Daniel Ambort

Sjoerd Van Der Post

Malin E V Johansson

Jenny Mackenzie

Elisabeth Thomsson

Ute Krengel

Gunnar C Hansson
email: gunnar.hansson@medkem.gu.se

Function of the CysD domain of the gel-forming MUC2 mucin

Keywords: collision-induced dissociation, CK, cystine-knot, EK, enterokinase, ESI, electrospray ionization, FCS, fetal calf serum, IgG2a, immunoglobulin G2a, IMDM, Iscove´s modified Dulbecco´s medium, LC, liquid chromatography, tandem mass spectrometry, PTS, proline threonine C-mannosylation, disulfide bonds, mass spectrometry, mucus, non-covalent dimer. Biochemical

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its Nand C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin-Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by mass spectrometry revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel thereby determining the pore sizes of the mucus.

INTRODUCTION

Mucins are large glycoproteins that coat the surfaces of cells in the respiratory, digestive, and urogenital tracts, and in some amphibia, the skin [START_REF] Perez-Vilar | The structure and assembly of secreted mucins[END_REF][START_REF] Thornton | Structure and function of the polymeric mucins in airways mucus[END_REF]. They function to protect epithelial cells from infection, dehydration, and physical injury, as well as to aid passage of materials on mucosal surfaces. The mucins have mucin domains with large amounts of O-linked oligosaccharides attached on the protein sequence rich in proline, threonine and serine (PTS domain) [START_REF] Lang | Gel-forming mucins appeared early in metazoan evolution[END_REF]. Moreover, these domains often have tandemly repeated amino acid sequences that vary in number, length, and sequence in different mucins [START_REF] Lang | Gel-forming mucins appeared early in metazoan evolution[END_REF]. There are two types of mucins, membrane-bound and secreted. In humans, six secreted mucins have been confirmed; the gelforming MUC2 [START_REF] Gum | Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor[END_REF], MUC5AC [START_REF] Li | Cloning of the aminoterminal and 5'-flanking region of the human MUC5AC mucin gene and transcriptional up-regulation by bacterial exoproducts[END_REF], MUC5B [START_REF] Desseyn | Genomic organization of the 3' region of the human mucin gene MUC5B[END_REF], MUC6 [START_REF] Toribara | Human gastric mucin. Identification of a unique species by expression cloning[END_REF], and MUC19 [START_REF] Zhu | Cloning and characterization of human MUC19 gene[END_REF] and the non-gel forming MUC7 [9].

MUC2 is the major gel-forming mucin of colon and organizes the mucus into two layers, an inner densely packed layer and an outer loosely adherent layer that is colonized by bacteria [START_REF] Johansson | The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria[END_REF]. MUC2 contains five distinct regions (Figure 1A): an N-terminal part with von Willebrand D1-D2-D´-D3 (VWD) domains and CysD domain, a small PTS domain, another CysD domain, a large PTS (tandem repeated) domain, and a C-terminal part with von Willebrand D4-B-C domains and cystine-knot (CK) domain [START_REF] Gum | Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor[END_REF]. MUC2 forms disulfide-linked dimers via its Cterminal parts in the endoplasmic reticulum [START_REF] Asker | Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by a N-glycosylation-dependent transfer of the mono-and dimers to the Golgi apparatus[END_REF][START_REF] Lidell | The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells[END_REF] and disulfide-linked trimers via its Nterminal part in the trans-Golgi network [START_REF] Godl | The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment[END_REF]. The concerted C-terminal dimerization and Nterminal trimerization of MUC2 monomers leads to the assembly of a polymeric net-like structure that can bind water via its heavily O-glycosylated mucin domains to form a gel. While the VWD domains and CK domain are involved in polymer formation, the function of the CysD domains remains elusive. Interestingly, multiple CysD domains have also been identified in other human gel-forming mucins, in MUC5AC [START_REF] Escande | Human mucin gene MUC5AC: organization of its 5'-region and central repetitive region[END_REF] and MUC5B [START_REF] Desseyn | Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family[END_REF], respectively. The small 110residue-long CysD domains with 10 invariant cysteine residues are found adjacent to or scattered within the heavily O-glycosylated central region of these mucins, at least nine in MUC5AC and seven in MUC5B. It is worth noting that the genes for human gel-forming mucins MUC2, MUC5AC and MUC5B are localized to the same chromosome locus, 11p15.5 [START_REF] Pigny | Human mucin genes assigned to 11p15.5: identification and organization of a cluster of genes[END_REF]. The high intra-and inter-species homologies suggest the CysD domains play critical, but yet undefined roles in mucus homeostasis. Mucin CysD domains show significant homologies with a protein domain in human cartilage intermediate layer protein [START_REF] Lorenzo | Cloning and deduced amino acid sequence of a novel cartilage protein (CILP) identifies a proform including a nucleotide pyrophosphohydrolase[END_REF] and with a protein domain in the protein Oikosin1 of the larvacean tunicate Oikopleura dioica [START_REF] Spada | Molecular patterning of the oikoplastic epithelium of the larvacean tunicate Oikopleura dioica[END_REF]. Neither the tertiary structure nor the specific functions of these two proteins are known, though it has been proposed that they have structural roles in the organization of the articular cartilage and the larvacean mucous houses, respectively. A potential C-mannosylation WXXW acceptor site [START_REF] Löffler | Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms[END_REF] is found in the CysD domains of MUC2, MUC5AC, MUC5B and in 11 repeats in Oikosin 1. It has been suggested that at least the CysD1 and CysD5 domains of MUC5AC are mannosylated [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF]. Moreover, Perez-Vilar et al. [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF] reported that the CysD domains, CysD1 and CysD5 of MUC5AC and CysD1 and CysD3 of MUC5B were monomers. The mucin domains of different mucins are similar in sequence and the localization of the cysteines identical (see http://www.medkem.gu.se/mucinbiology/databases/index.html). More recently, we found the CysD domain in a number of gel-forming mucins from other species such as mammals, frog, fish, fruit fly, sea urchin, sea squirt and lancelet, respectively [START_REF] Lang | Gel-forming mucins appeared early in metazoan evolution[END_REF].

We therefore aimed at studying the function and biochemical properties of the CysD domain. We selected to analyze the second CysD domain of human MUC2. Two recombinant fusion proteins were constructed, in which the CysD domain alone or including two copies of the general tandem repeat from the PTS domain was fused to a removable IgG-Fc region. The two

EXPERIMENTAL

Construction of expression vector pSMCysD-IgG2a/His and pSMCysD(2TR)-IgG2a/His

A PCR product termed Enterokinase (EK)-IgG2a/His was generated from pcDNA3/MUC1-IgG [START_REF] Bäckström | Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells[END_REF] using primers 5´-CGTCTAGAGGATCCGATGATGATGATAAAGCAGAGC-3´ and 5´-CGTCGCTAGCATGATGGTGATGGTGATGGTAG-3´. It encoded XbaI and NheI sites, an EK cleavage site, exon 1-3 of murine IgG2a Fc domain and a C-terminal histidine tag. This amplicon inserted into XbaI and NheI sites of pSM vector [START_REF] Lidell | The recombinant C-terminus of the human MUC2 mucin forms dimers in Chinese-hamster ovary cells and heterodimers with full-length MUC2 in LS 174T cells[END_REF] encoding a N-terminal mouse Ig N-chain signal sequence and a myc tag was named pSM-IgG2a/His. The CysD domain was amplified from cDNA made from mRNA of LS174T cells of human MUC2 with primers 5´-GACTAGTGCTAGCCCATGCGTGCCTCTCTGC-3´ and 5´-GGTCTAGAGTTCTCTGTGGTGGTGGTTGTCATG-3´. This amplicon encoding XbaI and NheI sites, the second CysD of human MUC2 (bases 5368-5703) and a N-terminal part of adjacent PTS domain (bases 5661-5703) was inserted in-frame into the XbaI site of pSM-IgG2a/His and was called pSMCysD-IgG2a/His. The annealed phosphorylated primer pair with primers 5´-GCTAGCCCAACAACGACACCCATCAGCACCACCACCATGGTGACCCCAACCCCAAC ACCCACTGGAACACAGACCT -3´ and 5´-GCTAGAGGTCTGTGTTCCAGTGGGTGTTGGGGTTGGGGTCACCATGGTGGTGGTGCT GATGGGTGTCGTTGTTGGG-3´ encoding the general tandem repeat of the PTS domain of human MUC2 was inserted sequentially twice in-frame into the XbaI site of pSMCysD-IgG2a/His and was called pSMCysD(2TR)-IgG2a/His.

Expression of CysD-IgG and CysD(2TR)-IgG

CHO-K1 and CHO-K1 Lec 3.2.8.1 [START_REF] Stanley | Selection of specific wheat germ agglutinin-resistant (WgaR) phenotypes from Chinese hamster ovary cell populations containing numerous lecR genotypes[END_REF] cells grown in 10% fetal calf serum (FCS) Iscove´s modified Dulbecco´s medium (IMDM) (Lonza) with 1% penicillin/streptomycin were transfected with pSMCysD-IgG2a/His or pSMCysD(2TR)-IgG2a/His using Lipofectamine 2000 (Invitrogen) and stable clones generated 2-3 days later by adding 250 Pg/ml Geneticin (Invitrogen). Clones were selected based on high secretion of fusion protein into culture medium as described previously [START_REF] Bäckström | Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins[END_REF]. One high-level expression clone for each construct was selected, recloned and used for protein production.

Protein production

Adherent CHO-K1 Lec 3.2.8.1 cells producing CysD-IgG were resuspended to 0.3 u 10 6 cells/ml in 2 % FCS ProCHO-4 medium (Lonza) with 4 mM L-glutamine, 1x ProHT and 250 ȝg/ml Geneticin (Gibco). Cells were adapted to serum-free suspension growth within 6 weeks at 90 rpm at 37°C in 5% CO 2 . A 1.5 l perfusion culture with spin filter separation (10 μm) was performed in a 3 l bioreactor (Applikon) controlled by an ADI 1010 Bio Controller and an ADI 1025 Bio Console at 37°C, pH= 6.9, dO 2 = 40% and 150-300 rpm. Oxygen and CO 2 was introduced by bubble-free aeration using 1mm thick 9 m silicone tubing. Perfusion rate was 0.

SDS-PAGE, BN-PAGE and Western blotting

SDS-PAGE was performed as described [START_REF] Lämmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. Precision protein standards (Bio-Rad) were used as markers. Silver staining was carried out as described [START_REF] Swain | A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate-polyacrylamide gels[END_REF]. For liquid chromatographyelectrospray ionization tandem mass spectrometry (LC-ESI MS/MS) non-reducing SDS gels were stained with Imperial Protein Stain (Thermo Scientific). Western blotting was performed as described [START_REF] Towbin | Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications[END_REF]. After transfer the membrane was blocked in 5% milk powder in PBS with 0.1% Tween 20 or in 10 mM Tris-HCl pH 7.5, 1% BSA, 100 mM NaCl, 0.1% (w/v) Tween 20 and 0.05% (w/v) NaN 3 (for biotin-labeled antibody) and incubated with anti-Myc mAb (clone 9E10.2, American Type Culture Collection, CRL-1729) or biotin-labeled rat anti-mouse IgG2a (R19-15, BD Biosciences). The anti-Myc mAb was cultured by the Mammalian Protein Expression core facility at the University of Gothenburg. The membrane was then treated with AP-conjugated goat anti-mouse IgG1 (Southern Biotech) or streptavidin (Southern Biotech) and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Promega). Blue Native PAGE (BN-PAGE) allows studies of proteins under native conditions [START_REF] Schägger | Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form[END_REF]. BN-PAGE was performed as described [START_REF] Schägger | Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form[END_REF] on ready-made NativePAGENovex 4-16% Bis-Tris Gels (Invitrogen). NativeMark Unstained Protein Standards (Invitrogen) were used as markers. BN gels were stained by silver [START_REF] Swain | A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate-polyacrylamide gels[END_REF].

Protein G purification of CysD-IgG, CysD(2TR)-IgG or EK-cleaved CysD-IgG

Spent culture medium (9.5 l) containing CysD-IgG fusion protein was filtered (0.45 μm Mini Capsule, PALL) and concentrated by Tangential Flow Filtration (Pellicon™-2 system (Millipore)) with two 5 kDa PLCC filters (four times 1 l PBS reduced to 500 ml). For protein G purification of CysD(2TR)-IgG fusion protein serum-containing medium (130 ml) was dialyzed (Spectra/Por Dialysis Membrane, M r 6-8 000 cutoff, Spectrum Laboratories) three times against 20 mM sodium phosphate buffer pH 7 and filtered (Durapore Membrane Filter, 0.22 Pm GVWP, Millipore Corp.). The concentrate or filtered medium was loaded onto a HiTrap Protein G HP column (1.6 u 2.5 cm, Amersham Biosciences) equilibrated with 20 mM sodium phosphate buffer pH 7 at 1 ml/min. The column was rinsed with same buffer. Bound components were eluted (100 mM glycine-HCl pH 2.7), collected in 1-ml neutralized fractions (100 mM Tris-HCl pH 9) and analyzed by SDS-PAGE and silver staining. EK-cleaved CysD-IgG was directly loaded onto a protein G column and the flow through and eluate fractions analyzed.

Sialidase and Enterokinase treatment

Purified CysD-IgG and CysD(2TR)-IgG fusion protein was cleaved with EKMax for 15 h at 37°C in EKMax Reaction Buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl 2 and 0.1% (v/v) Tween-20 (Invitrogen)) at 1:100 (enzyme to subsrate ratio) and reaction stopped with 2 mM PMSF. The CysD(2TR)-IgG fusion protein was treated with sialidase (1:100 (Sigma)) and EKcleaved (1:100) for 18 h at 37°C in 50 mM sodium phosphate buffer pH 6.0. Desialylated and/or EK-cleaved samples were analyzed by gel filtration, SDS-and BN-PAGE.

LC-ESI MS/MS, in-gel digestion and MS data analysis

CysD-IgG-and CysD(2TR)-IgG-containing bands excised from non-reducing SDS gels were destained (three times in 50% acetonitrile, 25 mM Tris-HCl pH 7.8), dried and digested with Asp-N (0.01 Pg/Pl in 25 mM Tris-HCl pH 7.8 (Roche)). Peptides were extracted and partly dried by centrifugation under vacuum. The digest of one sample per condition was reduced (50 mM tris(2-carboxyethyl)phosphine-HCl (TCEP-HCl)) after which the peptides from both conditions were extracted using C18 ZipTips (C18 ZipTips (Millipore)), eluted (60% ACN, 40% H 2 O, 0.2 % trifluoroacetic acid), partly dried and solubilized (0.2% formic acid). Samples were analyzed by LC-ESI MS/MS (LTQ Orbitrap XL, Thermo Scientific). Sample injection and LC were performed as described [START_REF] Andersch-Björkman | Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle[END_REF]. Data were acquired in a data-dependent mode automatically switching between MS and MS/MS acquisition. MS scans were obtained in the Orbitrap at m/z 400-2 000, 2 microscans, maximum 500 ms injection and a AGC of 500 000. MS/MS was performed in linear ion-trap on the six most abundant multiple charged ions for each scan (1 microscan, max. 200 ms injection and a AGC of 30 000) using collision-induced dissociation (CID) fragmentation at 30% normalized collision energy. After fragmentation peptides were excluded for 10 s for further acquisition. Peaklists were generated from raw data using extract_msn.exe (Thermo Scientific). Data was interpreted using Mascot (version 2.2, Matrix Science) searched against IPIhuman database (version 3.52) with addition of CysD-IgG or CysD(2TR)-IgG fusion construct. A second search was performed against a specific cross-linked database generated for CysD-IgG or Cys(2TR)-IgG using xComb (version 1.1) [START_REF] Panchaud | xComb: a cross-linked peptide database approach to protein-protein interaction analysis[END_REF]. Search parameters for reduced samples against IPIhuman were as follows: (i) one missed cleavage Asp-N; (ii) tolerance 5 ppm (precursor), 0.5 Da (fragment ions); (iii) charge state 2 + , 3 + , 4 + ; (iv) oxidation Cys, Met (variable). Search parameters for non-reduced samples were as follows: (i) "do_not_cleave" after amino acid "J" for non-cleavage; (ii) tolerance 5 ppm (precursor), 0.5 Da (fragment ions); (iii) charge state 2 + , 3 + , 4 + ; (iv) dehydro Cys (fixed), oxidation Met (variable), gain of water Asp (variable). The Mascot cutoff score was set to 25.

RESULTS

Construction and expression of recombinant CysD-IgG fusion protein

A plasmid expressing a myc tag, the second CysD of human MUC2 (residues 1782 to 1878 [START_REF] Gum | Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor[END_REF]), an Enterokinase-cleavage site, exon 1-3 of murine IgG-Fc region and a C-terminal histidine tag, was constructed and called pSMCysD-IgG2a/His (Figure 1B). IgG was chosen as this gives high protein expression. The CysD domain contains 97 amino acids and lies between the small and large PTS domains of human MUC2. The plasmid was transfected into CHO-K1-Lec3.2.8.1 cells [START_REF] Stanley | Selection of specific wheat germ agglutinin-resistant (WgaR) phenotypes from Chinese hamster ovary cell populations containing numerous lecR genotypes[END_REF]. Stable clones were generated and selected for the secretion of maximum levels of CysD-IgG, as determined by immunoassay [START_REF] Bäckström | Purification and characterization of a membrane-bound and a secreted mucin-type glycoprotein carrying the carcinoma-associated sialyl-Lea epitope on distinct core proteins[END_REF] and Western blotting (data not shown). One high-level expression clone was selected and adapted to suspension culture in protein-free medium.

Purification of CysD-IgG fusion protein

To obtain CysD-IgG fusion protein, the supernatant from a perfusion culture was concentrated by ultrafiltration and then loaded onto a protein G column. The bound proteins were eluted and fractions analyzed by SDS-PAGE and silver stained (Figure 1C andD). The fusion protein migrated at a50 kDa under reducing (Figure 1C, -EK) and at a150 kDa under non-reducing conditions on SDS gels corresponding to the CysD-IgG mono-and dimer, respectively (Fig. 1D, -EK). To separate the CysD from IgG, the fusion protein was cleaved with Enterokinase. The IgG part showed a a30 kDa band under reducing conditions (Figure 1C, + EK) and a dimeric a60 kDa band under non-reducing conditions that were both labeled by the IgG-specific antibody (Figure 1D, + EK). Three distinct bands of a20-22 kDa were found under reducing SDS-PAGE that were identified as the CysD part with the anti-myc tag antibody (Figure 1C, + EK). When the Enterokinase-cleaved samples were analyzed by non-reducing SDS-PAGE most of the CysD-containing material disappeared (Figure 1D, + EK). The disappearance of the CysD protein under non-reducing conditions on SDS-treated samples led us to speculate that it had a high tendency to form insoluble aggregates and the CysD was further studied under native conditions.

Gel filtration and Native PAGE of CysD-IgG fusion protein

The intact fusion protein displayed four major bands at a200 kDa, a400 kDa, a600 kDa and a800 kDa for the tetra-, octa, dodeca and hexadecamers, respectively, on silver stained BN gels (Figure 2A, -EK). The same material analyzed in reducing SDS gels gave single bands of a50 kDa (Figure 1C, -EK). The EK-cleaved fusion protein showed two strong bands of a35 kDa for the CysD dimer and a120-180 kDa for the IgG2 dimer, respectively, in BN gels (Figure 2A, + EK). Next, EK-cleaved fusion protein was loaded onto a protein G column and analyzed by BN-PAGE. The CysD fusion part found in the flow through migrated as a a35 kDa CysD dimer on BN gels as the calculated CysD monomeric mass is 16 kDa (Figure 2B, Flow through). When the IgG-containing eluate was separated on native gels, three bands of a120-180 kDa, a200 kDa and a400 kDa, respectively, were found (Figure 2B, Eluate), the first band due to dimeric IgG that was cleaved off its fusion partner and the two latter due to oligomeric forms of remaining noncleaved fusion protein.

Gel filtration was then used to further investigate the oligomeric state of CysD and its fusion protein. The CysD-IgG fusion protein eluted as two major peaks at a200 kDa and a800 kDa, respectively (Figure 3A). This suggests the higher oligomeric tetra and hexadeca forms as observed on the native gels as analysis of these two peaks on reducing SDS gels showed the expected a50 kDa CysD-IgG band in both peaks. When the IgG part was analyzed by gel filtration, a single peak eluted at a35 kDa (Figure 3B). a dimeric form as it migrated with an apparent mass of 20-22 kDa on reducing SDS gels (Figure 3C).

Gel filtration of CysD(2TR)-IgG fusion protein

To further prove the dimeric nature of the CysD domain and that this was not caused by the absence of a flanking mucin domain with a typical glycosylation, a second fusion protein with two human MUC2 tandem repeats (23 amino acids each) were also analyzed (Figure 1A andB). The plasmid pSMCysD(2TR)-IgG2a/His was transfected into CHO-K1 cells that gives normal sialylated N-glycans and largely sialyl-T O-glycans (NeuAcD2-3GalE1-3GalNAc-) [START_REF] Bäckström | Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells[END_REF]. One high-level expression clone was selected for protein production. The protein G-purified fusion protein migrated at a75 kDa on a reducing SDS-PAGE gel (data not shown). The IgG part of the EK-cleaved fusion protein showed as for the first protein a a30 kDa band under reducing and a a60 kDa band under non-reducing SDS-PAGE (Figure 4A). Three distinct bands of a40-45 kDa due to the CysD(2TR) part were labeled by the myc tag antibody in a reducing SDS gel (Fig. 4A). When the same EK-cleaved material was analyzed under non-reducing SDS-PAGE, most of the CysD-containing material was absent as before (Fig. 4A). The EK-cleaved fusion protein eluted from a gel filtration column as two distinct peaks at a113 kDa and a35 kDa, respectively. The 113-kDa peak was due to dimeric CysD(2TR) whereas the 35-kDa protein form was due to IgG as confirmed by SDS-PAGE and Western blotting (Figure 4B, -sialidase). Desialylation had no influence on the elution time of the IgG part from gel filtration column or migration behaviour in reducing and non-reducing gels (Figure 4A,B). Desialylated dimeric CysD eluted at a95 kDa from the gel filtration column (Figure 4B, + sialidase) and migrated as a monomer at a50 kDa, slightly larger than sialylated, when analyzed by reducing SDS-PAGE (Figure 4B, + sialidase). The sialic acids are probably attached to the N-linked glycans as the CysD domain of MUC2 contains three potential N-glycosylation sites and this also explains the presence of three separate bands on reduced SDS gels when the protein was sialylated. It can thus be concluded that the CysD had an estimated mass inferred from gel filtration and BN-PAGE experiments as for a dimer. The IgG formed covalent dimers and when fused to the CysD that also formed dimers, larger oligomeric ladders were found. The CysD dimer were not possible to quantitatively reveal on non-reduced SDS gels, something that might suggest disulfide stabilization. This was not the case as shown below.

Effect of pH and calcium on the dimeric state of the CysD domain

To reveal if the CysD dimer was affected by pH and calcium, recombinant CysD was isolated and analyzed by gel filtration at different pH in the presence or absence of calcium. The isolated CysD-containing material eluted as a 30-kDa dimer in buffers at low or high pH (Figure 5A) and when analyzed in the presence or absence of calcium (Figure 5B). A tiny shift towards a smaller size, less than expected for a change in oligomeric status, was observed at low pH and high calcium as for the conditions in the secretory vesicles. Different pH and calcium conditions were tested to dissociate the 30-kDa CysD dimers into monomers (described in Experimental), but no conditions were able to do this (data not shown).

Disulfide bond pattern of the CysD domain determined by LC-ESI MS/MS

To reveal if the CysD dimer were held together by disulfide bonds, the band of interest containing CysD-IgG fusion protein was excised from a non-reducing SDS gel, in-gel digested with Asp-N and then analyzed by LC-ESI MS/MS and database searching as described [START_REF] Panchaud | xComb: a cross-linked peptide database approach to protein-protein interaction analysis[END_REF]. All 6A). Three unambiguous Cys-pairs were allocated as follows: Cys32-Cys36, Cys63-Cys73 and Cys92-Cys100, respectively. Mass spectrometry analysis revealed that the non-Cyscontaining peptide at m/z 934.96 2+ was partly acetylated at Lys 46. The last peptide was observed as a triply charged ion at m/z 924.07 3+ . This peptide included the four remaining cysteines that formed two internal disulfide bonds as the observed molecular mass (2769.17 Da) had lost four hydrogen atoms as for two disulfide bonds. There were no fragmentation spectra that could reveal the exact disulfide bond pattern, but the most likely disulfide bond arrangement in this peptide was Cys116-Cys126 and Cys125-Cys128, respectively. The peptide DVPIGQLGQTVVCDVSVGLICKNE, including an Asp-N cleavage site at Asp 93 within the Cys92-Cys100 disulfide bridge, eluted as two distinct peaks due to a partial cleavage at this site (Figure 6B). The first peptide at m/z 835.09 3+ eluted at 37.5 min from the column whereas the second peptide at m/z 1242.63 2+ eluted at 52.5 min. The first peak originated from cleavage at Asp93 within the loop formed by the Cys92-Cys100 disulfide pair. In the second peak, the noncleaved form of the same peptide was found with an intact loop. Proteolytic cleavage within the loop of Cys92-Cys100 had a profound effect on the hydrophobic properties of this peptide as the elution times for the two different forms when separated on the hydrophobic C18 column shifted dramatically upon opening the loop.

In the peptide DLSSPCVPLCNWTGWL at m/z 894.90 2+ the intramolecular disulfide bridge (Cys32-Cys36) was still intact after fragmentation and peptide sequence information was only obtained for the neighbouring amino acids that were flanking the intact Cys pair (Figure 7A). Upon reduction of the samples with the reducing agent TCEP-HCl the intact disulfide bridge was chemically cleaved (Figure 7B) and almost complete y and b ion series were obtained. The WXXW peptide motif in this peptide was not C-mannosylated as predicted on the first Trp residue when analyzed in its reduced or non-reduced form (Figure 7A andB). Thus all disulfide pairs were within the CysD and no indication of any intermolecular disulfide bonds could be found.

DISCUSSION

The function of CysD domains found in gel-forming mucins has not been understood so far. However, we have been able to show that the CysD domain of human MUC2 forms noncovalent dimers. The a50-kDa form of the CysD-IgG fusion protein observed in reducing SDS gels migrated at a200-kDa, a400-kDa, a600-kDa and a800-kDa in BN-PAGE and eluted at a200 kDa and a800 kDa in gel filtration due to IgG covalent and CysD non-covalent dimers. Furthermore, the CysD part with a calculated mass without glycans of 16 kDa eluted at 30 kDa on gel filtration and at 35 kDa BN-PAGE, suggesting that CysD forms dimers. The Fc region of IgG from mouse is known to form three intermolecular disulfide bridges via residues Cys237, Cys240 and Cys242, respectively, in the hinge core by connecting two IgG chains to a covalent dimer [START_REF] Harris | Refined structure of an intact IgG2a monoclonal antibody[END_REF]. In line with this, we observed that the a30-kDa IgG part formed a a60 kDa dimer in non-reducing SDS gels. The dimeric nature of the CysD domain was confirmed by analyzing a second recombinant CysD fusion protein that included two 23 amino acid tandem repeats of the MUC2 mucin. The CysD(2TR) protein formed three bands at a40-45 kDa on reducing SDS gels and eluted as a a113 kDa peak in gel filtration. Mass determination by gel filtration depends on the hydrodynamic (Stokes) radius of the protein and overestimation of mass is not atypical for glycoproteins [START_REF] Andrews | The gel-filtration behaviour of proteins related to their molecular weights over a wide range[END_REF]. Nevertheless, comparative gel filtration analysis of glycoproteins and its desialylated forms revealed that the apparent molecular masses of desialylated forms were smaller than expected just from removal of the mass of sialic acid [START_REF] Alhadeff | Gel filtration of sialoglycoproteins[END_REF]. Thus the aberrant behavior of glycoproteins in gel filtration is largely contributed to the negative charges of sialic acids [START_REF] Alhadeff | Gel filtration of sialoglycoproteins[END_REF]. Desialylated CysD(2TR) protein migrated at a50 kDa in reducing SDS gels and eluted as a a95 kDa peak in gel filtration. These findings strongly supported that the CysD domain formed dimers. The three bands of a20-22 kDa of recombinant CysD and a40-45 kDa of the CysD(2TR) protein, respectively, found in reducing SDS gels originated from differentially sialylated glycoprotein forms. For the latter protein we showed that the three distinct bands were shifted to one a50 kDa band by sialidase treatment. The sialic acids are probably attached to the N-linked glycans as the CysD domain of MUC2 contains three N-glycans.

Perez-Vilar et al. have previously studied two of the CysD domains in the MUC5AC mucin and then concluded that these are monomeric [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF]. In contrast to this publication we have made quantitative biochemical analyses at protein levels instead of only using radioactive traces. Although, we have not worked with the same mucin CysD, we have not found evidence for the conclusions made by Perez-Vilar et al. [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF]. Cross-linking experiments as made by Perez-Vilar et al., [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF] after simple purification does not prove that CysD are monomers. In fact, there are substantial amounts of dimers in their gels and we believe that they also had dimers.

In conclusion, CysD forms non-covalent dimers and the a200-kDa tetrameric complexes of the CysD-IgG2a/His fusion protein are build up by disulfide-bonded IgG dimers that interact via its fused CysD domain(s) with another identical dimer (Figure 8A). Further oligomerization (dimerization) of the a200-kDa complexes is mediated via non-occupied free CysD domains thus forming stable a400-kDa octameric (Figare 8B), a600-kDa dodecameric, and a800-kDa hexadecameric complexes.

To further understand the nature of the CysD dimer and the bonds formed by the 10 cysteines in the CysD domain, we cleaved the CysD-IgG fusion protein with Asp-N and analyzed the peptides by LC-ESI MS/MS. All 10 cysteine residues of the CysD domain were involved in intramolecular disulfide-bonds as follows: Cys32-Cys36, Cys63-Cys73, Cys92-Cys100, Cys116-Cys126 and Cys125-Cys128, respectively. Although the exact disulfide pattern for the peptide with four cysteine residues could not be elucidated from the MS data, the Biochemical Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t disulfide bonds Cys116-Cys126, Cys125-Cys128 are most likely. As all the five disulfide bonds were pair wise within four peptides and no other cysteine-containing peptides were found, it can be concluded that the disulfide bonds were intramolecular and that the CysD dimers were held together with non-disulfide bonds.

A high hydrophobicity of the loop generated by the Cys92-Cys100 disulfide bridge (CDVSVGLIC) was observed as long as the loop was intact. This hydrophobicity was much higher than that of the open peptide itself and a cleavage between the two cysteines (Figure 6B) gave the same loss of hydrophobicity as reducing the disulfide bond of the intact peptide. A similar conserved hydrophobic amino acid sequence stretch between these two cysteines can be found in all CysD domains of gel-forming mucins indicating that the formation of a hydrophobic loop at this site is important for the CysD function. It may be speculated that the 'disappearance' of the CysD protein under non-reducing conditions upon SDS-PAGE is due to this (and maybe other) hydrophobic patch present in a highly stabilized globular protein. It is tempting to speculate that the hydrophobic loop stabilized by the Cys92-Cys100 pair is exposed at the surface of the molecule thereby forming the very stable CysD dimers that were observed by gel filtration. The observation that one of the lysines could be acetylated could make the CysD even more hydrophobic.

Beside gel-forming mucins, the CysD domain has also been identified in the protein Oikosin1 of the larvacean tunicate Oikopleura dioica [START_REF] Spada | Molecular patterning of the oikoplastic epithelium of the larvacean tunicate Oikopleura dioica[END_REF]. This protein contains 13 tandemly repeated CysD domains. Larvaceans feed on dissolved organic carbon and microorganisms by filtering seawater through a transparent structure called the house. Importantly, Oikosin1 is the major structural component [START_REF] Spada | Molecular patterning of the oikoplastic epithelium of the larvacean tunicate Oikopleura dioica[END_REF] and is probably involved in the assembly of the secreted house. Due to the high number of adjacent tandemly repeated CysD domains, it may be speculated that they function in "sticking" the house together.

A potential C-mannosylation WXXW acceptor site [START_REF] Löffler | Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms[END_REF] is found in the CysD domains of MUC2, MUC5AC, MUC5B and in 11 repeats in Oikosin 1. Although it was postulated based on mutational studies that at least the CysD1 and CysD5 domains of MUC5AC are mannosylated [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF], we did not find such a modification on the tryptophan of the WXXW peptide motif in the peptide DLSSPCVPLCNWTGWL. In our analysis the tryptophan residue was not modified as determined by ESI-MS/MS. The mannosylation is easily identified by mass spectrometry [START_REF] Wang | Post-translational modification of thrombospondin type-1 repeats in ADAMTS-like 1/punctin-1 by C-mannosylation of tryptophan[END_REF]. The two different CysD variants studied by us were produced in CHO-K1 and CHO-K1-Lec3.2.8.1 cells, respectively. Other studies on C-mannosylated proteins have expressed the protein in the CHO-K1 cell line where the tryptophan in the WXXW peptide motif was modified [START_REF] Krieg | C-Mannosylation of human RNase 2 is an intracellular process performed by a variety of cultured cells[END_REF]. Perez-Vilar et al. [START_REF] Perez-Vilar | C-Mannosylation of MUC5AC and MUC5B Cys subdomains[END_REF] claim CysD mannosylation something we could not verify in at least the CysD we studied. In their report indirect experimentation using mutagenesis were done and no biochemical proof supporting such a conclusion was provided.

The pH along the secretory pathway is shifting gradually, from 7.2 in the ER [START_REF] Wu | Organelle pH studies using targeted avidin and fluorescein-biotin[END_REF], to 6.0 in the trans-Golgi network [START_REF] Demaurex | Mechanism of acidification of the trans-Golgi network (TGN). In situ measurements of pH using retrieval of TGN38 and furin from the cell surface[END_REF], to 5.2 in the secretory granules [START_REF] Miesenbock | Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins[END_REF] and upon release rises up to 8.0 in the colon lumen [START_REF] Guyton | Medical Physiology[END_REF]. In addition, mucin packing into storage granules is accompanied by an increase in intragranular calcium concentration [39]. Gel filtration analysis of isolated CysD dimer indicated that it was not dissociating at different pH conditions (pH of 5, 6, 7, 8) or calcium concentrations (0, 10 or 50 mM) and is probably due to the hydrophobic nature of the disulfide-bond-stabilized loop. The mucins stored in the mucus granulae are expanded 1,000-fold upon release into the lumen. It is difficult to envisage how CysD dimers could exist in the secretory granulae and still allow this large volume expansion. Although low pH or high calcium conditions do not dissociate the CysD dimers, it is tempting to speculate that the CysD domains As the second CysD domain is localized to the large middle part of MUC2 flanked by the small and large mucin domains [START_REF] Gum | Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor[END_REF], this CysD domain will insert additional cross-links into the net-like gel (Figure 8C). While such a role is theoretical and based on in vitro experiments of recombinant proteins, it is tempting to speculate that the CysD may act as a biomolecular "glue" sticking neighbouring polymer chains together. By glueing adjacent CysD domains together the overall pore size of the mucus gel meshwork will decrease. Interestingly, the three gel-forming mucins MUC2 [40], MUC5AC [START_REF] Escande | Human mucin gene MUC5AC: organization of its 5'-region and central repetitive region[END_REF] and MUC5B [START_REF] Desseyn | Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family[END_REF] do not differ in their N-terminal and Cterminal parts, but they differ in the number of CysD domains and how these are distributed within the central part of the molecules. The MUC2 mucin have two CysD domains, whereas MUC5AC has at least 9 CysD domains [START_REF] Escande | Human mucin gene MUC5AC: organization of its 5'-region and central repetitive region[END_REF] and MUC5B 7 CysD domains in [START_REF] Desseyn | Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family[END_REF], interspersed by relatively short PTS domains. The MUC2 mucin has one large mucin domain (about 2,300 amino acids) that does not contain any additional CysD domains [START_REF] Gum | Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor[END_REF]. As the number of and the distance between the CysD domains varies among different gel-forming mucins, gels with different porosity will be produced. This suggests a much denser polymer in MUC5AC and MUC5B than in MUC2. Whereas MUC2 is the major gel-forming mucin of the intestine [START_REF] Gum | Molecular cloning of human intestinal mucin (MUC2) cDNA. Identification of the amino terminus and overall sequence similarity to prepro-von Willebrand factor[END_REF], MUC5AC and MUC5B are the main components of secreted mucin in the stomach (only MUC5AC) and airways [START_REF] Davies | Respiratory tract mucins. In Cilia and Mucus: from Development to Respiratory Defense[END_REF]. The different expression patterns of MUC2 and MUC5AC/MUC5B may point to different functions of the gels formed. Analysis of mouse colonic mucus [START_REF] Johansson | The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria[END_REF] showed that it consists of two layers, a densely packed firm layer devoid of bacteria and a movable loose layer. As both layers are mainly composed of Muc2, these findings indicate a barrier function of the Muc2 colonic mucus likely controlled by the pore sizes. However, the MUC2 mucin still needs to have relatively large pore size as it should still allow nutrients to pass in the small intestine. In contrast, the mucus of the stomach shall only allow ions to pass and in the lungs only gases. It may thus be proposed that denser MUC5AC/MUC5B gels are important for trapping small particles in the lungs before being removed by the mucociliary system. Interestingly, in a previous review by Hollingsworth and Swanson [42] it was discussed that the mucus gel may act like a molecular sieve allowing passage of small molecules but excluding large molecules and organisms due to steric hindrance.

In conclusion, the CysD domain of human MUC2 forms non-covalent dimers and probably has an important role in the assembly and properties of a mucus gel. However, additional biochemical studies are necessary to shed light on the role of the many CysD domains of other gel-forming mucins.

Figure 3 Gel filtration of CysD-IgG fusion protein

(A) Purified fusion protein was analyzed on a Superose 6 gel filtration column. The Superose 6 column had a void volume of 0.843 ml, and the standards thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa) and ovalbumin (43 kDa) eluted at 1.315 ml, 1.494 ml, 1.629 ml and 1.753 ml, respectively. (B and C) EK-cleaved fusion protein was loaded onto a protein G column and the bound material (B) and flow through (C) analyzed on a Superose 12 gel filtration column. The Superose 12 column had a void volume of 0.831 ml, and the standards aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa) and ribonuclease A (13.7 kDa) eluted at 1.236 ml, 1.315 ml, 1.382 ml and 1.584 ml, respectively. UV absorbance units are given in mAU. The content of collected fractions from major peaks (indicated by arrows) was analyzed on reducing SDS gels (shown as insets) with Silver staining. M, molecular mass standards in kDa. The gel lanes shown together come from the same gel.

Figure 4 Analysis of EK-cleaved CysD(2TR)-IgG fusion protein by SDS-PAGE and gel filtration

(A) EK-cleaved CysD(2TR)-IgG fusion protein was analyzed by SDS-PAGE and Western blotting under reducing (Red) and non-reducing (NonRed) conditions before (-) and after (+) sialidase treatment. The same material as in (A) was also analyzed on a Superose 12 gel filtration column (B). Elution times of standards were specified in Figure 3B andC. UV absorbance units are given in mAU. The content of collected fractions from major peaks (indicated by arrows) before (-) and after (+) sialidase treatment was analyzed by reducing SDS-PAGE and Western blotting (shown as insets). The D-myc tag antibody labeled the CysD(2TR) part whereas BDMIgG antibody recognized the IgG part. M, molecular mass standards in kDa. The gel lanes shown together come from the same gel.

Figure 5 Effect of pH and calcium on the dimeric state of the CysD domain

The CysD-containing fraction of EK-cleaved CysD-IgG fusion protein was analyzed on a Superose 12 gel filtration column at pH 8 and pH 5 with 5 mM EDTA (A) or at pH 8 with and without 10 mM CaCl 2 (B). UV absorbance units are given in mAU. Elution times of standards were specified in Figure 3B andC.

Biochemical

  Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t fusion proteins expressed in Chinese hamster ovary (CHO) cells were secreted into the culture medium. The purified fusion proteins were studied by gel filtration, denaturing and native gel electrophoresis, and the disulfide bond pattern was established by mass spectrometry. The CysD part formed pH-independent non-covalent dimers stabilized by intramolecular disulfide bonds.

3 -

 3 Biochemical Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t 0.8 dilutions/day of culture volume. The CysD(2TR)-IgG fusion protein was produced in T-175 flask cultures grown in 10% IMDM with 250 Pg/ml Geneticin (Invitrogen).

Gel filtration 5

 5 Pg of purified CysD-IgG, CysD, IgG or sialylated and desialylated EK-cleaved CysD(2TR)-IgG fusion protein was applied to a Superose 6 or Superose 12 PC 3.2/30 column (Amersham Biochemical Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t Biosciences) eluted at 0.02 ml/min using an Ettan Monitor UV-900 (Amersham Biosciences) as detector with UV absorbance at 215 nm, 254 nm and 280 nm. Standards were chromatographed at pH 7 (50 mM phosphate buffer, 150 mM NaCl). The purified CysD-IgG and CysD(2TR)-IgG (EK-or sialidase-treated) was either directly injected or was buffer-exchanged by ultrafiltration (10 000 g at 4 °C; VIVASPIN 6 PES, M r 10 000 cutoff, Sartorius) to pH 5.2 (100 mM acetic acid), pH 7 (50 mM phosphate buffer), pH 7.4 (100 mM Hepes) or pH 8.3 (100 mM sodium bicarbonate) containing 150 mM NaCl with or without 50 mM CaCl 2 . The CysD-(flow through) or IgG-containing (eluate) protein parts isolated by protein G purification of EK-treated fusion protein were either directly injected or buffer-exchanged by ultrafiltration (10 000 g at 4°C; VIVASPIN 6 PES, M r 10 000 cutoff, Sartorius) to pH 5 (20 mM acetic acid), pH 6 (20 mM Mes), pH 7 (20 mM Tris-HCl), pH 7.4 (10 mM phosphate buffer) and pH 8 (20 mM Tris-HCl) containing 150 mM NaCl and 5 mM EDTA or 10 mM CaCl 2 .

  Fusion of the CysD domain to Biochemical Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t

  Reducing SDS-PAGE and silver staining confirmed the presence of the a30 kDa IgG form. The CysD protein eluted at 30 kDa suggesting Biochemical Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Biochemical

  Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited 10 cysteines of the CysD domain formed five intramolecular disulfide bonds within four peptides (Figure

Biochemical

  Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited exist in a momomeric form within the secretory granules. The putative monomeric state of the CysD domains may be achieved by a "capping" protein.

Biochemical

  Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Biochemical Journal Immediate Publication. Published on 22 Feb 2011 as manuscript BJ20102066 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20102066 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited

Figure 1

 1 Figure 1 Domain organization of MUC2 and the two CysD fusion constructs (A) MUC2 is made up of D1-D2-D´-D3-CysD-PTS-CysD-PTS (tandem repeated)-D4-B-C-CK domains. (B) The two CysD fusion constructs named pSMCysD-IgG2a/His and pSMCysD(2TR)-IgG2a/His is made up by an N-terminal myc tag, CysD and a C-terminal IgG-Fc and His 6 -tag. The second fusion construct included two repeats (2TR) of the general tandem repeat of the PTS domain. Both fusion proteins included an EK cleavage site. (C, D) Analysis of protein G purified CysD-IgG fusion protein by SDS-PAGE and Western blotting under reducing (Red) and non-reducing (NonRed) conditions before (-) and after Enterokinase cleavage (+). M, molecular mass standards in kDa. The D-myc tag antibody labeled the CysD part whereas BDMIgG antibody recognized the IgG part. Silver, Silver staining.

Figure 2

 2 Figure 2 Native-PAGE of purified CysD-IgG fusion protein (A) BN gel of purified fusion protein before (-) and after Enterokinase cleavage (+). (B) EK-cleaved fusion protein was loaded onto a protein G column and the flow through and eluate analyzed by BN-PAGE. M, molecular mass standards. Silver staining. The gel lanes, including standards, shown together come from the same gel.

Figure 6

 6 Figure 6 Analysis of disulfide bonds in the CysD domain by LC-ESI MS/MS (A) Disulfide bond pattern in the CysD domain. Underlined numbers indicate Cys residues forming intramolecular disulfide bonds. Solid horizontal lines indicate unambiguous whereas dotted horizontal lines theoretical disulfide bonds. Solid vertical lines represent position of cleavage sites. (B) LC MS separation of the peptide DVPIGQLGQTVVCDVSVGLICKNE with internal disulfide bond. Peak number one represented the extracted ion chromatogram of the peptide cleaved N-terminal to Asp at position 93. The second peak belonged to the non-cleaved form causing an increase in hydrophobicity.

Figure 7

 7 Figure 7 LC-ESI MS/MS analysis of the CysD peptide with C-mannosylation motif

Figure 8

 8 Figure 8 Proposed model on CysD dimers in the fusion protein and the MUC2 gelThe CysD-IgG fusion protein is made up by two subunits and each is 50-kDa in size. These two subunits are covalently linked via the IgG Fc part (S-S bridges) and form a covalent 100-kDa dimer. (A) Two covalent 100-kDa CysD-IgG dimers form a non-covalent 200-kDa tetramer via non-covalent interactions of the CysD domain. (B) Four covalent 100-kDa CysD-IgG dimers form a non-covalent 400-kDa octamer via non-covalent interactions of the CysD domain. In both cases the CysD domains form non-covalent dimers. (C) The MUC2 mucin forms a covalent gel via its N-and C-terminal domains. The N-terminus of MUC2 forms covalent trimers whereas the C-terminus forms covalent dimers. The CysD domain forms non-covalent dimers. The CysD domain inserts non-covalent cross-links into MUC2 gel thereby determining its pore size and gel properties.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGEMENTS

We thank The Mammalian Protein Expression (MPE) Core facility for expression and production of recombinant fusion proteins.

FUNDING

This work was supported by the Swedish Research Council [grant numbers 7461, 21027, and 342-2004-4434], The Swedish Cancer Foundation, The Swedish CF Foundation, The Knut and Alice Wallenberg Foundation (KAW2007.0118), IngaBritt and Arne Lundberg Foundation, Sahlgren's University Hospital (LUA-ALF), EU-FP7 IBDase (#200931), Wilhelm and Martina Lundgren's Foundation, Torsten och Ragnar Söderbergs Stiftelser, The Swedish Foundation for Strategic Research -The Mucosal Immunobiology and Vaccine Center (MIVAC) and the Mucus-Bacteria-Colitis Center (MBC) of the Innate Immunity Program (2010-2014).