Andrea Fritzer

Birgit Noiges

Daniela Schweiger

Angelika Rek

Andreas J Kungl

Alexander Von Gabain

Eszter Nagy

Andreas L Meinke
email: ameinke@intercell.com

Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in

Keywords: Chemokine degradation by ScpC from S. pyogenes Streptococcus pyogenes, Interleukin 8, Spy0416, ScpC, CXC chemokine 1

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Streptococcus pyogenes belongs to Group A streptococci (GAS) and is mainly an extracellular pathogen capable of causing a wide variety of infections in humans ranging from asymptomatic colonization to life threatening invasive infections [START_REF] Cunningham | Pathogenesis of Group A streptococcal infections[END_REF]. Primary disease manifestations include pharyngitis, impetigo and soft tissue infections, which may lead to invasive diseases if untreated such as sepsis, toxic shock syndrome, rheumatic fever or necrotizing fasciitis [START_REF] Efstratiou | Group A streptococci in the 1990s[END_REF]. The versatility of this pathogen is explained by complex virulence mechanisms mediated by adhesins such as the M protein and fibronectin binding proteins, as well as powerful invasins e.g. streptolysin O and S [START_REF] Kehoe | Nucleotide sequence of streptolysin O (slo) gene: structural homologies between Slo and other membrane damaging thiol-activated toxins[END_REF][START_REF] Owens | Hemolytic mutants of group A Streptococcus pyogenes[END_REF], and further virulence factors such as streptokinase, hyaluronidase [START_REF] Wessels | Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci[END_REF] and streptodornase [START_REF] Alouf | Cellular constituents and extracellular proteins involved in the pathogenic capacity of Streptococcus group A[END_REF]. Additionally, pyrogenic exotoxins which can elicit strong inflammatory cytokine responses leading to endotoxic shock contribute to successful, host-specific survival of S. pyogenes [START_REF] Kotb | Bacterial pyrogenic exotoxins as superantigens[END_REF]. One of the various strategies the bacterium uses to survive in humans is the antiphagocytic behaviour mediated by binding to fibrinogen via M protein [START_REF] Cunningham | Pathogenesis of Group A streptococcal infections[END_REF][START_REF] Bisno | Alternate complement pathway activation by group A streptococci: role of M-protein[END_REF][START_REF] Horstmann | Antiphagocytic activity of streptococcal M protein: selective binding of complement control protein factor H[END_REF][START_REF] Kihlberg | Protein H, an antiphagocytic surface protein in Streptococcus pyogenes[END_REF]. A common molecular mechanism to survive in the human host described for several bacterial pathogens [START_REF] Rasmussen | Proteolysis and its regulation at the surface of Streptococcus pyogenes[END_REF][START_REF] Dubin | Extracellular proteases of Staphylococcus spp[END_REF] is the secretion of proteolytic enzymes, capable to degrade e.g. immunoglobulins, extracellular matrix and plasma proteins. The virulence factor C5a peptidase e.g. is able to avoid direct contact with phagocytes through destruction of complementderived chemotaxins [START_REF] Wexler | Mechanism of action of the group A streptococcal C5a inactivator[END_REF].

Recently, a novel evasion mechanism, the degradation of the neutrophil recruiting IL-8 chemokine was associated with S. pyogenes in patients suffering from necrotizing fasciitis, a severe soft tissue infection associated with high morbidity and mortality rates [START_REF] Edwards | Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes[END_REF][START_REF] Green | Necrotizing fasciitis[END_REF]. The site specific cleavage of IL-8 between Gln 59 and Arg 60 (converting the 8 kDa IL-8 to a 6 kDa species) was suggested to be responsible for the paucity of neutrophils at the site of infection [START_REF] Edwards | Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes[END_REF]. It was shown that the cell envelope serine proteinase (ScpC or Spy0416), partially purified from S. pyogenes, was capable of in vitro degradation of human IL-8, GCP-2 and GRO-, as well as murine keratinocyte-derived chemokine (KC) and murine MIP-2 [START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF][START_REF] Sumby | A chemokine-degrading extracellular protease made by group A Streptococcus alters pathogenesis by enhancing evasion of the innate immune response[END_REF]. These CXC chemokines are responsible for acute neutrophil priming and/or mobilisation and migration in their respective species. In contrast, the CC chemokine RANTES, which is responsible for T-cell and monocyte chemotaxis, was not degraded at all [START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF]. Furthermore, Spy0416/ScpC was identified as an in vitro expressed and surface exposed protein of GAS by analyses of its surface proteome [START_REF] Rodriguez-Ortega | Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome[END_REF]. The protein comprises 1647 amino acids and appeared to be a highly conserved major component of the cell surface in more than 70% of the analyzed S. pyogenes strains [START_REF] Rodriguez-Ortega | Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome[END_REF]. Although it was not possible to specifically delete the scpC gene, a double mutant in the scpA (encodes the related C5A peptidase) and scpC locus showed drastic reduction of virulence in a murine model of GAS necrotising fasciitis whereas the scpA mutant behaved similar to wild type cells [START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF]. It was further observed that ScpC promoted resistance to neutrophil killing [19] and contributed to virulence in murine infection models [START_REF] Sumby | A chemokine-degrading extracellular protease made by group A Streptococcus alters pathogenesis by enhancing evasion of the innate immune response[END_REF]19,[START_REF] Sjölinder | The ScpC protease of Streptococcus pyogenes affects the outcome of sepsis in a murine model[END_REF].

Here we demonstrate for the first time that the recombinantly expressed form of Spy0416 cleaves IL-8 in a site specific manner that leads to inactivation of this chemokine. Site-directed mutagenesis revealed that the amino acid residues in the N-terminal domain, predicted to form the active site of Subtilisin family serine proteases, are indeed essential for enzymatic activity. In addition, we show that the C-terminal domain of Spy0416 for which no function is assigned yet, is also required for proteolytic activity. Besides IL-8 and GCP-2, another CXC chemokine, GRO-, is also recognized by recombinant Spy0416 as a specific substrate. Its important function in immune evasion, the presence in all analyzed S. pyogenes strains, its high sequence conservation together with its capability to provide protection in animals against challenge with a lethal dose 1). The Spy0416B sequence was amplified using primers 2248 (5'-TAGTAGGAATTCGCATGTAGACCCACAAAAGGGC-3') and 2249 (5'-TAGTAG CTCGAGCGTTGATGGTAGGGCTTTTGC-3'). Both PCR products were cloned into the pET28b(+) expression vector.

Construction of active site mutants of Spy0416A. Two single amino acid mutants within Spy0416A were generated, Spy0416-D151A and Spy0416A-S617A (Table 1). For Spy0416A-D151A a 3-step PCR strategy was employed. First, a 369 bp fragment was amplified from genomic S. pyogenes SF370 DNA with the primers 3022 (5'-TAGTAGACATGTCTGCAGATGAGCTAAGCACAATG-3') (AflIII underlined) and 3023 (5'-CGGATCGATCCCTGTCGCAATAACTGCGACAAC-3'). The second PCR amplified a 2166 bp fragment using primers 2247 (5'-TAGTAG CTCGAGCTCTGAACCAAGAGTGACAAG-3') (XhoI underlined) and 3024 (5'-GTTGTCGCAGTTATTGCGACAGGGATCGATCCG-3'). The third PCR used the two amplicons as templates together with primers 3022 and 2247 generating a 2528 bp fragment. The Spy0416-S617A mutant was constructed similarly using the oligonucleotides 3027 (5'-CTGAGGAGAGGCCATCGCTGTTCCTGTTTGGCT-3') and 3022 as well as 2247 and 3028 (5'-AGCCAAACAGGAACAGCGATGGCCTCTCCTCAG-3'), amplifying 1748 bp and 750 bp fragments, respectively. The third PCR using primers 3022 and 2247 resulted in a 2528 bp fragment containing the mutation S617A. This PCR was performed with 2 mM MgCl Construction of deletion mutants Spy0416 and Spy0416A1. The Spy0416 fragment contained a deletion of 585 bp, the PA domain (Table 1, Fig. 1). For its construction, PCR was carried out with primers 3022 (5'-TAGTAG ACATGTCTGCAGATGAGCTAAGCACAATG-3') and 5596 (5'-AATAGCCATCAGAAGTATGGTCAGATCCATAGA-3') amplifying 1098 bp from the Spy0416 N terminus and primers 2247 (5'-TAGTAG CTCGAGCTCTGAACCAAGAGTGACAAG-3') and 5597 (5'-TCTATGGATCTGACCATACTTCTGATGGCTATT-3') amplifying 878 bp of Spy0416A's C terminus. This PCR was carried out with 1.5 mM MgCl 2 and required first five cycles with an annealing temperature of 45°C. For the ligation PCR, the previously generated amplicons containing the 3' and 5' overhangs were used as templates. The ligation PCR required optimized conditions: 2 mM MgCl 2 , 5 cycles at an annealing temperature of 47°C and 35 cycles with 50°C as annealing temperature. The final PCR product (1943 bp) was cloned directly into the pET28b(+) expression vector. Spy0416A1 was amplified with primers 2246 (5'-TAGTAGGAATTCGGCAGATGAGCTAAGCACAATG-3', EcoRI site is underlined) and 2263 (5'-TAGTAGCTCGAGTGCCCCTTGCTGACGCGGTG-3', XhoI site underlined) under standard conditions.

Construction of single site IL-8 mutants. The IL-8 mutant IL-8 F17K,F21K was cloned and the encoded recombinant protein purified as described recently (Wabitsch et al., manuscript submitted).

SDS-PAGE and Western blotting.

Proteins were separated by SDS-PAGE using 4-20% gradient gels (PAGEr ® Duramide Precast Gels, Cambrex Bio Science, USA) and 15% nongradient gels. For Western blot analysis, proteins were transferred via semi dry blot (BioRad ® , USA) onto nitrocellulose membranes. Nitrocellulose membranes were blocked with 5% (w/v) milk powder in PBS at 4°C over night or at room temperature for one hour. Incubation with primary and secondary antibodies was carried out at room temperature for one hour. All histagged recombinant proteins were detected using monoclonal mouse anti-Penta-His TM antibodies diluted 1:5,000 in 5% (w/v) milk powder in PBS and 1:5,000 diluted anti-mouse-IgG/HRP antibodies in PBS. For the detection of IL-8 after degradation by Spy proteins, anti-human CXCL-8 antibodies (1:1,000 dilution) were used. For the detection of MCP-1, IP-10 and I-TAC in Western blot the respective primary antibodies (1:1,000) were used and rabbit anti-goat IgG/HRP was utilized at a 1:5,000 dilution. For the degradation experiments of IL-8 F17K,F21K , PF-4, GRO-, NAP-2 and SDF-1 , polyclonal rabbit anti-human IL-8 antibody at 1:5,000, antihuman PF-4 at 1:2,500, anti-human GRO-at 1:2,500, anti-human NAP-2 at 1:2,500 and antihuman SDF-1 at 1:500, respectively, were used as primary antibodies. Anti-rabbit IgG/HRP (1:160,000 dilution) and anti-goat IgG/HRP (1:50,000 dilution) acted as secondary antibodies. All proteins were visualized with the ECL system. For the quantification of IL-8 on nitrocellulose membrane, Chemiglow TM (Alpha Innotech Corporation; USA) was used as substrate for the measurement of chemiluminescence with the FluorChem SP Imaging System and a CCD digital camera.

Expression and purification of recombinant proteins. For expression of recombinant proteins, E. coli BL21 Star TM One Shot ® cells, harbouring the pET28b(+) vector with the respective gene, were cultivated overnight. The culture was diluted 1:40 with fresh LB medium containing 50 µg/ml Kanamycin and incubation continued with shaking (150 rpm) at 37°C until an OD 600 nm of 0.8 was reached. Protein expression was induced by 0.5 mM IPTG for three hours and cells harvested by centrifugation (4,400 rpm/30 min/4°C). All recombinant proteins were purified from the soluble fractions via affinity chromatography using Ni-Sepharose TM beads (GE Healthcare Biosciences AG, UK) according to the instructions of the manufacturer. Spy0416B was purified from the insoluble fraction, adding 1 mM DTT and using binding buffer devoid of Imidazole. Washing was performed with 20 mM Imidazole and 40 mM Imidazole in 50 mM Tris/ HCl pH 8.0, 1% Triton X114. Eluted fractions were dialyzed against PBS overnight. The identity of the purified recombinant proteins was verified by Western blot analysis using mouse anti-Penta-His TM antibodies.

CXC chemokine degradation assays. Chemokine degradation assays were carried out at 37°C. IL-8 was used at a final concentration of 6.6 µg/ml in PBS containing 0.1% BSA. The chemokines IL-8 F17K,F21K , PF-4, and NAP-2 were used at a final concentration of 6.6 µg/ml in PBS, corresponding to 150 ng chemokine per assay sample (22.8 µl). GRO-and SDF-1 were used at a final concentration of 26.3 µg/ml in PBS (600 ng per 22.8 µl). MCP-1, IP-10 and I-TAC were utilized at a final concentration of 8.8 µg/ml (200 ng per 22.8 µl). The Spy proteins were used at a concentration of 22 µg/ml unless otherwise stated. Degradation was visualized via Western blot analysis. BSA, present in the IL-8 solution at 100 µg/ml (228 ng per sample), served as a standard showing use of equal amounts of IL-8.

Native-PAGE and immunodetection. 4.5 g of human IL-8 and different amounts of the S. pyogenes proteins Spy0416A, Spy0416B, mutant Spy0416A1 were incubated on ice for 30 minutes. After mixing 1:1 with final sample buffer (10 mM Tris, 1 mM Na 2 EDTA, 20% (v/v) glycerol), probes were loaded onto a 15% polyacrylamide gel, prepared in 10 mM Tris/HCl pH 7.4 supplemented with 1 mM Na 2 EDTA (Spy0416A/Spy0416B had no activity under these conditions). Protein samples were run for 3 h at 100 V towards the cathode in 40 mM Tris/acetate pH 8.3 and 1 mM Na 2 EDTA as running buffer. Transfer onto nitrocellulose membrane was followed by standard Western blotting. Detection of IL-8 was performed using polyclonal rabbit anti-human IL-8 antibody at 1:5,000 and horseradish peroxidase conjugated secondary anti-rabbit antibody at 1:160,000 using ECL detection. Visualisation of IL-8 antibody reactive bands was accomplished by exposing Kodak X-Omat LS films (Sigma-Aldrich, USA).

Protein interaction studies by Surface Plasmon Resonance (SPR).

Binding experiments were performed using a BIAcore X-100 biosensor system (Biacore, GE Healthcare). FC2 (flow cell 2) of a BIAcore C1 sensor chip-surface was initially activated with a mixture of 0.4 M EDC and 0.1 M NHS. IL-8 was immobilized via amine coupling at a concentration of 25 µg/ml in 10 mM acetate puffer pH 5.5. At a flow rate of 10 µl/min and a temperature of 25°C the injection time was 7 min, resulting in an immobilization level of 342 RU after blocking for 7 min with ethanolamine. FC1 served as control surface that underwent the same chemistry except immobilization of IL-8. Analyses were performed by injecting different concentrations of Spy proteins (from 0 -1 µM in PBST buffer) over the IL-8 surface for 3 min at a flow rate of 30 µl/min. Considering the high stability of the formed complexes, the dissociation time was set to 7 min, followed by a regeneration step with 0.5% SDS at the end of each cycle. The sensorgrams were analyzed using the BIAcore X-100 evaluation software. Therefore reference subtracted association and dissociation phases of all binding curves were simultaneously fitted to different binding models.

Isothermal Fluorescence Titration (IFT)

. 39 nM of Spy0416A or Spy0416B were titrated with IL-8 in the presence of 1 M bis-ANS (Molecular Probes, Invitrogen, USA). Excitation was set at 395 nm and fluorescence emission spectra were collected between 450 nm and 550 nm. Fluorescence quenching of bis-ANS emission was detected due to competition of IL-8 for Spy0416 binding. The excitation-/ emission slit widths were set to 15 nm and 20 nm, respectively; the temperature was adjusted to 22°C and a 430 nm cut off filter was inserted into the emission path to avoid stray light. After background correction of the spectra, areas were integrated and mean values of the fluorescence intensity, F, resulting from two independent experiments plotted against the volume corrected concentration of the IL-8 ligand. The resulting binding isotherms were analysed by nonlinear regression as described previously [START_REF] Halden | Interleukin-8 binds to syndecan-2 on human endothelial cells[END_REF][START_REF] Moser | Interleukin-8 antagonists generated by N-terminal modification[END_REF]. The fitted parameters were F max and K D.

Gene conservation studies. The genes encoding Spy0416 were amplified and sequenced from S. pyogenes strains by standard PCR with the following 4 primer pairs: 4588 (5'-TGAGTTAATGATTAACATTAAACTGGT-3') and 4591 (5'-TGACATAAGCAAATTGATGCG-3'); 4591 (5'-CCATCTATTCAGAGTCTGTCGAC-3') and 4595 (5'-CCTTGTCACTAGCATGGTAGAC-3'); 4596 (5'-TTGCAGCCTTCAAAGGTG-3') and 4599 (5'-AAGACACATTACCAGCTCTATCTTC-3'); 4600 (5'-CAGATGGTTCTTACACCATTTC-3') and 4603 (5'-AATCTCAAAGAAAGGTCAGACTG-3'). Contigs were analysed using Sequencher Version 4.7 and sequence analysis was done using the ClustalW interface.

Preparation of human neutrophils and intracellular [Ca 2+

]-measurement. Human whole blood was obtained from healthy volunteers by venipuncture into tubes containing K 3 EDTA (Vacuette, GBO, Austria). Neutrophils were isolated by Ficoll-Paque centrifugation (400x g, 30 min, 4°C) followed by dextran sedimentation of the red blood cell (RBC) pellet containing PMNs for 1 h at RT. The PMN-rich supernatant was aspired, washed twice with HBSS(-) (PAA, Austria) at 600x g for 10 min followed by hypotonic lysis of the RBCs and 2 further washing steps. Finally, cells were diluted with HBSS(+) at 10 7 cells/ml, considering that only 60% of the counted cells were neutrophils. Fluorescence measurements were performed on a Perkin Elmer LS50B fluorometer (UK). The diluted neutrophils were supplemented with 0.5% BSA and loaded with 3 µM FURA-2AM for 30 min at 37°C in the dark. Subsequently, cells were washed twice with HBSS(+) (500x g, 4 min) and stored in HBSS(+)/0.5% BSA. Immediately before measurement, 1 ml aliquots were centrifuged for 3 min at 500x g and resuspended in 1 ml HBSS(+). Cells were placed in a continuously stirred cuvette maintained at 37°C. After an equilibration period of 10 min, a stable fluorescence signal was reached. The fluorescence was monitored at an excitation of 345 nm and an emission of 495 nm, as determined in an initial measurement. The excitation/emission slits were set at 10/4. Cells were stimulated with 100 nM protein 60 sec after pre-equilibration, and maximum and minimum fluorescence were determined using the following solutions: Minimum buffer (100x): 0.

Results

Recombinant Spy0416 possesses IL-8 cleavage activity. The identification of Spy0416 as a prominent antigen (unpublished data) using the ANTIGENome technology [START_REF] Meinke | Antigenome technology: a novel approach for the selection of bacterial vaccine candidate antigens[END_REF] together with the recent report showing that Spy0416 of invasive S. pyogenes strains possesses human chemokine degrading activity [START_REF] Edwards | Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes[END_REF][START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF][START_REF] Sumby | A chemokine-degrading extracellular protease made by group A Streptococcus alters pathogenesis by enhancing evasion of the innate immune response[END_REF], has prompted us to further investigate its function. Despite several attempts, expression and purification of the full length recombinant protein was not possible, as correct amplification of the gene was problematic and cloning into an E. coli expression vector not successful. Therefore we generated two subfragments, Spy0416A & B overlapping by 132 amino acids (Fig. 1). The design of these fragments was supported by bioinformatics analyses of Spy0416 showing a complex domain structure, indicating that the Nterminal proteinase activity might be modulated and/or regulated by its C-terminal accessory domains. The Subtilisin-like protease domains of Spy0416 are intervened by a protease associated domain [START_REF] Brown | Structure of the streptococcal cell wall C5a peptidase[END_REF][START_REF] Siezen | Multi-domain, cell-envelope proteinases of lactic acid bacteria[END_REF]. The PA domain of ScpB from S. agalactiae was postulated to be involved in substrate recognition [START_REF] Mahon | The PA domain: a protease-associated domain[END_REF] or in an interaction with an unknown cellular receptor [START_REF] Brown | Structure of the streptococcal cell wall C5a peptidase[END_REF]. The catalytic domain of Spy0416 is followed by the Fn1 region of unknown function, which shows homology to the A domain of proteinases such as PrtP of Lactococcus lactis or the Fn1 domain of C5A of S. pyogenes, and is believed to be involved in the regulation of the catalytic activity [START_REF] Siezen | Multi-domain, cell-envelope proteinases of lactic acid bacteria[END_REF][START_REF] Chen | Complete nucleotide sequence of the streptococcal C5a peptidase gene of Streptococcus pyogenes[END_REF][START_REF] De Vos | Cloning and expression of the Lactococcus lactis subsp. cremoris SK11 gene encoding an extracellular serine proteinase[END_REF]. The C-terminal region of Spy0416 entails two repetitive regions of unknown function, RI and RII, identified in C5A peptidase as Fn2/Fn3 and Fn2'/Fn3', respectively, and postulated to contribute to regulation of enzymatic activity as well [START_REF] Brown | Structure of the streptococcal cell wall C5a peptidase[END_REF]. The Y region located at the C terminus between Fn3´and the cell wall anchor LPXTG motif shows homology to similar regions in proteinases such as CspA of S. agalactiae [START_REF] Brown | Structure of the streptococcal cell wall C5a peptidase[END_REF] and to sigma A factors of Flavobacteriaceae (app. 30% identity with the N-terminal 100 amino acids).

We first investigated whether recombinant Spy0416A displayed the predicted IL-8 degrading activity, but did not observe any IL-8 cleavage as assessed by immunoblotting with anti-CXCL-8 antibodies (detecting active IL-8, but not or with much lower sensitivity degradation products of IL-8) (Fig. 2A). Surprisingly, IL-8 cleavage was only detected when both Spy0416A and Spy0416B were present (Fig. 2A). The observed IL-8 cleavage activity was specific for Spy0416, since Spy0416B and two other unrelated S. pyogenes proteins with unknown function, Spy0488 and Spy1727 (chosen from a list of S. pyogenes proteins identified as antigens, unpublished data), caused no IL-8 degradation (Fig. 2A &B). IL-8 cleavage was time-(Fig. 2E &F) and Spy0416A/B concentration-dependent (Fig. 2E &G). Co-incubation of Spy0416A1 or Spy0416A with Spy0416B did not result in degradation of IL-8 (Fig. 2D), indicating that the Fn1 and PA domains were both required to fully retain the functional role of the N-terminal fragment.

Since purified Spy0416B showed several degradation products and in order to evaluate possible contributions of the predicted individual domains within Spy0416B to IL-8 degrading activity, three additional subfragments were expressed: Spy0416B Fn2Fn3 (Phe 819 -Thr 1127 ), Spy0416B Fn2´Fn3´ (Leu 1128 -Asp 1390 ), and Spy0416B Fn2´Fn3´+Y (Leu 1128 -Ala 1600 ). None of these proteins showed in combination with Spy0416A any IL-8 cleavage activity, suggesting that Spy0416B in its entirety is essential for activity (data not shown).

Having shown that recombinant Spy0416A possesses IL-8 degrading activity, we wanted to affirm that the activity was attributed to its predicted active site. In analogy to Subtilisin-like proteinases [START_REF] Carter | Dissecting the catalytic triad of a serine protease[END_REF], the three conserved catalytic site residues within Spy0416 (Asp 151 , His 279 and Ser 617 ) were expected to form the catalytic triad. Site-directed mutagenesis of single catalytic triad residues in Subtilisin from Bacillus amyloliquefaciens [START_REF] Carter | Dissecting the catalytic triad of a serine protease[END_REF] or ScpA from S. pyogenes [START_REF] Cleary | Immunization with C5a peptidase from either group A or B streptococci enhances clearance of group A streptococci from intranasally infected mice[END_REF] resulted in drastically reduced or abolished protease activity. Using the two active site mutants Spy0416A D151A and Spy0416A S617A , we confirmed that IL-8 cleavage by Spy0416A was indeed dependent on the catalytic triad (Fig. 2C).

Influence of IL-8 degradation by Spy0416A/B on Ca 2+ -release of neutrophils. It was shown recently that cleavage of IL-8 by ScpC reduces the migration of neutrophils in response to the cytokine [START_REF] Edwards | Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes[END_REF]. As it was previously also described that one can distinguish between IL-8 mediated activation and migration of neutrophils [START_REF] Clark-Lewis | Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs[END_REF][START_REF] Webb | Binding to heparan sulfate or heparin enhances neutrophil responses to interleukin 8[END_REF] we investigated whether IL-8 cleavage by Spy0416 also abrogates human neutrophil activation. We thus assessed the Ca 2+ -release of neutrophils before and after pre-incubation of the chemokine with Spy0416A/B. Neutrophils showed the expected Ca 2+ -release after injection of native IL-8 (Fig. 3A). However, prior incubation of IL-8 with Spy0416A/B had no effect on neutrophilic Ca 2+ -release (Fig. 3B), rather a signal of similar intensity was detected after an incubation time of 3.5 hours, indicating that the degradation product of IL-8 (devoid of the 13 C-terminal amino acids) is able to stimulate neutrophils, most probably due to the ELR motif at the N terminus which is mainly responsible for recognition and activation via CXCR1 and CXCR2.

Binding of IL-8 by Spy0416 is mainly mediated by the N-terminal domain. In order to further evaluate the essential contribution of Spy0416B to the enzymatic activity of Spy0416, we assessed whether IL-8 binding occurs via the catalytic Spy0416A domain or the catalytically inert Spy0416B domain. Since immunoprecipitation studies failed for technical reasons, we applied isothermal fluorescence using soluble proteins and the fluorophore bis-ANS to evaluate binding of IL-8 to Spy0416A and B. Titration of IL-8 to samples containing Spy0416A or B and bis-ANS, should induce displacement of the fluorophore upon binding of IL-8 to S. pyogenes proteins, resulting in fluorescence quenching of bis-ANS emission. These analyses showed that both proteins bound IL-8, however Spy0416A had an approximately 20 times higher affinity for IL-8 (K D = 2.7x 10 -7 M) compared to Spy0416B (Fig. 4A). In order to confirm the large differences in affinities of Spy0416A and Spy0416B towards its IL-8 ligand, we performed SPR experiments in which IL-8 was immobilised via amine coupling to a BIAcore C1 chip. By this means, a kinetically-derived K D value of 13.1 nM was determined for the interaction of Spy0416A with IL-8, but no interaction of IL-8 with Spy0416B was detected. We also evaluated the binding of the Spy0416A1 mutant protein to IL-8 and detected a 70-fold lower, but still significant K D value of 926 nM for the interaction with Spy0416A1 (Fig. 4B). The difference in K D values for Spy0416A interacting with IL-8 derived by the two different methods (IFT and SPR) relates to the fact that potentially induced oligomerization and/or cooperative structural effects induced by protein-protein binding lead to different results, if one of the interacting partners is immobilised.

The interaction of Spy0416A and Spy0416B competes with binding of Spy0416A to IL-8.

Since the isothermal fluorescence experiment was performed by an indirect displacement using a non-covalently attached fluorophore, we were not able to measure the interaction of three proteins -Spy0416A, Spy0416B and IL-8 -in one assay. We thus applied a gel depletion assay to investigate how the two fragments of Spy0416 influence binding to IL-8. In this assay, binding of a Spy0416 protein to IL-8 would shift the resulting complex towards the opposite charge-flow direction as compared to IL-8 alone, resulting in depletion of IL-8 from the gel. Despite the intermediate binding affinity between IL-8 and the Spy0416 domains (low micromolar range), we were able to detect significant shifts due to the chemokine's quaternary structure that tends to increase from a dimeric to an oligomeric structure upon ligand binding. A 1:10 molar ratio of the Spy0416 proteins and IL-8 used in the gel depletion assays led to chemokine oligomerization and thus to a complete depletion of IL-8 by the stronger binding to Spy0416A (Fig. 5, lane 1), whereas Spy0416B alone was not capable of fully depleting the chemokine (Fig. 5, lane 2). The Spy0416A D151A protein, which was shown to be devoid of catalytic activity, was also able to deplete IL-8, indicating significant binding to the chemokine even in the absence of proteolytic activity (Fig. 5, lanes 3 & 4). Interestingly, in the presence of Spy0416B, Spy0416A and Spy0416A D151A were unable to completely deplete IL-8 from the gel (Fig. 5, lanes 5 & 6) suggesting that the interaction between the N-and C-terminal parts of Spy0416 modulates substrate binding.

Mutation of single residues of IL-8 reduces degradation by Spy0416.

The G protein-coupled (GPC) receptor binding activity of IL-8 has been assigned to the N-terminal amino acids Glu 4 -Leu-Arg 6 [START_REF] Moser | Interleukin-8 antagonists generated by N-terminal modification[END_REF], whereas cleavage by ScpC was reported to occur between Gln 59 and Arg 60 of mature IL-8 [START_REF] Edwards | Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes[END_REF] (Fig. 6A). It was thus of interest to determine whether the cleavage activity of Spy0416 could be influenced by mutations within IL-8 and whether the specificity could be assigned to the cleavage residues itself or residues outside of the cleavage site. It was shown recently that the interaction of IL-8 with the GPC receptor peptide derived from CXCR1 involves phenylalanine residues at position 17 and 21 of the chemokine [START_REF] Skelton | Structure of a CXC chemokine-receptor fragment in complex with interleukin-8[END_REF]. Interestingly, degradation of mutant IL-8 F17K,F21K by Spy0416 is abolished as compared to wild type IL-8 (Fig. 6B). Further IL-8 mutants containing other than Lys substitutions at the Phe 17 and Phe 21 positions (e.g. Arg) exhibited a similar reduction in cleavage by Spy0416 (data not shown), indicating that changes within IL-8 distinct from the cleavage site strongly influence the interaction with Spy0416.

Recombinant Spy0416 recognizes the human CXC cytokines IL-8 and GRO-. So far IL-8, GCP-2 and GRO-were the human cytokines that were reported to be degraded by ScpC from culture supernatants [START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF][START_REF] Sumby | A chemokine-degrading extracellular protease made by group A Streptococcus alters pathogenesis by enhancing evasion of the innate immune response[END_REF], which prompted us to analyse these and further human chemokines of the CXC family for activity exerted by the recombinantly produced Spy0416. The chemokines PF-4, NAP-2, SDF-1 , GRO-P and I-TAC were selected as they belong to the same chemokine (CXC) family and display very similar overall three-dimensional folds. Furthermore MCP-1 was selected from the CC-chemokines for the degradation assay. Except for GRO-the combined Spy0416A & B domains were not capable to readily degrade any of the human CXC chemokines nor the CC chemokine tested (Fig. 6C; data not shown).

Discussion

The cell envelope proteinase ScpC has been shown to be a mediator of a specific and novel mechanism employed by Group A streptococcus to escape the innate immune response upon infection [START_REF] Edwards | Specific C-terminal cleavage and inactivation of interleukin-8 by invasive disease isolates of Streptococcus pyogenes[END_REF][START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF]. Deletion of the scpC gene clearly showed its important role as a virulence factor, whether inactivation of the scpC gene occurred in a M14 GAS strain and only in a scpA negative background [START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF] or in a M1 or M49 strain [17, 19, 20, unpublished results]. Degradation of host CXC chemokines (human IL-8, GCP-2, GRO-and murine KC and MIP-2) by ScpC is assumed to prevent recruitment of polymorphonuclear leukocytes to the site of infection, allowing GAS to escape from the immune response.

Our studies aiming at the identification of novel candidates for a Group A streptococcal vaccine have selected Spy0416 as one of several protective antigens. The previously provided evidence that IL-8 cleavage is mediated by Spy0416 [START_REF] Hidalgo-Grass | A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues[END_REF] and its selection as vaccine candidate warranted further characterization. It was first necessary to establish an in vitro system to measure IL-8 cleavage activity. Using recombinant Spy0416 protein we could show that it mediates proteolysis of IL-8 in vitro and that the predicted active site residues are essential for enzymatic activity. Surprisingly the predicted catalytic domain alone (Spy0416A) was not sufficient, but instead activity could only be restored by separate addition of the C-terminal Spy0416B. Many of the known cell envelope proteinases possess multiple domains [START_REF] Siezen | Multi-domain, cell-envelope proteinases of lactic acid bacteria[END_REF], but most of their functions have not been elucidated. The S. pyogenes Spy0416 protein consists of at least 8 distinct regions (Fig. 1). The essential function of the non-catalytic Spy0416B for enzymatic activity may possibly be attributed to a structural change that it triggers in Spy0416A upon binding or by contributing to the binding of IL-8 itself, thereby supplying the substrate for proteolysis to the active site of the enzyme. Similarly, it was postulated for C5a peptidase from S. agalactiae, that binding of integrins to the Fn1-3 and PA domains would enable accessibility of the active site for its substrate [START_REF] Brown | Structure of the streptococcal cell wall C5a peptidase[END_REF]. The data from isothermal fluorescence studies indicated that binding of IL-8 was mainly mediated by the proteinase domain of Spy0416, but also showed that Spy0416B could contribute to binding of IL-8. In agreement with its higher affinity, only Spy0416A, but not Spy0416B was capable of significant binding to IL-8 as determined by gel depletion and surface Plasmon resonance assays. However when both proteins, Spy0146A and Spy0416B were supplied together in the gel depletion assay, Spy416B reduced binding of Spy0416A to IL-8. Apparently, under these conditions, the interaction of Spy0416A and Spy0416B dominates over Spy0416A binding to IL-8. Since Spy0416A binds with significantly higher affinity to IL-8 (yet without exhibiting proteolytic activity; see Fig. 3 and4), we hypothesise that IL-8 binds predominantly to the A domain of full-length Spy0416 which causes a conformational change leading the B domain to ´´lock´´ the IL-8 substrate in the catalytic site, which then triggers enzymatic activity. Under gel depletion conditions, however, Spy0416B prevents binding of IL-8 to Spy0416A by a strong interdomain (A-B) interaction which is somehow leaky in the case of the Spy416A-D151A mutant. The reduced binding of IL-8 by Spy0416A in the presence of Spy0416B may also indicate Spy0416B-induced changes in structure or binding of Spy0416A resulting in the observed reduction in IL-8 binding. The importance of the interactions between the two proteins, Spy0416A & B, for catalytic activity is also underlined by the abolishment of activity through removal of the auxiliary PA and Fn1 domains from Spy0416A, and of any of the separate domains from Spy0416B, which may possibly contribute to these interactions. This is also reflected in the reduced binding of IL-8 observed by SPR for the Spy0416A1 mutant protein, lacking the Fn1 domain. As most of the non-catalytic domains of serine proteinases have not been analyzed in more detail, functions can currently only be postulated for such domains. For example, the PA domain of C5a peptidase A c c e p t e d M a n u s c r i p t from S. agalactiae may be involved in recognition of a cellular receptor thus triggering enzymatic activity [START_REF] Brown | Structure of the streptococcal cell wall C5a peptidase[END_REF][START_REF] Kagawa | Model for Substrate Interactions in C5a Peptidase from Streptococcus pyogenes: A 1.9 A Crystal Structure of the Active Form of ScpA[END_REF]. We assume that the deletion of the PA region might have either disrupted the structure of the proteinase itself or that it is directly involved in substrate recognition or cleavage. The Fn1 region on the other hand might mediate interactions with Spy0416B and therefore be essential for IL-8 cleavage activity. Interestingly, based on the crystal structure of ScpA from S. pyogenes it was recently postulated that the PA and Fn2 domains are involved in substrate binding by ScpA [START_REF] Skelton | Structure of a CXC chemokine-receptor fragment in complex with interleukin-8[END_REF]. The successful in vitro reconstitution of IL-8 cleavage activity by recombinant Spy0416 proteins has also allowed us to further investigate the interaction of Spy0416 with the substrate IL-8, and to perform studies to identify possible interactions with other bacterial or host proteins.

It is known that IL-8 interacts via the distinct amino acid residues Glu 4 -Leu-Arg 6 with its receptor CXCR2 [35] and that mutation of the Phe 17 and Phe 21 positions results in deficiencies to promote neutrophil chemotaxis (data not shown). The reduced activity of Spy0416 towards the biological inactive IL-8 F17K,F21K mutant provides first evidence for the basis of the specificity of IL-8 cleavage activity by Spy0416. Since the IL-8 mutant is deficient in neutrophil chemotaxis, Spy0416 seems to have evolved a very efficient way to inactivate the biological active chemokine, whereas inactivation of IL-8 by mutation coincides with abolished cleavage activity by Spy0416. As can be seen from the molecular structure of the wild type IL-8 molecule (see Figure 6A), the proximal loop containing the amino acids Phe 17 and Phe 21 is in close proximity to the C-terminal helix containing the Spy0416 cleavage site Gln 59 -Arg 60 . Introducing basic amino acids like lysine into the proximal loop of the chemokine induced a structural change -as detected in far-UV CD spectroscopy (data not shown) -which prohibits interaction with the protease by sterical and/or electrostatic hindrance. While Spy0416 cleaved IL-8 is unable to promote neutrophil chemotaxis, our Ca 2+ -release data suggest that the cleavage of IL-8 does not affect its capability to activate neutrophils. This is in line with previous observations, that the ELR motif of IL-8 is mainly responsible for receptor recognition, whereas the C-terminal domain, cleaved off by Spy0416, is required for inducing neutrophil migration also because it is responsible for glycosaminoglycan (GAG) binding [START_REF] Clark-Lewis | Structure-activity relationships of interleukin-8 determined using chemically synthesized analogs[END_REF][START_REF] Webb | Binding to heparan sulfate or heparin enhances neutrophil responses to interleukin 8[END_REF]. Chemokine binding to cell-surface GAG molecules like heparan sulphate (HS) is a major prerequisite (1) for establishing the solid state chemotactic gradient along an inflamed endothelial tissue and (2) for inducing conformational changes and higher oligomerization states of the chemokine in order to fully activate the protein. The impact of Spy0416 on IL-8 biological activity is apparently to impair the chemokine's GAG binding activity thereby interrupting the chemotactic response of neutrophils.

Using the newly established in vitro Spy0416 assay, we could furthermore successfully confirm that Spy0416 exerts another activity possibly contributing to virulence and/or evasion of the immune system. The human cytokine GRO-was very efficiently degraded in vitro by Spy0416. Despite the strong structural similarity to IL-8, with IL-8, NAP-2 and GRO-all binding to the same human receptor CXCR2 [36], the human cytokine NAP-2 was not degraded in vitro by Spy0416 (Fig. 5C). Interestingly, it was recently described that the related C5A peptidase from S. agalactiae also exerts distinct specificity for individual chemokines, by degrading NAP-2 and GRO-, but displaying no activity for IL-8 [37]. From a linear sequence comparison, the cleavage site within IL-8, Gln 59 -Arg 60 , is not conserved among the investigated CXC chemokines. However, sequence alignment between IL-8 and GRO-showed that GROcontains isofunctional amino acids at IL-8-homologous positions, namely Lys 94 -Lys 95 . Since both NAP-2 (Lys 114 -Lys 115 ) and PF-4 (Lys 114 -Lys 115 ) contain similar isofunctional sequence deviations from IL-8 as GRO-, we hypothesise that the difference in degradability by Spy0416 is, in addition to sequence-specificity, dependent upon the different oligomerization behaviour of the different chemokines under investigation.

The lack of proteolytic activity of the individual domains of Spy0416 and the observed participation of the non-catalytic domains of Spy0416B to CXC chemokine degradation in vitro also argues for the essential contribution of several, if not all of the domains of Spy0416 for enzymatic activity and thus for its contribution to virulence. This most likely also explains the high conservation level that we determined for this large cell envelope proteinase throughout the entire amino acid sequence. PCR analyses showed that Spy0416 is present in at least 98% of more than 300 tested Group A streptococcal strains. Subsequent sequencing of the Spy0416 gene from more than 60 strains, including 26 distinct emm types, revealed that the protein shows more than 95% sequence identity within the analyzed Group A streptococcal strains (data not shown). This high level of sequence conservation of Spy0416 gives reason to speculate that it may exhibit additional functions besides its degradation of the CXC chemokines IL-8 and GRO-. In this regard it is of interest that the protease domain of Group B streptococcal C5a peptidase has recently been shown to contribute to adhesion of host cells by binding to immobilized fibronectin [38]. Spy0416 may thus elicit its contribution to GAS virulence not only through degradation of IL-8, but may interact with other GAS or host proteins as well. It can thus be postulated that the entire sequence of Spy0416 needs to be conserved in order to exhibit CXC chemokine cleavage activity and to maintain potentially further roles contributing to escape from the human immune defence.

The described activities, the high sequence conservation together with our unpublished and the recently reported protection data by several research groups [START_REF] Sumby | A chemokine-degrading extracellular protease made by group A Streptococcus alters pathogenesis by enhancing evasion of the innate immune response[END_REF][START_REF] Rodriguez-Ortega | Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome[END_REF][19][START_REF] Sjölinder | The ScpC protease of Streptococcus pyogenes affects the outcome of sepsis in a murine model[END_REF] provide strong evidence that Spy0416 can contribute to a vaccine capable of preventing Group A streptococcal infections. shown in ball and stick representation are amino acids targeted by Spy0416 as well as sites which are, in addition, required for processing by Spy0416. (B) IL-8 F17K,F21K was incubated at 6.6 µg/ml with Spy0416A and Spy0416B (22 µg/ml each) at 37°C up to 72 hours. IL-8 F17K,F21K was visualized by Western blot analysis as described in Experimental. (C) PF-4 (6.6 µg/ml), NAP-2 (6.6 µg/ml), GRO-(26.3 µg/ml) and SDF-1 (26.3 µg/ml) were incubated with Spy0416A and Spy0416B (22 µg/ml each) at 37°C up to 72 hours. The control (C) represents chemokine without Spy0416A and Spy0416B incubated at 37°C for 72 hours.

Fritzer et al. Fritzer et al. Fritzer et al.

Figure 6

ExperimentalReagents.

  The listed reagents were purchased as following: Human chemokines NAP-2, SDF-1 GRO-MCP-1, IP-10 and I-TAC (PeproTech Inc., UK); recombinant human IL-8 and polyclonal goat anti-human CXCL8/IL-8, CXCL11/I-TAC and IP-10 antibodies (R&D Systems, USA); polyclonal rabbit anti-human IL-8, PF-4, NAP-2 and GRO-antibodies (Abcam, UK); goat polyclonal antibodies to SDF-1 and MCP-1 (Santa Cruz Biotechnology, Inc., USA); antirabbit IgG Peroxidase conjugated antibody (Sigma, USA); polyclonal rabbit anti-goat IgG-HRP antibody (DakoCytomation, Denmark); monoclonal mouse penta-his antibody (Qiagen, The Netherlands); Ni-Sepharose TM 6 Fast Flow beads (GE Healthcare Biosciences AG, UK); Wide Range Marker (Sigma-Aldrich, USA); PageRuler™ pre-stained protein ladder (Fermentas, Canada); Simply Blue Safe Stain Solution (Invitrogen, USA); Ponceau S stain (Sigma-Aldrich, USA); ECL system (Amersham Pharmacia Biotech, UK). Genomic DNA from S. pyogenes isolates was purified using Wizard ® Genomic DNA Purification Kit (Promega, USA). All PCRs were performed with the Expand High Fidelity PCR System from Roche (Switzerland) unless otherwise stated.

  18 M EGTA, 1.4 M Tris, 10% Triton X-100 and Maximum buffer (100x): 1M HCl, 0.5M CaCl 2 . 7 Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090278A 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.

  NH2-terminal residues and evidence for uncoupling of neutrophil chemotaxis, exocytosis, and receptor binding activities. J. Biol. Chem. 266, 23128-23134 36 Katancik, J.A., Sharma, A. and de Nardin, E. (2000) Interleukin 8, neutrophil-activating peptide-2 and GRO-alpha bind to and elicit cell activation via specific and different amino acid residues of CXCR2. Cytokine 12, 1480-1488 37 Bryan, J.D., and Shelver, D.W. (2009) Streptococcus agalactiae CspA is a serine protease that inactivates chemokines. J. Bacteriol. 191, 1847-1854 38 Tamura, G.S., Hull, R.J., Oberg, M.D. and Castner, D.G. (2006) High-affinity interaction between fibronectin and the group B streptococcal C5a peptidase is unaffected by a naturally occurring four-amino-acid deletion that eliminates peptidase activity. Infect. Immun.

Figure legends Figure 1

 legends1 Figure legends

Figure 3 Figure 4

 34 Figure 3 Ca 2+ -release by neutrophilsNeutrophils were isolated as described under Experimental. IL-8 was incubated with Spy0416A and Spy0416B in PBS at 37°C and degradation was stopped after 3.5 hrs by freezing the sample at -20°C. Fluorescence was monitored for app. 3 min. (A) Calcium-release by neutrophils following injection of undigested IL-8 (840 ng) after 60 seconds (arrow). (B) Calcium-release of neutrophils after IL-8 (840 ng) injection previously incubated for 3.5 hrs with Spy0416A/B (described as IL-8d in this Figure)Figure 4 Binding of IL-8 to Spy0416 as measured by Isothermal Fluorescence Titration and Surface Plasmon Resonance (SPR) analysis(A) Isothermal fluorescence titrations of 39 nM Spy0416A ( ) or 39 nM Spy0416B ( ) was measured in the presence of 1 M bis-ANS. Excitation was set at 395 nm and fluorescence emission spectra were collected between 450 nm and 550 nm. The fluorescence quenching of bis-ANS emission upon addition of increasing IL-8 concentrations was background corrected and normalised for generation of the binding isotherms. K D values were fitted by non linear regression as described in Experimental. (B) Sensorgram overlay of Spy0416 variants binding to immobilized IL-8. Spy0416A ( ) or Spy0416A1 ( ) were injected over an IL-8 sensorchip at 0, 50, 100, 250, 500 and 750 nM concentration for 3 min at 30 µl/min flow rate. The RU (resonance unit) response was recorded as a function of time and kinetic rates were calculated from both association and dissociation phases.

Figure 5

 5 Figure 5 Interaction of Spy0416 variants and IL-8 evaluated by gel depletion assay Western Blot analysis of IL-8/ Spy0416-protein complexes after 15% native-PAGE. lane 1: 5.2 M Spy0416A + 54 M IL-8; lane 2: 4.9 M Spy0416B + 54 M IL-8; lane 3: 5.2 M Spy0416A-D151A + 54 M IL-8; lane 4: 2.6 M Spy0416A + 3.5 M Spy0416A-D151A + 54

Figure 6

 6 Figure 6 Degradation of the IL-8 mutant IL-8 F17K,F21K and the human CXC chemokine GRO-by Spy0416 (A) Ribbon drawing of the IL-8 backbone structure;shown in ball and stick representation are amino acids targeted by Spy0416 as well as sites which are, in addition, required for processing by Spy0416. (B) IL-8 F17K,F21K was incubated at 6.6 µg/ml with Spy0416A and Spy0416B (22 µg/ml each) at 37°C up to 72 hours. IL-8 F17K,F21K was visualized by Western blot analysis as described in Experimental. (C) PF-4 (6.6 µg/ml), NAP-2 (6.6 µg/ml), GRO-(26.3 µg/ml) and SDF-1 (26.3 µg/ml) were incubated with Spy0416A and Spy0416B (22 µg/ml each) at 37°C up to 72 hours. The control (C) represents chemokine without Spy0416A and Spy0416B incubated at 37°C for 72 hours.

Figure 1 Spy0416A

 1 Figure1

Figure 2

 2

  Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090278 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. © 2009 The Authors Journal compilation © 2009 Portland Press Limited Fritzer et al.

Figure 3

 3 Figure3

  Publication. Published on 24 Jun 2009 as manuscript BJ20090278 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090278 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. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

11

  Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090278

Table 1 : Summary for Spy0416 constructs and mutants generated for this study.

 1 The oligonucleotides, vectors and cloning sites used for cloning of the Spy0416 constructs are listed. Mw, calculated molecular weight of the expressed fusion protein. Size, number of bp derived from the Spy0416 gene (full length: 4944 bp).

	74, 5739-

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Figure 2 Degradation of Interleukin 8 by Spy0416 and its active site and deletion mutants

	All proteins were used individually at 44 µg/ml and in combination at 22 µg/ml of each protein
	and incubated with 6.6 µg/ml IL-8 at 37°C for up to 3 hour. Human IL-8 was detected by
	Western blot. (A) Spy0416A and/or B incubated with IL-8. (B) IL-8 incubated with Spy0488 and
	Spy1727. (C) Spy0416A-D151A (A-D151A) and Spy0416A-S617A (A-S617A) in combination
	with Spy0416B incubated with IL-8. (D) Spy0416A and Spy0416A1 in combination with
	Spy0416B incubated with IL-8. (E) Spy0416A and Spy0416B were incubated at increasing
	concentrations for each protein with IL-8 for various time intervals at 37°C. BSA as present in
	the IL-8 preparation was visualized as loading control by Ponceau red staining (bottom panel).
	(F) IL-8 degradation using each 22 µg/ml Spy0416A and Spy0416B at various time intervals and
	constant amounts of IL-8 (6.6 µg/ml). IL-8 was quantified by chemiluminescence. (G) IL-8
	degradation using increasing amounts of Spy0416A and Spy0416B and constant amounts of IL-8
	(6.6 µg/ml). IL-8 was quantified as under F.

, 5 cycles with an annealing temperature of 50°C followed by

cycles with an increased annealing temperature of 62°C. Both mutant constructs were cloned into the pET28b(+) expression vector.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Biochemical Journal Immediate Publication. Published on 24 Jun 2009 as manuscript BJ20090278 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20090278 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. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

We are grateful to Astrid Teubenbacher and Simon Rittmann for technical assistance. This work was supported by the Austrian Science Fund (project P17872-B09 to A.R.) and by the GEN-AU programme of the Austrian federal ministry for science and research (project APP-II) to A.J.K. The authors declare potential conflict of financial interest being employees of Intercell AG, a biotechnology company.