Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal 1 enzyme, SpyCEP 2

Abstract


Introduction
Group A Streptococcus (GAS) or Streptococcus pyogenes is a leading human pathogen that manifests clinically as a broad spectrum of diseases, ranging from less severe, usually self-limiting infections to life-threatening invasive diseases such as necrotizing fasciitis and toxic shock syndrome.
While much of the global health burden can be attributed to streptococcal auto-immune sequelae such as rheumatic heart disease, invasive diseases contribute greatly to S. pyogenes associated global mortality.Invasive infections account for an estimated 163,000 deaths worldwide per year and at least 663,000 new cases (Carapetis et al., 2005) with mortality rates remaining high despite intervention; indeed, 20% of patients with invasive S. pyogenes disease die within 7 days of infection (Ralph and Carapetis, 2013).
Several virulence factors contribute to pathogenesis in invasive S. pyogenes disease, chief among which is Streptococcus pyogenes cell envelope protease (SpyCEP), an immune-modulatory cell wall-associated protease.SpyCEP is responsible for the rapid and efficient cleavage of a distinct group of CXC chemokines comprising CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8 both locally at the site of an infection, and systemically, (Edwards et al., 2005, Kurupati et al., 2010, Turner et al., 2009a, Zingaretti et al., 2010).The ELR + chemokines, named for their conserved Nterminal glutamate-leucine-arginine motifs, specifically act upon neutrophils eliciting their recruitment and activation.SpyCEP inactivates these chemokines by cleaving the chemokine Cterminal α-helix, releasing a 13 amino acid peptide in the case of CXCL8 (Edwards et al., 2005).Specificity for binding the neutrophil chemokine receptors CXCR1 and CXCR2 is conferred to a large extent by the chemokine N-terminal ELR motif (Middleton et al., 1997), although the chemokine Cterminus is necessary for efficient receptor binding and activation (Goldblatt et al., 2019) in addition to chemokine translocation from tissues to endothelial lumen (Middleton et al., 1997).As such, SpyCEP cleavage of CXC chemokines results in a reduction of CXCR1 and CXCR2-mediated neutrophil chemotaxis and subsequent paucity of neutrophils at the site of S. pyogenes infection (Edwards et al., 2005).In addition to CXC chemokines, SpyCEP has recently been shown to cleave the human anti-microbial peptide, LL-37 (Biswas et al., 2021).Whilst cleavage does not affect the antimicrobial action of the peptide, it was reported to reduce LL-37 specific neutrophil chemotaxis (Biswas et al., 2021).
SpyCEP is expressed by S. pyogenes as a 1647 amino acid, 180 kDa subtilisin-like serine protease, the crystal structure of which was recently solved to 2.8 Å resolution (Jobichen et al., 2018) and further refined to 2.2 Å resolution (McKenna et al., 2020).It is a member of the S8 subtilase family, members of which are characterised by a catalytic triad consisting of an aspartate, histidine and serine residue each surrounded by a region of highly conserved amino acids (Siezen, 1999).SpyCEP is unique among streptococcal proteases in that, during maturation, it is autocatalytically cleaved between residues Q244 and S245 into 2 distinct polypeptides, a 30 kDa Nterminal polypeptide and a 150 kDa C-terminal polypeptide.The two polypeptides harbour the separate residues required for the formation of the catalytic site (Zingaretti et al., 2010); the Nterminal fragment contains the catalytic D151 and the C-terminal fragment contains the catalytic H279 and S617 residues.Upon cleavage, the two polypeptides re-associate non-covalently to reconstitute the active enzyme (Zingaretti et al., 2010).The recent crystal structures have shed further light upon the structure of SpyCEP, describing 9 separate domains, the first 5 of which contain the catalytic triad necessary for enzymatic activity (Jobichen et al., 2018).
SpyCEP has been included as a target antigen in several recent S. pyogenes vaccine designs due to its cell surface expression, highly conserved nature, and central role in S. pyogenes pathogenesis.
Immunisation with SpyCEP successfully elicits a SpyCEP-specific neutralising antibody response, providing protection against systemic bacterial dissemination and reducing disease severity in S. pyogenes intramuscular, skin infection models and non-human primate infection models (Rivera-Hernandez et al., 2019, Bensi et al., 2012, Pandey et al., 2015, Pandey et al., 2016, Rivera-Hernandez et al., 2016, Turner et al., 2009b).Data that demonstrate vaccine dependence on enzyme inhibition highlight the importance of understanding the enzymatic activity of SpyCEP and the potential to improve upon vaccine or inhibitor design.

Construction of recombinant S. pyogenes expressing histidine-tagged SpyCEP
To permit release of soluble His-tagged SpyCEP by S. pyogenes, the C-terminal anchor domain of SpyCEP was replaced by a stop codon in strain H292, a strain that makes abundant SpyCEP (Turner et al., 2009a).A 549 base pair region immediately upstream of the SpyCEP cell wall anchor motif was amplified from S. pyogenes H292 genomic DNA using the primers :5′GGGAATTCTGTTGTCAGGTAACAGTCTTATCTTGCC -3′ and 5′CCGAATTCACAACACTAGGCTTTTGCTGAGGTCGTTG -3′.EcoRI restriction sites were incorporated at the terminal ends of the primer sequences.The amplified DNA was cloned into the homologous recombination plasmid pUCMUT to produce the vector pUCMUT CEP which was transformed into One Shot TOP10 Chemically Competent E. coli (Thermo Fisher Scientific) according to the manufacturer's instructions.The nucleotide sequence of the SpyCEP C-terminus was subsequently further modified to encode a hexa-histidine tag by inverse PCR using pUCMUT CEP as the template and the primers 5'-TATCCTAGGTAGTGTTGTGAATTCGTAATCATGGTCATAG-3' and 5'-TATCCTAGGATGATGATGATGATGATGGGCTTTTGCTGAGGTCGTTG-3'.The amplification was preformed using GoTaq Long PCR Master Mix (Promega) according to the manufacturer's instructions.AvrII restriction sites, incorporated at the terminal ends of the primer sequences, EMBO Reports manuscript facilitated re-circulation of the amplified plasmid.The presence of a hexa-his sequence in the modified pUCMUT CEP construct (denoted pUCMUT CEP-HIS ) was confirmed by Sanger sequencing using the pUCMUT sequencing primers 5'-GACAGCAACATCTTTGTGAAAGATGG-3' and 5'-CATTAATGCAGCTGGCACGAC-3'.The pUCMUT CEP-HIS construct was introduced into H292 by electroporation and crossed into the chromosome by homologous recombination as previously described (Lynskey et al., 2013) to generate strain H1317.Secretion of His-tagged SpyCEP into the culture supernatant of H1317 was confirmed by western blotting (data not shown).To purify SpyCEP, S. pyogenes H1317 was grown in Todd-Hewitt broth (Oxoid) overnight at 37 o C with 5% C0 2 The culture was pelleted at 2500 xg for 10 minutes and the supernatant sterilised using Amicon 0.22 µM filters, then concentrated using 15 ml Amicon 100 kDa centrifuge filter columns and purified by nickel column affinity chromatography (Novagen His-Bind Resin) as per the manufacturer's instructions.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-page)
To visualise chemokine cleavage, 5 µM recombinant human CXCL1 and CXCL8 (R&D Systems) was incubated with full-length recombinant SpyCEP or C-terminal SpyCEP at a molar ratio of 1:5, 50, 500, 5000 in favour of chemokine for 2 hours at 37 O C. SpyCEP DASA , C-terminal SA and N-terminal constructs 34-244 were included as controls and assayed at the highest 1:5 molar ratio.Reactions were halted by the addition of Dithiothreitol (DTT) to a final concentration of 100 mM, 4X Bolt LDS sample buffer and heating to 70°C for 10 minutes.Samples were separated on pre-cast 4-12% MES buffered Bolt Bis-Tris gradient gels (Invitrogen) by SDS-PAGE gel electrophoresis at 165 V for 35 minutes with SeeBlue Plus 2 (Invitrogen) used for molecular weight ladder.Gels were stained with PageBlue protein staining solution (Thermo Fisher Scientific) overnight and de-stained in deionised water.
To visualise generation of both intact CXCL8 and the larger (N-terminal) CXCL8 cleavage product, a rabbit antiserum was raised against the neo-epitope (anti-ENWVQ) that is exposed following SpyCEP cleavage of CXCL8 (Edwards et al., 2007).SpyCEP constructs were incubated at 37 o C with 937.5 nM of recombinant human CXCL8 (R&D Systems) at a 1:50 molar ratio in favour of CXCL8.Digests were halted and separated by SDS-PAGE gel electrophoresis as described above.Proteins were transferred by iBlot2 (Thermo Fisher Scientific) onto 0.22 μm nitrocellulose membranes as per manufacturer's instructions.Membranes were subsequently blocked for 1 hour at room temperature in blocking buffer (PBS with 5% skimmed milk powder (Sigma-Aldrich) and 0.1% Tween), then blotted overnight at 4 o C with 2 primary antibodies, 1 µg/ml mouse anti-human CXCL8 (R&D Systems) and 1:1000 rabbit anti-ENWVQ.Membranes were washed in wash buffer (PBS with 0.05% Tween) and incubated with 1:7500 goat anti-rabbit IgG A680nm and 1:7500 goat anti-mouse IgG A790nm for 1 hour at room temperature before being visualised on LiCor Odyssey Fc (Invitrogen).
SpyCEP activity against LL-37 was assessed by an 16 hour, 37 o C incubation of 5.56 µM human LL-37 (R&D Systems) with SpyCEP constructs at a 1:10 molar ratio in favour of LL-37.Reactions were stopped, separated and blotted onto 0.22 μM nitrocellulose as above and incubated overnight at 4 o C in blocking buffer supplemented with 2 μg/ml polyclonal sheep IgG anti-LL-37 (R&D Systems).
Membranes were washed in wash buffer (PBS with 0.05% Tween) and incubated in blocking solution with rabbit anti-sheep IgG (Abcam) at 1:40,000 dilution for 1 hour at room temperature and visualised on LiCor Odyssey Fc (Invitrogen).

EMBO Reports manuscript
SpyCEP were incubated with an equal volume of 20 nM human CXCL8 and CXCL1 respectively (R&D Systems) and incubated at room temperature for 30 minutes.Reactions were halted at defined timepoints with the addition of concentration of Pefabloc (Sigma-Aldrich) to a final concentration of 2 mg/ml (8.34 mM).
Linear regression analyses of the initial five time points of CXCL1 and CXCL8 cleavage (0, 1, 2, 3 and 4 minutes) was utlised to determine the maximal rate of SpyCEP activity.

Mass spectrometry analysis of CXCL8 cleavage
Analysis of CXCL8 cleavage was assayed on a SCIEX API6500 triple quadrupole electrospray mass spectrometer coupled to a high-throughput robotic sample preparation and injection system, RapidFire200 (Agilent Technologies).CXCL8 substrate, the 13 amino acid CXCL8 SpyCEP cleavage product RVVEKFLKRAENS, and a heavy atom substituted internal standard of CXCL8 SpyCEP cleavage product RV[U13C5 15N-VAL]-EKF-[U-13C6 15N-Leu]-KRAENS) were monitored by mass spectrometry.The mass spectrometer was operated in positive electrospray MRM mode, and transitions (Q1/Q3) for each species were optimised to give m/z as follows: CXCL8, 1048.7/615.3,CXCL8 cleavage product 526.2/211.1,internal standard 530.5/211.1.A dwell time of 50 ms was used for the MRM transitions.The mass spectrometer was operated with a spray voltage of 5500 V and at a source temperature of 650 °C.
To assay CXCL8 cleavage dynamically, chemokine and SpyCEP constructs were loaded into a 384 well plate to the following final concentrations: 6.25-2000nM chemokine, 250 pM SpyCEP or 40 nM Cterminal SpyCEP.Reactions were stopped at desired time points, 0-240 minutes, by the addition of stop solution (1% formic acid) supplemented with 1 µM heavy atom substituted CXCL8 internal standard and centrifuged 2000 xg for 10 minutes.Assay plates were transferred onto the RapidFire200 integrated to the API6500 mass spectrometer.Samples were aspirated under vacuum directly from 384-well assay plates for 0.6 s.The samples were then loaded onto a C18 solid-phase extraction cartridge to remove non-volatile buffer salts, using HPLC -grade water supplemented with EMBO Reports manuscript 0.1% (v/v) formic acid at a flow rate of 1.5 mL/min for 4 s.The retained analytes were eluted to the mass spectrometer by washing the cartridge with acetonitrile HPLC-grade water (8:2, v/v) with 0.1% (v/v) formic acid at 1.25 mL/min for 4 s.The cartridge was re-equilibrated with HPLC-grade water supplemented with 0.1% (v/v) formic acid for 0.6 s at 1.5 mL/min.Results were normalised to the fixed internal standard and converted to molarity by interpolation from a standard curve of known cleaved CXCL8 concentrations.

Kinetic analysis
Linear regression analyses of the initial five time points of full length SpyCEP (0, 1, 2, 3, 4 minutes) and C-terminal SpyCEP (0, 15, 30, 45, 60 minutes) CXCL8 reactions were plotted against substrate concentration and kinetics derived from the Michaelis-Menton equation Y = V max *X/(K M + X) and the K cat equation Y = ET*k cat *X/(K M + X) where Et = enzyme concentration as fitted by Prism 8.0.2 (GraphPad).

Screening of human CXCL8 cleavage activity using fluorescent western blotting
To initially assess the activity of our recombinant SpyCEP constructs we screened them for CXCL8 cleavage activity using two-colour multiplex western blotting.Human CXCL8 was incubated for 2 hours at 37 o C with SpyCEP constructs, the reaction products were separated by SDS-PAGE and immunoblotted using seperate antibodies that detect either intact or cleaved CXCL8.Detection of full-length CXCL8 (8 kDa, green bands) or the CXCL8 neo epitope (ENWVQ), exposed after SpyCEP cleavage, (6 kDa, red bands) was evident with this system (Figure 1).Both native S. pyogenes SpyCEP and recombinant full-length SpyCEP successfully cleaved CXCL8 to completion (Figure 1).As expected, no cleavage was observed when using the catalytically dead mutant, SpyCEP DASA .As has been previously reported (Zingaretti et al., 2010), the SpyCEP N-and C-termini, when independently expressed and purified, can be re-associated to form an active enzyme, which successfully cleaved CXCL8.The N-terminal fragment of SpyCEP alone could not cleave CXCL8.Unexpectedly however, the C-terminal fragment of SpyCEP was able to cleave CXCL8, albeit not to completion.This independent catalytic activity was negated by mutation of the catalytic S617 to alanine (C-terminal SA construct, Figure 1).Cleavage of CXCL8 by the SpyCEP C-terminal fragment was enhanced when reassociated with the catalytically inert N-terminal mutant (N-terminal DA construct); indeed, activity was equivalent to that observed with the re-associated enzyme under these conditions, with cleavage of CXCL8 to near completion.However, the N-terminal DA construct was unable to restore catalytic activity to the C-terminal SA construct when the two were combined.
The results demonstrated for the first time that the SpyCEP C-terminal fragment alone is sufficient for catalytic cleavage of CXCL8 in this assay system.The data also established that, although the presence of the SpyCEP N-terminal fragment enhanced enzymatic activity, the N-terminal residue D151 was dispensable for enzymatic activity.We further sought to examine whether the C-terminal EMBO Reports manuscript displayed activity against the newly described substrate, LL-37 (Biswas et al., 2021).Western blot analysis confirmed cleavage of LL-37 by full-length recombinant SpyCEP, demonstrated by a reduction in band size, however the C-terminal fragment was unable to cleave LL-37 despite a high molar ratio of enzyme to LL-37 (1: 10) and a prolonged 16-hour incubation at 37 °C (Figure EV1).

Differntial cleavage of CXCL8 and CXCL1 by full-length and C-terminal SpyCEP
To determine whether the catalytic activity of the C-terminal SpyCEP fragment was reproducible over a shorter incubation period, we assessed CXCL8 cleavage by ELISA, where CXCL8 cleavage is detected through a reduction in substrate concentration.SpyCEP C-terminal constructs were incubated with CXCL8 at molar ratios ranging from 1:5 -1:250 (enzyme: chemokine) over a 60 minute timecourse at room temperature.Full-length recombinant SpyCEP and the inactive Cterminal fragment, C-terminal SA , were included as controls at a molar ratio of 1:1000 and 1:5, respectively.Under these conditions, near complete CXCL8 cleavage was observed for full-length SpyCEP and a dose-dependent increase in catalytic activity was observed for the C-terminal fragment (Figure 2).After 5 minutes incubation full-length SpyCEP cleaved over 50% of the starting CXCL8 input, with only 6.25% of CXCL8 remaining after 1 hour.At the highest concentration of C-terminal SpyCEP tested, a 1:5 molar ratio, the C-terminal fragment alone cleaved 9% of the starting CXCL8 by 5 minutes, 25% by 30 minutes and 42% by 60 minutes.Indeed, at the lowest concentration tested, a 1:250 molar ratio, the C-terminal of SpyCEP cleaved 9% of CXCL8 input by 1 hour.As demonstrated by immunofluorescent western blotting, the serine residue at position 617 was vital for SpyCEP catalytic function as no CXCL8 cleavage was observed using the C-terminal SA construct, even when employed at the highest enzyme: chemokine ratio.After a 1 hour incubation, full length SpyCEP and C-terminal contructs (assayed using a molar enzyme: chemokine ratio of 1:5, 1:25 and 1:50) cleaved significantly more CXCL8 compared to the C-terminal SA construct.SpyCEP constructs were co-incubated with CXCL8.Graphs show residual CXCL8 after a 60-minute room temperature incubation, using full length SpyCEP at a 1:1000 ratio to CXCL8; C-terminal at 1:5 -1:250 molar ratio to CXCL8; and C-terminal S617A mutant at a 1:5 molar ratio to CXCL8.Reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml.N=6 experimental replicates for each construct, data points show means, error bars represent SD. ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, at 60 minutes as determined by ordinary one-way ANOVA.
Further SDS-PAGE analysis of the catalytic activity of the C-terminal SpyCEP construct additionally showed that C-terminal activity was not restricted to the CXCL8 substrate.Over 2 hours at 37 o C using a 1:5 molar ratio of enzyme: substrate, the SpyCEP C-terminal construct was capable of cleaving human CXCL1 to near completion (Figure EV2).
To further assess the activity of SpyCEP and to interrogate reaction rates against separate chemokines, full-length SpyCEP was incubated with CXCL1 and CXCL8 and the remaining chemokine levels determined by ELISA.Human CXCL1 or human CXCL8 were incubated with SpyCEP over a 30 minute, room temperature timecourse, at 1:200 or 1:400 molar ratios respectively (enzyme: chemokine).When incubated in equal volumes, 50 pM SpyCEP rapidly and efficiently cleaved 20 nM CXCL8, with ~ 15% of the chemokine input remaining after 10 minutes of incubation (Figure 3A).This contrasted with CXCL1 cleavage, that required 100 pM SpyCEP to cleave just 25% of the chemokine input over the same 10-minute period.Indeed, by 30 minutes SpyCEP had yet to cleave half of the starting CXCL1 (Figure 3B).Utilising a linear regression of the initial 5 timepoints, where SpyCEP activity is maximal, we found that 5 fmol of SpyCEP was able to cleave 284 fmol of CXCL8 per minute, and 10 fmol of SpyCEP was capable of cleaving 62 fmol of CXCL1 per minute.These data confirmed the activity of recombinant SpyCEP and highlighted differential cleavage efficiency across the CXC substrate range -a feature which has been previously recognised but not quantified (Goldblatt et al., 2019).

Mass spectrometry-derived kinetics of active SpyCEP constructs
To directly compare the active full-length and C-terminal SpyCEP constructs and to understand relative catalytic efficiencies, a mass spectrometry approach to continuously assay the generation of the 13 amino acid peptide cleaved from the native substrate CXCL8, following incubation with enzyme, was employed.A range of CXCL8 concentrations (6.25-2000 nM) were incubated with a fixed concentration of enzyme, either 250 pM for SpyCEP or 40 nM for the C-terminal SpyCEP 245-1613    and the production of the 13 amino acid peptide monitored over time.250 pM full length SpyCEP cleaved CXCL8 to near completion over 30 minutes when substrate concentrations were less than 250 nM; incomplete CXCL8 cleavage was observed when substrate concentration was in excess of 250 nM (Figure EV3A.In contrast, 40 nM of the C-terminal SpyCEP 245-1613 cleaved CXCL8 to near completion over 4 hours when the CXCL8 concentration was 250 nM or less; incomplete CXCL8 cleavage was again observed when substrate concentration were over 250 nM (Figure EV3B).Under these conditions the C-terminal SpyCEP fragment maintained measurable catalytic activity thoughout, though with reduced efficacy compared to the full-length construct.A 160-fold increase in enzyme concentration and additional 3.5 hours reaction time were required to cleave a comparable amount of CXCL8.
Linear regression analyses of the initial 5 time points, where the rate of cleaved CXCL8 production was linear, were used to derive Michaelis-Menton plots (Figure 4) and K M and K cat values for each construct (Table 2).

Discussion
SpyCEP is a serine protease and a leading virulence factor of S. pyogenes, with a narrow range of substrate specificity, restricted to the family of ELR + CXC chemokines which modulate neutrophil mediated immune responses and the newly identified substrate, LL-37 (Biswas et al., 2021).
Autocatalytic processing of SpyCEP results in the generation of two individual fragments that reassemble to form an active enzyme (Zingaretti et al., 2010).Here, we describe the enzyme kinetics of full-length SpyCEP and report the K M of the enzyme for its natural substrate to be remarkably low, just 53.49nM, consistent with high efficiency.Furthermore, we demonstrate that the C-terminal SpyCEP fragment can catalyse the cleavage of CXCL1 and CXCL8 independent of the N-terminal fragment.Indeed, when K M values were compared, they were found to be similar, suggesting that substrate binding may be confined to the C-terminal domain of SpyCEP.The enzymatic activity of the C-terminal SpyCEP fragment was, however, markedly reduced compared to full-length SpyCEP.The N-terminal and N-terminal DA constructs were equally able to restore full catalytic activity of the Cterminal SpyCEP fragment.Collectively, this suggests that although the aspartate 151 of the Nterminal fragment may be dispensable for catalysis, the domain itself is important for optimal enzyme activity.
Serine proteases are ubiquitous and comprise up to one third of all proteolytic enzymes currently described.They are currently categorised by the MEROPS database (Rawlings et al., 2012) into 13 distinct clans, being differentiated into groups of proteins based on their evolution from the same common ancestor, with SpyCEP belonging to the S8 family of the SB clan.S8 serine proteases are typified by a classical catalytic triad composed of serine, histidine and aspartic acid residues that together contribute to the hydrolysis of a peptide bond within the substrate.It is recognised that a number of serine protease clans employ a variation on the S8 catalytic triad, utilising instead a triad of serine, histidine, and glutamic acid, or serine, glutamic acid, and aspartic acid residues.Other clans utilise catalytic dyads of lysine and histidine or histidine and serine for proteolytic activity.Our

EMBO Reports manuscript
data suggest that SpyCEP activity can reside in a catalytic dyad of histidine and serine, albeit at a reduced efficacy.It is likely this large gulf in efficiency explains the failure of previous studies to detect catalytic activity within the isolated C-terminal SpyCEP fragment (Zingaretti et al., 2010).
Within serine proteases there are additional features, beyond the catalytic triad residues, which can contribute to activity.The oxyanion hole for example, a pocket in the active site composed of backbone amide NH groups, may provide substrate stabilisation and help drive catalysis (Hedstrom, 2002).Additional residues located in close proximity to the catalytic pocket can also mitigate a loss of activity resulting from a missing residue, and water also has the potential to moonlight as a missing functional group (Hedstrom, 2002).These 'stand ins' can provide a possible substitute machinery to help drive catalytic function.Indeed, some studies have shown that, even with all three catalytic triad residues removed, serine proteases are still capable of catalysis at rates 1000fold greater than the background rate of hydrolysis (Corey and Craik, 1992, Hedstrom, 2002, Paul and James, 1988).
Mass spectrometry-based kinetics showed that full-length active SpyCEP has a K M of 53.49 nM and K cat of 1.34 molecules per second; values which are in agreement with our initial ELISA-based kinetic assessment (McKenna et al., 2020).In contrast, the C-terminal fragment of SpyCEP has a K M of 40.98 nM and K cat of 0.00068 molecules per second.These constructs both demonstrated low, nanomolar K M values, suggesting a high binding efficiency of SpyCEP for its natural substrate CXCL8, likely conferred by the C-terminal domain.This is in keeping with the fact that low nanomolar concentrations of CXCL8 are optimal for neutrophil recruitment (Goldblatt et al., 2019).Although K M values are often reported in the µM -mM range, nanomolar K M values are not without precedent for other serine proteases; human Kallikrein 6 has a reported K M of 300 nM and Factor Xa, a constituent of the prothrombinase complex, has a K M of 150 nM for prothrombin (Angelo et al., 2006, Luettgen et al., 2011).Enzyme specificity, a constant which measures the cleavage efficiency of enzymes, (K cat /K M ), for full length SpyCEP was estimated to be 2.46 x10 7 M -1 s -1 , a value in the EMBO Reports manuscript order of magnitude typical for serine proteases (Hedstrom, 2002).The specificity constant of the Cterminal fragment was ~1500-fold less, 1.67 x10 4 M -1 s -1 , and K cat ~2000 fold less, a reduction that is in line with previously reported aspartic acid mutants from a systematic mutational study of the Bacillus amyloliquefaciens subtilisin catalytic triad (Paul and James, 1988).
The kinetic assays attributed a marked increase in CXCL8 turnover to the additional presence of the N-terminal fragment.Although we did not specifically evaluate the role of the aspartate residue at position 151 on the kinetics of SpyCEP activity, this residue did not contribute appreciably to cleavage of CXCL8 when evaluated using immunoblotting.This raises a question as to whether the Nterminal fragment confers some additional structural contribution to enzyme activity.Our data strongly suggest that substrate binding is likely to be attributed to the C-terminal fragment, a finding consistent with related cell envelope proteinases of Lactococci (Siezen, 1999) and the closely related streptococcal protein, C5a peptidase (Kagawa et al., 2009).
The implications of our findings relating to activity of the C-terminal SpyCEP fragment in S. pyogenes pathogenesis are currently unclear; without the N-terminus, the enzymatic activity detected may be too low to be of consequence for chemokine cleavage in vivo, and in nature both N-and C-terminal fragments are likely to co-exist.SpyCEP has been the focus of S. pyogenes vaccine development since 2006 (Rodriguez-Ortega et al., 2006), used either in isolation or combination with other antigenic targets (Bensi et al., 2012, McKenna et al., 2020, Pandey et al., 2016, Reglinski et al., 2016, Rivera-Hernandez et al., 2016).Vaccine-induced SpyCEP specific antibodies appear not to act through traditional opsonic means (Rivera-Hernandez et al., 2019) and so likely act through inhibition of SpyCEP activity.A majority of vaccine preparations evaluated have been based on 'CEP5'; a polypeptide spanning residues 35 -587 of SpyCEP which contains the N-terminal fragment and only part of the C terminal fragment (Turner et al., 2009b).Our findings relating to enzyme function highlight the possibility that antibodies targeting the C-terminal fragment of SpyCEP are more likely to provide greater neutralizing activity, and potentially improve vaccine efficacy.

Figure 1 .
Figure 1.Cleavage activity of recombinant SpyCEP constructs assayed by immunoblot.Two colour immunoblot showing cleavage of 150 ng CXCL8 incubated for 2 hours at 37 o C either alone (2 nd lane) or with panel of SpyCEP constructs at a 1:50 molar ratio (SpyCEP:CXCL8).Green bands represent intact CXCL8 (anti-CXCL8 antibody); red bands represent cleaved CXCL8 (anti-ENWVQ).A 17 kDa molecular weight marker is shown in blue, 8 kDa and 6 kDa molecular weights are highlighted by arrows.Figure is representative of 2 independent immunoblots.

Figure 2 .
Figure 2. Cleavage activity of SpyCEP and C-terminal SpyCEP 245-1613 constructs using CXCL8 ELISA.SpyCEP constructs were co-incubated with CXCL8.Graphs show residual CXCL8 after a 60-minute room temperature incubation, using full length SpyCEP at a 1:1000 ratio to CXCL8; C-terminal at 1:5 -1:250 molar ratio to CXCL8; and C-terminal S617A mutant at a 1:5 molar ratio to CXCL8.Reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml.N=6 experimental replicates for each construct, data points show means, error bars represent SD. ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, at 60 minutes as determined by ordinary one-way ANOVA.

Figure 3 .
Figure 3. Cleavage of CXCL8 and CXCL1 by recombinant full length SpyCEP.30-minute, room temperature cleavage time course of: A. 20nM CXCL8 by an equal volume of 50pM SpyCEP and B. 20nM CXCL1 by an equal volume of 100pM SpyCEP.Cleavage reactions were halted at specified timepoints by the addition of Pefabloc to a final concentration of 2 mg/ml and the remaining chemokine measured by ELISA.N=6 experimental replicates for each data point, error bars represent SD of mean values.

Figure 4 .
Figure 4. Michaelis-Menton analysis of full length and C-terminal SpyCEP construct cleavage of CXCL8.Data points represent the mean change in cleaved CXCL8 production over time (M s -1 ) of, A. full-length SpyCEP and B. C-terminal SpyCEP).Error bars represent the standard error of the mean of 5 reactions.

Figure EV1 .
Figure EV1.Western blot of SpyCEP specific LL3-7 cleavage.Immunoblot of 500 ng human LL-37 incubated for 16 hours at 37 o C either alone (lane 7) or with a panel of SpyCEP constructs at a 10:1 molar ratio in favour of LL-37.4.5 kDa full length LL-37 band and 3.5 kDa cleaved LL-37 band are indicated by arrows and were detected with 2 μg/ml sheep IgG polyclonal LL-37 antibody and rabbit anti-sheep IgG antibody (1:40,000).Figure is representative of two experiments.

Table 1 . SpyCEP constructs used in this study.
HiLoad Superdex 200 or 75 prep grade column (GE Healthcare) dependent on molecular weight.The resultant panel of recombinantly expressed SpyCEP constructs is shown in Table1.The reassociation of the SpyCEP N and C-termini to create full-length constructs was carried out by equimolar co-incubation at 37 o C for 30 minutes in 40 mM Tris-HCL pH 7.5, 0.1 mM CHAPS, 1 mM DTT, 0.1% BSA, 75 mM NaCl.Constructs were expressed recombinantly in E. coli except for s.pSpyCEP that was expressed in, and purified from S. pyogenes .
Catalytic activities of SpyCEP constructs were measured through detection of remaining intact CXCL8 or CXCL1 substrate following incubation with SpyCEP by ELISA (R&D Systems human CXCL8 and CXCL1 DuoSet ELISA) as per manufacturer's instructions.For cleavage time courses 50 pM or 100 pM .CC-BY 4.0 International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

Table 1 .
SpyCEP constructs used in this study.Constructs were expressed recombinantly in E. coli except for s.pSpyCEP that was expressed in, and purified from S. pyogenes.

Table 2 . Kinetic parameters of full length and C-terminal SpyCEP construct activity in cleavage of CXCL8
. K cat and K M ± SD and K cat / K M for full length SpyCEP and C-terminal SpyCEP derived from Michaelis-Menton graphs Y = V max *X/(K M + X) and K cat equation Y = ET*k cat *X/(K M + X) where Et = enzyme concentration, 2.5 x10 -10 for full length SpyCEP and 4 x10 -8 for C-terminal SpyCEP