________________________________________________

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The bioactivity of human bone morphogenetic protein-15 is sensitive to C-terminal modification: characterization of the purified untagged processed mature region

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Introduction

Bone morphogenetic protein-15 (BMP15) also called growth differentiation factor-9B (GDF9B) is an oocyte-derived growth factor and a member of the transforming growth factor-β (TGF-β) superfamily discovered in 1998 by two independent research groups [START_REF] Dube | The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes[END_REF][START_REF] Laitinen | A novel growth differentiation factor-9 (GDF-9) related factor is coexpressed with GDF-9 in mouse oocytes during folliculogenesis[END_REF]). BMP15 and its closest homolog growth differentiation factor-9 (GDF9) [START_REF] Mcpherron | GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines[END_REF][START_REF] Mcgrath | Oocyte-specific expression of growth/differentiation factor-9[END_REF][START_REF] Dube | The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes[END_REF][START_REF] Hayashi | Recombinant growth differentiation factor-9 (GDF-9) enhances growth and differentiation of cultured early ovarian follicles[END_REF][START_REF] Jaatinen | Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B[END_REF]) play crucial roles in the regulation of fertility (Gilchrist et al. 2004a;McNatty et al. 2005c). Mutations in these genes lead to reproductive defects in animals [START_REF] Mcnatty | The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology[END_REF]) and with the recombinant forms of these growth factors the actions of the oocyte can be mimicked (Gilchrist et al. 2004b). Further, mutations in the human BMP15 or GDF9 genes are associated with polycystic ovary syndrome A c c e p t e d M a n u s c r i p t 2 (PCOS), premature ovarian failure (POF) or dizygotic twinning (Teixeira Filho et al. 2002;[START_REF] Di Pasquale | Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene[END_REF][START_REF] Dixit | Mutational screening of the coding region of growth differentiation factor 9 gene in Indian women with ovarian failure[END_REF][START_REF] Di Pasquale | Identification of new variants of human BMP15 gene in a large cohort of women with premature ovarian failure[END_REF][START_REF] Dixit | Expansion of the germline analysis for the INHA gene in Indian women with ovarian failure[END_REF][START_REF] Laissue | Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure[END_REF][START_REF] Palmer | Novel variants in growth differentiation factor 9 in mothers of dizygotic twins[END_REF]). Furthermore, both proteins have been shown to regulate ovulation rate in sheep [START_REF] Galloway | Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner[END_REF][START_REF] Juengel | Effects of immunization against bone morphogenetic protein 15 and growth differentiation factor 9 on ovulation rate, fertilization, and pregnancy in ewes[END_REF]. One approach to the evaluation of the role of BMP15 on granulosa cells has been the use of unpurified wild type recombinant BMP15 produced in mammalian cells. Such studies show that the functions of BMP15 vary between species (McNatty et al. 2005b;McNatty et al. 2005a) and that BMP15 enhances oocyte developmental competence [START_REF] Hussein | Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins[END_REF][START_REF] Hussein | Oocyte-secreted factors enhance oocyte developmental competence[END_REF]).

An alternative approach for the characterization of the effects of BMP15 on granulosa cells has utilized purified engineered recombinant forms of BMP15 (reviewed in [START_REF] Mottershead | Oocyte peptides as paracrine tools for ovarian stimulation and oocyte maturation[END_REF]. In particular, quite a number of studies have utilized a recombinant human BMP15 (rhBMP15) tagged with a Flag epitope at the C-terminus of the mature region and purified using anti-Flag M2 Affinity Gel (Otsuka et al. 2001a;Otsuka et al. 2001b;Otsuka and Shimasaki 2002b;Otsuka and Shimasaki 2002a;[START_REF] Moore | Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[END_REF]McMahon et al. 2008;[START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF]Tibaldi et al. 2010). One of these recent studies using the C-terminally Flag tagged rhBMP15 showed that the 17 kDa rhBMP15 is O-glycosylated and the 16 kDa form is phosphorylated [START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF]). Further, it was shown that the phosphorylation of the 16 kDa form is essential for rhBMP15 bioactivity [START_REF] Mcmahon | Phosphorylation of bone morphogenetic protein-15 and growth and differentiation factor-9 plays a critical role in determining agonistic or antagonistic functions[END_REF]. In contrast to these reports, the recent study by [START_REF] Li | Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15[END_REF]) describes the production and isolation of recombinant hBMP15 tagged with a Flag epitope at the N-terminus of the mature region and purified using anti-Flag M2 Affinity Gel.

We have recently characterized recombinant human GDF9 (hGDF9) signaling in ovarian granulosa cells and found that the position of the affinity purification tag crucially effects GDF9 bioactivity [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF]. We found that a C-terminal His 6 tag destroys GDF9 bioactivity, the purified untagged mature region of recombinant human GDF9 being biologically active, exhibiting the ability to activate a Smad3/4 specific transcriptional reporter in human granulosa-luteal cells. Like other members of the superfamily, BMP15 and GDF9 are produced as precursor proteins with the biologically active portion of the protein residing in the C-terminus.

However, BMP15 and its closest homologue GDF9 are unusual within the TGF-β superfamily, as they lack the fourth conserved cysteine residue involved in dimer formation [START_REF] Laitinen | A novel growth differentiation factor-9 (GDF-9) related factor is coexpressed with GDF-9 in mouse oocytes during folliculogenesis[END_REF] found in most TGF-β family members [START_REF] Daopin | Crystal structure of transforming growth factor-beta 2: an unusual fold for the superfamily[END_REF][START_REF] Schlunegger | Crystallization and preliminary X-ray analysis of recombinant human transforming growth factor beta 2[END_REF][START_REF] Griffith | Threedimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily[END_REF]. In this current study we have characterized the purified recombinant human BMP15wt and -C-Flag (similar to the one in published literature [START_REF] Otsuka | Bone morphogenetic protein-15. Identification of target cells and biological functions[END_REF]) mature regions and we find, that like in the case of GDF9, the bioactivity of rhBMP15 is crucially affected by the position of an affinity purification tag.

Materials and methods

Expression vector construction

To prepare human BMP15 expression constructs the coding region of hBMP15 was first amplified from human genomic DNA with following primers: the exon 1 encoding region 5´-AAATGGTCAGAGTGACGTCCCTT and 5´-AGGTACCTCTGTGAGGCCTTGCCAC and the exon 2 encoding region 5´-GGGCTGATTATAGCTATCAGTC and 5´-TCTCCTCCAGTTTCCTGGGAAA.

The amplified fragments were first subcloned into pGTE vector and thereafter transferred to the pEFIRES-P expression plasmid [START_REF] Hobbs | Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins[END_REF]. Flag and His 6 tagged vectors were constructed by introducing the affinity tag into the pro-or mature region by PCR and by subcloning the pro-and the mature regions into the pEFIRES-P expression vector. Human BMP15 NFlag6H pro-region was amplified with following primers: (5' primer) 5' GAAGATCTCACACCATCACCATCACCATG AACACAGGGCCCAAATGG 3' and (3' primer) 5'GGACTAGTTTATCATCGACGGCGCCGGA GAAGAGATTCCCT 3'. Human BMP15 C-Flag mature-region was amplified with following primers:

(5' primer) 5' TAGATCTCGGCGCCGCCGACAAGCAGATG GTATCTCA 3' and (3' primer) 5' AACTAGTTTATCACTTATCGTCGTCATCCT TGTAATCTCTGCATGTACAAGACTC 3'.

Protein expression and analysis

Development of a HEK-293T cell line expressing mGDF9wt protein has been previously described [START_REF] Kaivo-Oja | Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells[END_REF]. Cell lines expressing epitope tagged processed human BMP15 were developed by a similar protocol and A c c e p t e d M a n u s c r i p t 3 were used as sources of recombinant hBMP15 proteins. The purified proteins were analysed on silver gels and Western blots. Reduced protein fractions (with 10 mM DTT) were run in 15% SDS-PAGE gels, and stained with silver nitrate and blotted onto a Hybond C nitrocellulose membrane as described previously (Kaivo-Oja et al. 2003). Blotted membranes were treated with BMP15 specific mAb-28 (1:10,000) and a secondary antibody, peroxidase-conjugated anti-IgG (Jackson ImmunoResearch Laboratories, Inc.; 1:20,000). Immunoreactive proteins were detected using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) or alkaline phosphatase based detection (Bio-Rad). The purified proteins were quantified by measuring absorbance at 280 nm divided by extinction coefficient of the protein.

Generation of monoclonal antibody for hBMP15

A synthetic peptide corresponding to a region near to the C-terminus of the human BMP15 was selected as the immunogen (prepared by Sigma-Genosys Ltd, UK). The sequence was S

-A-E-V-T-A-S-S-S-K-H-S-G-P-E-N-N-Q-C.
Five female outbred Tyler Original (T/O) mice (Southend on Sea, Essex, U.K.) were primed with BCG vaccine. Four weeks later, the mice were initially immunised with the immunogen (human BMP15 synthetic peptide/ PPD conjugate) in Freund's incomplete adjuvant. After that, the mice received 3x monthly subcutaneous boosts of 50 g of the immunogen in Freund's incomplete adjuvant. The animals were killed and the spleens removed for fusion to Sp2/0 murine myeloma cells, following standard procedures. The hybridoma supernatants were initially screened by ELISA against the recombinant hBMP15 coated to Nunc immunoplates by standard protocol [START_REF] Groome | Monoclonal and polyclonal antibodies reactive with the 1-32 amino terminal sequence of the alpha subunit of human 32K inhibin[END_REF]). Reactive clones were expanded and recloned by limiting dilution. These were then rescreened against recombinant BMP15 and the best reacting clones selected prior to expansion and isotyping. As each clone was found to be IgG1, all were purified on a protein G column using a high-salt protocol [START_REF] Harlow | Antibodies : a laboratory manual[END_REF]) before assessment.

Reagents and growth factors

TGF-β1 was purchased from R&D Systems (Minneapolis, MN). E. coli produced BMP2 was a kind gift from Dr. Peter Mace (University of Otago, NZ). Fetal calf serum (FCS) was purchased from Euroclone Ltd. (Devon, UK). DMEM and Ham's F-12 were purchased from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). Heparin (Fragmin) was purchased from Pharmacia & Upjohn (Stockholm, Sweden). BSA was purchased from Roche (Mannheim, Germany).

Peroxidase-conjugated rabbit antimouse IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). BMPR2ecd-Fc was produced in mammalian cells and purified in house [START_REF] Myllymaa | Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice[END_REF]).

Reporter gene constructs

The usage of the pGL3BREluciferase reporter plasmid [START_REF] Korchynskyi | Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter[END_REF] and generation of the recombinant adenovirus BRE-Luc has been previously described [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF].

2.6. hGL cell cultures hGL cells were obtained with informed consent from women undergoing in vitro fertilization (IVF) treatments. For each experiment, cells from one to six patients were pooled, enzymatically dispersed, and separated from red blood cells by centrifugation through Ficoll-Paque as previously described [START_REF] Eramaa | Inhibin/activin subunit mRNA expression in human granulosa-luteal cells[END_REF]. Thereafter, hGL cells were plated at a density of 3-4x10 4 cells/well on 24-well plates (Cellstar, Greiner Bio-one, Frickenhausen, Germany; final concentration, 3-4x10 4 cells/ml). hGL cells were cultured in DMEM supplemented with 10% FCS, 2 mM l-glutamine, and antibiotics (100 IU/ml penicillin, 100 g/ml streptomycin and 0.25 g/ml amphotericin-B; GIBCO). Cells were cultured 1-2 d before adenovirus infections and 2-3 d before ligand stimulation experiments.

Adenovirus infections

We have previously optimized the usage of a reporter BRE-Luc adenovirus [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF] which was generated accordingly to provided protocol using AdEasy system as described previously [START_REF] He | A simplified system for generating recombinant adenoviruses[END_REF].

Viruses were amplified and titrated in transcomplemental 293A cells and purified with cesium chloride gradient ultracentrifugation as described previously [START_REF] He | A simplified system for generating recombinant adenoviruses[END_REF]. The use of recombinant adenoviruses in hGL cultures has been previously optimized (Bondestam et al. A c c e p t e d M a n u s c r i p t 4 2002). The hGL cells were infected by incubating the cells with viruses at 37º C in serum-free DMEM supplemented with l-glutamine and antibiotics for 45 min, and DMEM containing 2% FCS was added on top to stop the infection. The cells were then incubated for 24 h before continuing the luciferase-assay experiments.

2.8. Transient transfections and luciferase assays COV434 cells were plated on 24well plates and cultured in DMEM supplemented with 10% FCS, 2 mM l-glutamine, 100 IU/ml penicillin, and 100 g/ml streptomycin at 37˚ C in 5% CO 2 . When the cells became 40-50% confluent, the transfections were performed in 0.5 ml medium with 100 ng/well of the BMP response element (BRE)-luciferase reporter construct [START_REF] Korchynskyi | Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter[END_REF] and 20 ng/well ß-galactosidase reporter plasmid using the Fugene 6 transfection reagent (Roche Applied Science). Twenty-four hours later COV434 cells were starved 6-8 h in 0.2% FCS DMEM and treated with BMP2 or BMP15 for 24 h. The recombinant BMPRII receptor protein that was used for the neutralization assays consist of a human receptor extracellular domain (ECD) fused to the Fc region of human IgG via a polypeptide linker (receptor ECD/Fc chimera proteins). The purification of BMPR2ecd-Fc is described in our recent study [START_REF] Myllymaa | Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice[END_REF]. Ligand and receptor proteins were incubated in 0.2 FCS DMEM in RT for 45 minutes, before adding to the cells. Ad-BRE-luc infected hGL cells were treated with BMP2 or BMP15 in 2% FCS/DMEM for 24 h. The cells were then lysed into 1x passive lysis buffer, and luciferase activity was measured with luciferase assay reagent (Promega Corp., Madison, WI) and normalized to ß-galactosidase activity. Data are the mean ±sem of triplicate determinations from representative experiments, relative to an adjusted value of 1.0 for the mean of the control wells. In each assay, each treatment was applied to 3 replicate wells. The bioassays were repeated at least 3 times with COV434 cells or with 3 separate pools of granulosa cells and the data are from representative experiments.

2.9. Analysis of Smad phosphorylation COV434-and hGL cells were cultured on 24 well plates in DMEM supplemented with 10% FCS, 2 mM l-glutamine, and antibiotics (100 IU/ml penicillin, 100 g/ml streptomycin and additionally 0.25 g/ml amphotericin-B for hGL cells; GIBCO). When COV434 cells reached 80% confluency and hGL cells were cultured 2-3 days, the COV434 cells were incubated with 0.2% FCS DMEM and hGL cells with 1% FCS DMEM for 4-6 hours before stimulations. Subsequent stimulation times varied from 15 to 90 minutes, and cells were thereafter recovered in ice-cold PBS and subsequently lysed in reduced (10 mM DTT) SDS-PAGE sample buffer. The samples were sonicated for 10 sec (amplitude, 5 µm) as described previously [START_REF] Kumar | Nodal signaling uses activin and transforming growth factor-beta receptor-regulated Smads[END_REF], boiled 3 minutes, run in 10% SDS-PAGE gels, and blotted onto a Hybond C nitrocellulose membrane. The membranes were blocked for 30 minutes at RT in a 5% (wt/vol) nonfat dried milk in Tris-buffered saline/Nonidet P-40 [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Nonidet P-40] and incubated with the primary antibody in 5% milk in Tris-buffered saline/Nonidet P-40 overnight at 4˚ C (dilution, 1:1 000 for pS1). After washing, the membranes were incubated with the secondary antibody, a peroxidase conjugated anti-IgG (Jackson ImmunoResearch Laboratories, Inc.; 1:20,000), for 1 h in RT. Immunoreactive proteins were detected using enhanced chemiluminescence reagents (Pierce Biotech). Specificity of the antibodies for phospho-Smads has been previously reported [START_REF] Persson | The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation[END_REF]).

Measurement of DNA synthesis

Determination of the effects of BMP15 on [ 3 H]-thymidine incorporation of rat granulosa cells was performed as described (McNatty et al. 2005a). All experiments involving rats were approved by the Wallaceville Animal Ethics committee. Briefly, granulosa cells were collected from all surface visible follicles approximately 46 h after i.p. administration of 20 IU eCG (Intervet Ltd, Auckland NZ) to 23-26 day old Sprague-Dawley rats. Isolated cells (20,000 cells per well) were incubated in M199 (Earle's salts; Sigma) with 100 U/ml penicillin (Invitrogen), 100 g/ml streptomycin (Invitrogen), 2mM GlutaMax-1 (Invitrogen), 0.23 mM sodium pyruvate (Sigma) 0.3 mg/ml polyvinyl alcohol (Sigma) purified hBMP15wt or -C-Flag tagged mature region at varying concentrations (0.2-400 ng/ml). After 18h of culture, methyl-[ 3 H]-thymidine (Perkin Elmer) was added to each well and cells were harvested for determination of [ 3 H]-thymidine incorporation A c c e p t e d M a n u s c r i p t 5 6 hours later. In each assay, each treatment was applied to 4 replicate wells. The bioassay was repeated with 3 separate pools of granulosa cells. The average cpm for each treatment was calculated as described for each pool of granulosa cells (McNatty et al. 2005a). Differences between controls and treatments or between treatments were analyzed using the paired t-test function of Microsoft Excel 2003. Data were transformed (natural log) prior to analyses.

Inhibin B ELISA Assay

We have previously developed an improved Inhibin B ELISA assay [START_REF] Ludlow | Development of a new antibody to the human inhibin/activin betaB subunit and its application to improved inhibin B ELISAs[END_REF]. The ELISA consists of monoclonal antibody '46A/F' (recognising the B subunit) as the capture antibody and 'R1_FAb' (recognising the  subunit) as the detection antibody. Briefly, human granulosa cells were treated with growth factors in 24 well plates in triplicate and media was harvested after 48hours or every 24 hours (24-96h) in time course assay. These samples were pipetted in duplicate to Nunc Maxisorp plates coated with antibody '46A/F' and incubated o/n. The following day the plates were washed and R1 FAb was added and incubated for 1 h. Plates were washed again, and streptavidin HRP was added for 1 h. Finally plates were washed again, and detection was carried out using TMB substrate, with colour development being detected with iEMS Microplate reader.

2.12. Reversed phase chromatography, in-gel digestion, protein sequencing and mass spectrometry Protein separation by reversed phase chromatography was performed on a 1 x 150mm Jupiter C4, 5µm, 300Å (Phenomenex Inc, USA) column using a SMART™ System (Pharmacia Biotech, Uppsala, Sweden) instrument. Proteins were eluted with a linear gradient of acetonitrile (0-100% in 60min) in 0.1% trifluoroacetic acid. Flow rate was 50µl/min and detection at 214nm. For in-gel digestion the P16 and P17 protein bands from SDS-PAGE [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] were separately in-gel digested with trypsin [START_REF] Bamford | Constituents of SH1, a novel lipidcontaining virus infecting the halophilic euryarchaeon Haloarcula hispanica[END_REF] and the resulting peptides subjected to peptide mass fingerprint analysis by MALDI-TOF mass spectrometry.

N-terminal protein sequencing, after electroblotting from SDS-PAGE to PVDF membrane [START_REF] Matsudaira | Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes[END_REF]) was performed using a Procise 494A Sequencer (Applied Biosystems, Foster City, CA, USA). MALDI-TOF mass spectrometry was performed in the reflector-or linear ion mode using an Ultraflex TOF/TOF instrument (Bruker-Daltonik GmbH, Bremen, Germany) equipped with a 337 nm UV nitrogen laser. Sinapic acid, α-cyano-4hydroxycinnamic acid or 2.5-dihydroxybenzoic acid were used as the matrix. The instrument was externally calibrated using a standard mixture of proteins or peptides (Bruker-Daltonik). Electrospray mass spectrometry was performed using a Q-TOF instrument (Micromass Ltd, Manchester UK) externally calibrated with horse heart myoglobin (MW=16952Da).

Results

Human BMP15 produced by HEK-293T cells is sensitive to C-terminal modification

The different forms of recombinant BMP15 derived from the expression constructs shown in Fig. 1A were produced in stable HEK-293T cell lines. These epitope tagged and untagged versions of BMP15 were detected in medium conditioned by these cell lines utilizing monoclonal antibody 28 (mAb-28), which is specific for an epitope in the human BMP15 protein (see the materials and methods section). The specificity of this antibody for BMP15 was demonstrated via western blot analysis of various conditioned media containing either BMP15 or GDF9 (Fig. 1B). The western blot analysis in Fig. 1B shows that the form 1 is expressed at lower level than the wild type protein or form 2. The various forms of unpurified BMP15 in the conditioned media were tested for bioactivity via monitoring Smad1/5/8 activation. This was accomplished by transducing human granulosa luteal (hGL) cells with an adenovirus encoding the Smad1/5/8 responsive BRE-luciferase reporter containing BMP Smad binding sites derived from the Id1 promoter [START_REF] Korchynskyi | Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter[END_REF][START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF]. Similarly to mouse GDF9 (mGDF9) [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF], hBMP15 is produced by HEK-293T cells in an active form, but the bioactivity is lost by Cterminal modification (Fig. 1C). As shown in figure 1B, there is less form 1 than the wild type protein in the conditioned media. This is consistent with the bioactivity response of form 1 A c c e p t e d M a n u s c r i p t 6 being lower than the response of the wild type protein (Fig. 1C).

3.2. The C-terminally Flag-tagged rhBMP15 mature region does not show any bioactivity as the purified mature region Initially, an N-terminally His 6tagged BMP15 mature region was produced and purified, which was found to activate the Smad1/5/8 responsive (BRE-luc) reporter in hGL cells in a dose dependent manner (data not shown). This is consistent with a recent study by [START_REF] Li | Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15[END_REF]. A preferable situation however, is to be able to purify the protein of interest (in this case hBMP15) without modification by the addition of any epitope tags. We determined in a previous study [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF]) that the purified untagged mature region of recombinant human GDF9 is biologically active. The approach used in that study was subsequently used to purify rhBMP15 from conditioned media. This approach was also used to purify a C-terminally Flag tagged rhBMP15 mature region for comparative purposes since previously in the literature a number of studies have used such a protein (Otsuka et al. 2001a;Otsuka et al. 2001b;Otsuka and Shimasaki 2002b;Otsuka and Shimasaki 2002a;[START_REF] Liao | Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep[END_REF][START_REF] Moore | Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[END_REF][START_REF] Mcmahon | Phosphorylation of bone morphogenetic protein-15 and growth and differentiation factor-9 plays a critical role in determining agonistic or antagonistic functions[END_REF][START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF]Tibaldi et al. 2010). The purified rhBMP15wt and -C-Flag mature regions appear as two distinct bands corresponding to approximately 16 and 17 kDa (further referred to as P16 and P17 respectively) on a silver stained SDS-PAGE gel or a Western blot probed with mAb-28 (Fig. 2A). The purified rhBMP15wt was found to activate the Smad1/5/8 signaling pathway in hGL cells in a dose dependent manner, whereas the C-terminally Flag-tagged rhBMP15 was inactive (Fig. 2 B). As the hGL cells have undergone a process of terminal differentiation in response to a luteinizing surge of gonadotropins, we wanted to characterize the effect of the purified rhBMP15 on granulosa cells which have not become terminally differentiated. An immortalized human granulosa cell line COV434 was chosen as it has retained the ability to produce estradiol upon FSH stimulation, thus demonstrating the presence of functional FSH receptors (Zhang et al. 2000). The COV434 cells were transiently transfected with the Smad1/5/8 responsive transcriptional reporter construct, BRE-luciferase [START_REF] Korchynskyi | Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter[END_REF] and treated with the purified rhBMP15wt mature region, which activated BRE-luciferase reporter in a dose responsive manner (Fig. 2 C). The purified untagged hBMP15 was found to be very stable as storage at 4˚C for up to four months did not have any effects on its activity in the COV434 transcription reporter (BRE-luc) assays (data not shown). Longer storage periods than four months were not tested.

Human BMP15wt activates the phosphorylation of Smad1

To further confirm the activation of the BMP signaling pathway by the purified rhBMP15 we investigated the time course of Smad activation via phospho-Smad western analysis (Fig 3). Lysates of hGL and COV434 cells stimulated with BMP2 or purified rhBMP15wt were subjected to SDS-PAGE and immunostained after transfer to membranes using phospho-Smad1 (pS1) antiserum that specifically recognises the phosphorylated C-tail of Smad1 [START_REF] Persson | The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation[END_REF]. Following 90 minutes stimulation of hGL cells or 15 minutes stimulation of COV434 cells with rhBMP15wt (200 ng/ml), there was a pronounced increase in phospho-Smad 1, which was also observed in the lysates treated with BMP2 (100 ng/ml). Lysates from untreated (control) cells stimulated with DMEM containing 1% FCS (hGL) or 0.2% FCS (COV434) did not show detectable Smad 1 phosphorylation. All the membranes were probed with anti-β-actin antibody (Sigma-Aldrich) to show equal loading.

Human BMP15 stimulates rat granulosa cell [ 3 H]-thymidine

incorporation, which is antagonised by BMPR2ecd-Fc.

Previously we have used the incorporation of [ 3 H]-thymidine in primary cultures of rat granulosa cells as an additional in vitro model system for monitoring oocyte-derived growth factor effects in granulosa cells, and have reported that GDF9 is active in such a bioassay [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF]. We also find that our purified rhBMP15wt is highly active in this assay (Fig 4A). Consistent with the observed stimulation in DNA synthesis being due to the presence of the purified rhBMP15wt, we found that pre-incubation of the sample with the BMPRII ectodomain-Fc fusion molecule (BMPR2ecd-Fc) caused a dose dependent inhibition of bioactivity (Fig. 4B). The purified rhBMP15-C-Flag protein was completely inactive in the rat granulosa cell [ 3 H]-thymidine A c c e p t e d M a n u s c r i p t 7 incorporation assay (Fig. 4C) clearly demonstrating that the stimulation of DNA synthesis by the purified rhBMP15wt sample (Fig. 4A) was due to the presence of the rhBMP15wt protein and the epitope tag at the C-terminus of the rhBMP15 mature region destroys the bioactivity associated with this protein.

Human BMP15 stimulates inhibin B production in cultured human granulosa luteal cells

We have previously shown that GDF9 stimulates the production of inhibin B by human granulosa luteal cells [START_REF] Kaivo-Oja | Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells[END_REF][START_REF] Roh | Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells[END_REF][START_REF] Kaivo-Oja | Adenoviral gene transfer allows Smadresponsive gene promoter analyses and delineation of type I receptor usage of transforming growth factor-beta family ligands in cultured human granulosa luteal cells[END_REF]. In this study, we find that the purified recombinant human BMP15wt protein also stimulates inhibin B production in human granulosa cells in dose responsive manner (Fig 5A). Further, a time course effect on the inhibin B production in human granulosa cells by rhBMP15wt protein was seen (Fig 5B).

3.6. Physio-chemical characterization of the purified rhBMP15 mature region.

Our purified BMP15 consists of two related peptide species P16 and P17 (Fig. 2A) and efforts to resolve these via reverse phase HPLC were not successful. For further characterization of the P16 / P17 mixture we subjected a sample to molecular mass determination by mass spectrometry. The results indicated the presence of essentially two peaks with molecular masses of 13917.0 Da and 14864.0 Da. Tryptic peptide mass fingerprint analysis of the in-gel digested P16 and P17 showed with a reasonable peptide coverage (>85%) that both proteins represent BMP15. In the mass fingerprint of P16 a unique peptide with a protonated m/z =1433.6 Da corresponding to the blocked N-terminal tryptic peptide <QADGISAEVTASSSK (MW=1432.695 Da) was found. This peptide mass could not be found in the tryptic peptide mass fingerprint from P17 but instead a unique peptide with a protonated mass m/z = 2380.9 Da could be found which is suggested to represent the O-linked glycosylated N-terminal tryptic peptide of P17 [START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF]. When analysing the tryptic peptides from P16 in negative ion reflector mode, a peptide mass m/z=1511.69 Da corresponding to the phosphorylated N-terminal tryptic peptide was detected in low amounts together with the corresponding non-phosphorylated peptide.

Discussion

In the current study we show that although BMP15 is produced by HEK-293T cells in an active form, the bioactivity is lost by Cterminal modification of the protein. The main purpose of this study was to produce and purify recombinant human BMP15 in sufficient quantities for subsequent characterization of the protein. First, we introduced the His 6 tag at the amino terminus of the BMP15 mature region. Specifically, the tag was inserted between the 4 th and 5 th amino acid after the furin consensus sequence with the aim of minimizing the impact of the tag on the processing of the protein (Wolfraim et al. 2002). Conditioned media containing this his6 tagged modified BMP15 and the respective purified form of the protein were both biologically active, indicating that Nterminal modification of the mature region of BMP15 does not overly impair bioactivity (data not shown), which is consistent with the recent study by [START_REF] Li | Stable expression and characterization of N-terminal tagged recombinant human bone morphogenetic protein 15[END_REF]. In a separate study we discovered that a C-terminal His 6 tag destroys GDF9 bioactivity, and although human GDF9 is produced by 293T cells in a latent form, the purified untagged mature region of human GDF9 is biologically active [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF]. Hence, in the current study we characterized untagged and C-terminally Flagtagged BMP15 that were produced and purified via the same protocol that was previously used to obtain the purified GDF9. Both the purified and unpurified untagged BMP15 were potent activators of the Smad1/5/8 (BRE-luc) responsive transcriptional reporter in human granulosa cell bioassays. In contrast, both the purified and unpurified BMP15-C-Flag were inactive in the BRE-luc reporter assay.

In this article, we report that the bioactivity of rhBMP15 is lost by C-terminal modification, which is not in agreement with the published literature in the field, since part of the previous studies have been done using rhBMP15 Flag-tagged at the C-terminus of the mature region [START_REF] Otsuka | Bone morphogenetic protein-15. Identification of target cells and biological functions[END_REF]Otsuka et al. 2001a;Otsuka et al. 2001b;Otsuka and Shimasaki 2002b;Otsuka and Shimasaki 2002a;[START_REF] Liao | Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep[END_REF][START_REF] Moore | Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[END_REF][START_REF] Mcmahon | Phosphorylation of bone morphogenetic protein-15 and growth and differentiation factor-9 plays a critical role in determining agonistic or antagonistic functions[END_REF][START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF]Tibaldi et al. 2010). It needs to be emphasized that the majority of the recombinant A c c e p t e d M a n u s c r i p t 8 BMP15 secreted from our 293T cells is as a noncovalent complex of the processed mature region bound to the corresponding proregion. This is also the case with the BMP15 produced by the Shimasaki group, however the two constructs, i.e. ours and that used by the Shimasaki group are not identical. In particular our BMP15 Form 2 expression plasmid (with a C-terminal Flag tag, see Fig. 1A) incorporates a Flag tag at both the Nterminus of the pro-region, as well as the Cterminus of the mature region. The Flag tag is highly negatively charged, and an explanation of the difference between previously published results and ours would be that in our case the Nterminal Flag tag repels the C-terminal Flag tag, which we suggest tends to cause separation of the pro-region from the mature region as the protein is secreted into the culture medium. Under these circumstances we suggest that the C-terminal Flag tags on the mature region cause separation of the mature region dimer, and hence inactivation of the protein. This scenario is consistent with the very poor yields we obtain of the C-Flag BMP15 compared to untagged BMP15 using the purification procedure of Biotechvisions, even though the C-Flag form is expressed at least as well as the untagged form.

In the case of the previously published C-Flag BMP15 [START_REF] Otsuka | Bone morphogenetic protein-15. Identification of target cells and biological functions[END_REF]Otsuka et al. 2001a;Otsuka et al. 2001b;Otsuka and Shimasaki 2002b;Otsuka and Shimasaki 2002a;[START_REF] Liao | Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep[END_REF][START_REF] Moore | Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[END_REF][START_REF] Mcmahon | Phosphorylation of bone morphogenetic protein-15 and growth and differentiation factor-9 plays a critical role in determining agonistic or antagonistic functions[END_REF][START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF]Tibaldi et al. 2010), after purification the pro-region remains present with the mature region [START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF] and we suggest that the mature region bioactivity is stabilized by the presence of the pro-region. In contrast our purified BMP15 (both Forms 1 and 2) is free of pro-region, which in the case of Form 1 (no Flag tag) is biologically active, whereas Form 2, the Flag tagged form completely lacks bioactivity. Our studies highlight the effect of such a modification as a C-terminal Flag tag to the structural integrity of BMP15, and such modifications can not be recommended. Indeed, we show that they are not necessary as an untagged form of the human BMP15 mature region is possible to produce and purify, and is highly bioactive. Another recombinant form of BMP15 that has also appeared in the published literature is a BMP15 with a C-terminal Myc-His6 tag [START_REF] Di Pasquale | Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene[END_REF]). Although this form has been shown to be biologically active [START_REF] Di Pasquale | Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene[END_REF][START_REF] Fabre | Regulation of ovulation rate in mammals: contribution of sheep genetic models[END_REF][START_REF] Elis | Expression and biological effects of bone morphogenetic protein-15 in the hen ovary[END_REF] it is likely that this form is also stabilised by the presence of the pro-region, and may exhibit compromised bioactivity if the modified mature region alone was used.

In the present study, we monitored the effect of rhBMP15 on granulosa cell proliferation in primary rat granulosa cell cultures. It has been shown in previous studies, that rhBMP15 C-Flag directly stimulates the mitosis of rat GCs [START_REF] Otsuka | Bone morphogenetic protein-15. Identification of target cells and biological functions[END_REF][START_REF] Moore | Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[END_REF]. The mitosis of rat GCs by rhBMP15 C-Flag has been shown to be antagonized by BMPR2ecd-Fc [START_REF] Moore | Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells[END_REF]. However, we find in our study that the purified rhBMP15 C-Flag is completely inactive, whereas the wild type rhBMP15 is a potent stimulator of mitogenesis in rat granulosa cells and this effect is antagonized by BMPR2ecd-Fc. These results are all consistent with the above explanation of the basis for the difference between the Biotechvisions C-Flag BMP15 preparation and the previously published C-Flag BMP15 form.

Previous studies have shown that GDF9 (a close homolog of BMP15) stimulates inhibin B production in human granulosa cells [START_REF] Kaivo-Oja | Growth differentiation factor-9 induces Smad2 activation and inhibin B production in cultured human granulosa-luteal cells[END_REF][START_REF] Roh | Growth differentiation factor-9 stimulates inhibin production and activates Smad2 in cultured rat granulosa cells[END_REF][START_REF] Kaivo-Oja | Adenoviral gene transfer allows Smadresponsive gene promoter analyses and delineation of type I receptor usage of transforming growth factor-beta family ligands in cultured human granulosa luteal cells[END_REF][START_REF] Huang | Effects of growth differentiation factor 9 on cell cycle regulators and ERK42/44 in human granulosa cell proliferation[END_REF]Shi et al. 2009a;Shi et al. 2009b). Here we are showing that rhBMP15wt protein stimulates inhibin B production in human granulosa cells in a dose responsive manner. We also find that TGF-ß stimulates inhibin B production in human granulosa cells (Fig. 5A and [START_REF] Kaivo-Oja | Adenoviral gene transfer allows Smadresponsive gene promoter analyses and delineation of type I receptor usage of transforming growth factor-beta family ligands in cultured human granulosa luteal cells[END_REF]) and this response is seen at much lower levels of the ligand. However, it cannot be extrapolated from these results that TGF-ß would be the more significant factor in vivo in the regulation of inhibin B levels as TGF-ß is stored in vivo as an inactive complex. Hence to reveal the bioactivity of the TGF-ß mature fragment in vivo activation of the complex must take place [START_REF] Annes | Making sense of latent TGFbeta activation[END_REF]. Further, our observation that BMP15 stimulates inhibin B production is well in line with our previous report indicating that activation of both the activin and BMP Smad pathways results in significantly stimulated inhibin B production in human granulosa cells [START_REF] Bondestam | Engagement of activin and bone morphogenetic protein signaling pathway Smad proteins in the induction of inhibin B production in ovarian granulosa cells[END_REF].

The calculated average molecular mass of the rhBMP15wt mature form starting from <Q(1) and ending with R(125) is 13923.9 Da. Our results indicated the mixture of P16 and P17 was 13917.0 Da and 14864.0 Da respectively. Assuming that rhBMP15wt contains 3 disulfide bridges (6 x Cys residues) the calculated molecular mass is 13917.9 Da. Thus P16 Clearly, different preparations of purified rhBMP15 have different characteristics. Thus, it has become apparent that the field of reproductive biology requires a consistent, accurate, and reliable source of recombinant rhBMP15 which can be used in studies by workers in this field. We conclude that the wild type rhBMP15 mature region protein is the most reliable form of rhBMP15 for biological studies.

In conclusion, our results demonstrate that the unpurified rhBMP15 wild type protein activates the Smad1/5/8 signaling pathway, but the bioactivity is lost by C-terminal modification. Similarly to GDF9 [START_REF] Mottershead | Characterization of recombinant human growth differentiation factor-9 signaling in ovarian granulosa cells[END_REF], the bioactivity of rhBMP15 is crucially affected by the position of an affinity purification tag. Understanding the molecular mechanisms behind the full biological activity of rhBMP15 will help clarify its functions in the ovary, having implications for fertility management and the treatment of ovarian diseases. Fig. 2. Purified BMP15wt mature region activates the Smad1/5 pathway in ovarian granulosa cells in a dose dependent manner, whereas the C-terminally Flag-tagged rhBMP15 mature region does not show any bioactivity as the purified mature region. A. The purified human BMP15wt and -C-Flag mature regions were analyzed on a silver gel and on a western blot probed with the BMP15 specific mAb-28. The samples were reduced with 10 mM DTT before running into SDS-PAGE gels. B. hGL cells transduced with an adenovirus encoding the Smad1/5 (BRE-luc) reporter were incubated for 24 h in the absence (cont) or presence of BMP2 (20 ng/ml), rpHPLC purified BMP15wt or rhBMP15 C-Flag mature region. C. COV434 cells transfected with the Smad1/5 (BRE-luc) reporter were incubated for 24 h in the absence (an adjusted value of 1.0 for the mean of the control wells) or presence of BMP2 (100ng/ml) or various concentrations of rpHPLC purified rhBMP15wt mature region. Human BMP15 activated the Smad1/5 signaling pathway in hGL and COV434 cells in a dose dependent manner. Fig. 3. Purified rhBMP15wt mature region phosphorylates Smad1 in human ovarian granulosa cells. Before Smad activation experiments, the primary human granulosa luteal (hGL) cells were first cultured 2-3 days or COV434 cells were cultured until they reached 80% confluency. After 4-6 h low serum starvation period (1% FCS DMEM for hGL and 0.2% FCS DMEM for COV434 cells), the cells were treated with BMP2 (100ng/ml) or purified rhBMP15wt protein (200ng/ml) for the indicated time points (minutes). The cell lysates from hGL and COV434 cells were subjected to SDS-PAGE immunoblotting using the pS1 antiserum. Treatment with purified rhBMP15wt protein caused a rapid phosphorylation of Smad1.

  s c r i p t represents the unmodified polypeptide <Q(1)-R(125) without any modifications other than the three disulfide bridges. The larger mass 14864.0 Da then represents P17 with a modification (947 Da) which according to Saito et al.[START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF]) is a mucin-type O-glycan present at a threonine (T10) residue. The presence of the Oglycan on the N-terminal tryptic peptide from P17 was further demonstrated as a m/z=2380.914 Da peptide from the tryptic peptide mass fingerprint. However, the physiological significance of the Oglycosylation is not known. Saito et al.[START_REF] Saito | Characterization of the post-translational modification of recombinant human BMP-15 mature protein[END_REF] found that part of C-terminally tagged rhBMP15 P16 was modified by phosphorylation. Our peptide mass fingerprint analyses demonstrated the presence of low amounts of phosphorylation on the N-terminal tryptic peptide of P16. In our study only a minor part of P16 was phosphorylated as indicated by the lack of the mass of the phosphorylated protein in the direct mass spectrometric analysis. Despite the low level of phosphorylation of P16, it may well serve a regulatory role. Like the C-terminally Flag tagged rhBMP15 in[START_REF] Mcmahon | Phosphorylation of bone morphogenetic protein-15 and growth and differentiation factor-9 plays a critical role in determining agonistic or antagonistic functions[END_REF] (McMahon et al. 2008), our untagged rhBMP15 mature region also stained using Pro-Q diamond phosphoprotein stain (data not shown). It can be suggested that part of the protein is phosphorylated and it might serve as a regulatory component for hBMP15 bioactivity as shown in previous studies[START_REF] Mcmahon | Phosphorylation of bone morphogenetic protein-15 and growth and differentiation factor-9 plays a critical role in determining agonistic or antagonistic functions[END_REF]).

  Figures Figure 1.

  Figure 2.

Fig. 1 .

 1 Fig. 1. Analysis of unpurified forms of recombinant BMP15. A. Schematic of the two epitope tagged proteins produced in this study: 1 has only the N-terminal Flag and His6 tags, whereas form 2 also has a Cterminal Flag tag (SS: signal sequence, diagonal lines: Pro-region, Black: Proteolytic processing site, 6H: His6-tag, F: Flag-tag, Grey: Mature region). B. Recombinant proteins produced from stable HEK-293T cell lines and subjected to SDS-PAGE immunoblotting (ECL). 1: BMP15wt, 2: BMP15 Form 1, 3: BMP15 Form 2 (with C-terminal Flag tag), 4: GDF9 wt (recogniced by the GDF9 specific mAb-53 (Gilchrist et al. 2004b)), 5: TGF-ß 200 ng. All samples were reduced with 10 mM DTT before running into the gels. The specific BMP15 mAb-28 reveals the 16-17 kDa mature human BMP15 protein. The mAb-28 does not crossreact with GDF9, showing the specificity of the antibody to BMP15. C. Bioactivities of unpurified recombinant BMP15 forms on human granulosa-luteal cells, transduced with an adenovirus encoding the Smad1/5 (BRE-luc) reporter, were incubated for 24 h in the absence (cont) or presence of BMP2 (50 ng/ml), BMP15 wild type conditioned medium (CM), BMP15 Form 1 CM or BMP15 Form 2 CM (with C-Flag). Enzyme activity was measured in the cell extract and is expressed as fold change compared with the control (an adjusted value of 1.0 for the mean of the control wells), i.e. the activity level in the absence of ligand. (N = N-terminus, F = Flag-tag, 6H = His6-tag, C = C-terminus.)

Fig. 4 .

 4 Fig.4. Human BMP15wt stimulates rat granulosa cell [ 3 H]-thymidine incorporation, which is antagonised by BMPRII ectodomain-Fc fusion molecule (BMPR2ecd-Fc). A. Human BMP15wt protein stimulates rat ovarian granulosa cell [ 3 H]-thymidine incorporation in a dose dependent manner, B. which is neutralized with 1 µg/ml of BMPR2ecd-Fc at the control level. C. The C-terminally Flag-tagged rhBMP15 is inactive. (* P<0.05, ** P<0.01 compared to untreated control; ## P<0.01 compared to 20 ng/ml BMP15wt with 0 ng/ml BMPR2ecd-Fc.) Fig.5. Human BMP15 stimulates inhibin B production in cultured human granulosa luteal cells. Three different cell pools were treated with 0.3-300 ng/ml hBMP15wt protein for 48 hours or with 150 ng/ml hBMP15wt protein for 24, 48, 72 or 92 hours and the spent media were harvested for the measurement of inhibin B concentrations with an inhibin B ELISA. Data was similar in three different human granulosa luteal cell pools (each pool contains cells from 2-6 women) and representative experiments are shown. A. Human BMP15wt protein stimulates human granulosa luteal cell inhibin B production in a dose dependent manner. B. Time course of hBMP15wt action on inhibin B production in human granulosa luteal cells. The

Acknowledgments

A special thanks goes to Marko Hukka, Elina Ahola-Iivarinen and Gunilla Rönnholm for their contribution in protein chemical characterization of BMP15. The skillful technical assistance of Ms. Marjo Rissanen is kindly thanked.

These studies were supported by grants from the Academy of Finland, the Finnish Funding Agency for Technology and Innovation, the Juselius Foundation, the Jalmari and Rauha Ahokas Foundation, the Novo Nordisk Foundation, the Orion-Farmos research foundation, the Helsinki University research funds the Paulo Foundation, the Jenny and Antti Wihuri Foundation and New Zealand Foundation for Research Science and Technology.

Disclosure statement

The authors have nothing to disclose.

A c c e p t e d M a n u s c r i p t 15 15 Shi, F. T., A. P. Cheung, H. F. Huang and P. C. Leung (2009a). "Effects of endogenous growth differentiation factor 9 on activin A-induced inhibin B production in human granulosa-lutein cells." J Clin Endocrinol Metab 94( 12): 5108-16.

Shi, F. T., A. P. Cheung and P. C. Leung (2009b). "Growth differentiation factor 9 enhances activin a-induced inhibin B production in human granulosa cells." Endocrinology 150(8): 3540-6.