Porphyromonas gingivalis Gingipains Display Transpeptidation Activity

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Porphyromonas gingivalis Gingipains Display Transpeptidation Activity Lianyi Zhang, Paul D Veith, N Laila Huq, Yu-Yen Chen, Christine A Seers, Keith J Cross, Dhana G Gorasia, and Eric C Reynolds J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00286 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Journal of Proteome Research

Revised Submission to Journal of Proteome Research

Porphyromonas gingivalis Gingipains Display Transpeptidation Activity Lianyi Zhang¶, Paul D. Veith¶, N. Laila Huq, Yu-Yen Chen, Christine A. Seers, Keith J. Cross, Dhana G. Gorasia and Eric C. Reynolds*

Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.

Running title: Gingipain transpeptidation

* Corresponding author: [email protected]; +61 3 9341 1547

1 ACS Paragon Plus Environment

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Abstract Porphyromonas gingivalis is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arggingipains (RgpA and RgpB) are major virulence factors and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalysed transpeptidation. Purified gingipains and P. gingivalis whole cells were used to digest selected substrates including human haemoglobin in the presence or absence of peptide acceptors. Mass spectrometric analysis of the substrates digested with gingipains in the presence of GlyGly showed that transpeptidation outcompeted hydrolysis while the trypsin-digested controls exhibited predominantly hydrolysis activity. The transpeptidation levels increased with increasing concentration of GlyGly. Purified gingipains and whole cells exhibited extensive transpeptidation activities on human haemoglobin. All haemoglobin cleavage sites were found to be suitable for GlyGly transpeptidation and this transpeptidation enhanced haemoglobin digestion. The transpeptidation products were often more abundant than the corresponding hydrolysis products. In the absence of GlyGly, haemoglobin peptides produced during digestion were utilised as acceptors leading to the detection of up to 116 different transpeptidation products in a single reaction. P. gingivalis cells were able to digest haemoglobin faster when acceptor peptides derived from human serum albumin were included in the reaction suggesting that gingipain-catalysed transpeptidation may be relevant for substrates encountered in vivo. The transpeptidation of host proteins in vivo may potentially lead to the breakdown of immunological tolerance culminating in autoimmune reactions.

Key words: Gingipains; Hydrolysis; Transpeptidation; Mass spectrometry; Glycylglycine; Peptide substrates; Haemoglobin; Porphyromonas gingivalis


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INTRODUCTION An increasing array of enzyme catalysed peptide rearrangements are continuing to be discovered 1-4. These protease-mediated ligations or more commonly termed transpeptidation reactions result in the formation of new peptide bonds that can occur between two distinct peptides or within one single peptide. Transpeptidation also describes a reaction involving the transfer of one or more amino acids from one peptide chain to another 5. Cyclisation can be achieved by the head-to-tail linkage of the termini of a single peptide chain 6. Recently the role of papain in the gelation of mammalian fibrinogen 1 has been finally clarified with the description of the transpeptidation mechanism. These transpeptidase-derived macrocyclisation and cross-linking processes are examples leading to stability-enhanced proteins and peptides. In contrast the discovery of proteasome-catalysed peptide splicing demonstrated that the transpeptidation process enables a larger number of antigenic peptides that can be derived from a single protein, that are capable of evoking a wider cellular immune response 7. There are increasing reports of transpeptidation products observed during digestion of proteins by various proteases not classified as transpeptidases. These proteases include trypsin 8-12

, Staphylococcus aureus V8 protease 8, 9, and Lys-C endoprotease 9. The observed

transpeptidation activity ranges from the rearrangement of terminal amino acid residues within peptides to substitution with di- or tri- peptides. One well characterised transpeptidase is the γ-glutamyl transpeptidase that functions primarily as a hydrolase and is also capable of catalysing transpeptidation reactions involving amino acids in vivo 13-18. Nitroanilide-containing chromogenic substrates are commonly used to monitor proteolytic reactions due to the release of the yellow coloured para-nitroaniline (pNA) leaving group after hydrolysis. However the fate of the acyl group N-terminal to the nitroaniline, is not usually monitored. In vitro assays utilising γ-glutamyl-p-nitroanilide (GpNA) as the substrate for γ-glutamyl tanspeptidase revealed that the release of pNA was



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enhanced by the presence of glycine (Gly), glycylglycine (GlyGly), and glycylglycylglycine (GlyGlyGly) playing a role as acceptor molecules for the acyl group 19. During the transpeptidase reaction the cleaved glutamyl residue is transferred to these acceptor molecules resulting in the formation of glutamyl-Gly, glutamyl-GlyGly, and glutamyl-GlyGlyGly transpeptidation products. The first salient feature of these hydrolysis and transpeptidase reactions is that the affinity for the substrate (Km = 0.96 mM, 0-4 mM GpNA, 20 mM GlyGly) is much greater than the affinity for the acceptor molecules (Km = 6.65 mM, 5-50 mM GlyGly) 19. The second characteristic is that the transfer of the γ-glutamyl intermediate to such an acceptor is faster than the hydrolysis of the covalent bond of the substrate γ-glutamylnitroanilide thus resulting in a substantial increase in the rate of pNA release 20. Porphyromonas gingivalis is a keystone periodontal pathogen with three cell-surface located cysteine proteases known as gingipains: Kgp with Lys-specific activity and RgpA and RgpB with Arg-spcific activity 21-25. These proteases are known to cleave several host proteins including haemoglobin and the glycine-rich collagen 26-30. Early studies revealed that the trypsin-like proteolytic activities within crude gingipain extracts were stimulated by glycine containing compounds including glycinamide, GlyAsn, GlyGly, and GlyAla 31. GlyGly was demonstrated to stimulate the enzymatic digestion of chromogenic substrates in vitro by both purified RgpB and Kgp 32, 33 however a 105 kDa complexed form of Kgp was reported to be inhibited by GlyGly at high concentration 34 The dose-dependent stimulation by GlyGly was first reported for purified RgpB 35. Consistent with this previous work we recently demonstrated a dose-dependent stimulation of P. gingivalis whole cell proteolytic activity by GlyGly 32. The current study shows that the GlyGly enhancement could be attributed to gingipaincatalysed transpeptidation of the substrates with the GlyGly dipeptide functioning as an acylacceptor molecule. Both purified gingipains and whole P. gingivalis cells were found to


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extensively cleave and transpeptidate human haemoglobin utilising a wide range of natural peptides as acyl-acceptors. P. gingivalis may therefore produce an extensive number of transpeptidation products in-vivo (the “transpeptidome”) with potential implications for host immune responses and autoimmunity.

EXPERIMENTAL PROCEDURES Reagents The recombinant Kgp catalytic domain (rKgp) was purified from the P. gingivalis W50 strain and RgpB was purified from P. gingivalis HG66 as described previously 32. The chromogenic substrates L-BApNA (Nα-Benzoyl-L-arginine 4-nitroanilide) and GPKNA (N(p-tosyl)-Gly-Pro-Lys p-nitroanilide) and synthetic peptides SDGRG (≥90% pure with ≥70% peptide content), GRGDS (≥97% pure with ≥60% peptide content), SLIGKV-NH2 (≥95% pure with unknown peptide content) and HCKFWW (≥95% pure with ≥60% peptide content) and other chemical reagents were all purchased from Sigma-Aldrich.

Enzymatic digestion Enzymatic reactions were performed in a buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl and 5 mM CaCl2, in a total reaction volume of 200 or 100 µL. The chromogenic and other selected commercially available lysine or arginine containing peptide substrates (1 mM) were incubated with purified rKgp or RgpB (64 nM or otherwise indicated) in the presence of 5 mM cysteine and varied concentrations of GlyGly (0-200 mM). The reactions were terminated by adjusting the pH to lower than 4 with 2 M acetic acid immediately after incubation for 2 h. P. gingivalis whole cells (2.5 x 107 cells/mL) of strains W50 and ATCC 33277 were also used as a source of gingipains in the digestions under the same conditions. Trypsin was used as a protease control and equivalent reactions were carried out under the



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same conditions. The enzymatic digests were diluted with 0.1% formic acid and subjected to mass spectrometric analysis. Enzymatic activities were calculated as the initial rates according to the changes in absorbance at 405 nm from digestion of the chromogenic substrates, BApNA for RgpB and GPKNA for rKgp, monitored at 37 oC using a PerkinElmer 1420 Multilabel Counter VICTOR3TM. Haemoglobin (Hb) was used as a biologically relevant substrate. In preparation of the samples for mass spectrometric analysis, Hb (6 µM) was digested in the presence or absence of 10 mM GlyGly for 24 h at 37oC and the digests were analysed with SDS-PAGE at different time points. The samples at 24 h were analysed by mass spectrometry. In order to investigate possible in vivo effects of host protein digested peptides on the digestion of Hb with gingipains, human serum albumin (HSA) was cleaved with trypsin and the resulting peptide mix (0-2.5 mM) was included as an alternative to GlyGly in the Hb digestion. In preparation of the HSA peptide mix, HSA (20 mg/mL) was reduced by incubation with 10 mM DTT in 50 mM ammonium bicarbonate (ABC) containing 8 M urea. The cysteine residues were then alkylated with iodoacetamide. Trypsin (1 in 200, w/w) digestion was performed overnight in ABC plus 0.8 M urea. The resulting peptide mix was cleaned with a SepPak C18 cartridge (Waters) and the total peptide concentration was estimated from the absorbance at 280 nm determined with NanoDrop Lite (Thermoscientific).

Enzyme kinetics of gingipains Varied concentrations of chromogenic substrates BApNA and GPKNA (0-2 mM) were digested in combination with varied concentrations of GlyGly (0-200 mM). Vmax and Km were obtained by fitting the curves of apparent gingpain activity vs substrate concentration at given concentrations of GlyGly against Michaelis-Menten equations using the fitting tool KaleidaGraph (Synergy Software). The ratios of kcat to Km were then calculated accordingly.


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Mass spectrometry The enzymatic digests of the chromogenic substrates and synthetic peptides were analysed by ESI-TOF LC-MS in MS mode only (details below). The identity of selected products were confirmed by orbitrap LC-MS/MS (details below). All protein digestions were also analysed by orbitrap LC-MS/MS (details below). MS/MS fragmentation products were predicted with an online sequence mining proteomics tool ProteinProspector (http://prospector.ucsf.edu/prospector/mshome.htm) and exact monoisotopic masses were calculated via online servers (http://www.chemcalc.org and http://www.peptidesynthetics.co.uk/tools/ ). Experimental data were assessed against the calculated masses. ESI-TOF analyses was performed on an Agilent 6220 Accurate-mass TOF LC-MS system (USA) in positive ionization mode, coupled with an Agilent 1200 HPLC system (Germany). Capillary voltage, drying gas flow, gas nebulizer pressure and gas temperature were set at 4000 V, 9 L min−1, 50 psig and 350 ºC, respectively. Separation of the product species (20 µL via standard auto-injection) was performed on a Phenomenex C5 column (surface area: 440 m2/g; particle size: 5 µm; pore size: 100 Å) or an SGE Protecol C8 column (2.0 mm ID x150 mm; particle size: 5 µm; pore size: 120 Å, used for quantitation with A214) with 0.1% formic acid aqueous solution as buffer A and 95% acetonitrile in 0.1% formic acid as buffer B with flow rate of 100 uL/min. After equilibration with buffer A for 4 min, the buffer B was applied with gradients 5-60% for 12 min and 60-90% for 2 min followed by 2 min clean up at 90% before re-equilibration. Data were collected from 100 to 3200 m/z with internal reference masses of 121.0508 and 922.0097 for calibration. For product quantitation using absorption at 214 nm, an Agilent 1200 DAD SL detector (Germany) was coupled with the LC-MS system to monitor the UV absorption in-situ and for better resolution 56 min was



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used for the first gradient (5-60% buffer B) followed by 10 min for the second gradient (6090% buffer B). Mass spectrometric data were processed with Agilent Mass Hunter Qualitative Analysis software (B.05). LC-MS/MS analyses were conducted using a Q Exactive Plus Orbitrap Mass Spectrometer coupled to an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Buffer A was 0.1% formic acid, 2% acetonitrile in H2O, and buffer B was 0.1% formic acid in acetonitrile. Sample volumes of 5 µL were loaded onto a PepMap C18 trap column (75 µm ID x 2 cm, 3 µm, 100 Å, Thermo Fisher Scientific) and desalted at a flow rate of 2 µL min-1 for 15 min using buffer A. The samples were then separated through a PepMap C18 analytical column (75 µm ID x 15 cm, 2 µm, 100 Å, Thermo Fisher Scientific) at a flow rate of 300 nL min-1, with the percentage of solvent B in the mobile phase changing from 2% to 10% in 1 min, from 10% to 35% in 50 min, from 35% to 60% in 1 min and from 60% to 90% in 1 min. The columns were then cleaned using solvent B at 90% for 7 min before decreasing the percentage of solvent B to 2% in 1 min and re-equilibrating for 6 min. The spray voltage was set at 1.8 kV and the temperature of the ion transfer tube was 250°C. The S-lens was set at 50%. The full MS scans were acquired over a m/z range of 300-2000, with a resolving power of 70,000, and an automatic gain control (AGC) target value of 3.0x106 and a maximum injection time of 30 ms. Dynamic exclusion was set at 90 s. Higher-energy collisional dissociation MS/MS scans were acquired at a resolving power of 17,500, AGC target value of 5.0x104, maximum injection time of 120 ms, isolation window of m/z 1.4 and NCE of 25% for the top 15 most abundant ions in the MS spectra. The LC-MS/MS data have been deposited to the PRIDE Archive (https://protectau.mimecast.com/s/i3MeCZYMPyC0PgE9hykTt2?domain=ebi.ac.uk) via the PRIDE partner repository with the data set identifier PXD009950 and 10.6019/PXD009950. Username: [email protected] 8 ACS Paragon Plus Environment

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Password: pgy5OKIi .

Identification and quantitation of GlyGly transpeptidation during haemoglobin digestion The orbitrap LC-MS/MS data of haemoglobin digested in the presence of 10 mM GlyGly were searched against the SwissProt 2018 database with taxonomy restricted to human (20,391 sequences) using Mascot v 2.2.04. The search settings were enzyme = trypsin; fixed modifications = cysteinyl (C); variable modifications = Cys (C-term) and GlyGly (C-term); mass tolerance = 5 ppm (MS) and 0.4 Da (MS/MS); missed cleavages = 3. The Mascot decoy searches (p 0.98 for rKgp and > 0.96 for RgpB, consistent with a Michaelis-Menten mechanism for these reactions (Figs. 6A and 6B). The kcat and Km values increased in parallel for both enzymatic reactions producing a kcat:Km ratio that was relatively constant. (Figs. 6C and 6D).



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Fig 6. Kinetics of gingipain catalysis with varied concentrations of GlyGly. (A&B) Digestion of GPKNA and BApNA with Kgp and RgpB, respectively. Apparent activities were an average from triplicate measurements. The solid curves are the fits against MichaelisMenton equations (KaleidaGraph) with R = 0.98-1.00 for rKgp and 0.96-1.00 for RgpB. (C&D) Corresponding fold changes of kcat and Km relative to the values calculated in the absence of GlyGly. The error bars represent the standard error of the mean (SEM). Enzyme: 64 nM; Substrate: 0-2 mM; Cys: 5 mM; GlyGly: 0-200 mM; Reaction Temperature: 37 oC.

Gingipain transpeptidation of synthetic non-chromogenic peptides To investigate whether the gingipains could transpeptidate peptides containing only natural amino acids, RgpB and rKgp were used to digest various synthetic peptides containing Arg or Lys residues. ESI-TOF analysis of the RgpB digest of SDGRG (m/z 491) showed that the inclusion of GlyGly resulted in the formation of transpeptidation product SDGRGG of m/z 548 (Fig 7A, Table 1).


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Fig 7. ESI-TOF analysis of synthetic peptide transpeptidation. Spectra of the RgpB (A-B) and rKgp (C-F) digests of the four selected substrates in the absence or presence of GlyGly. 19 ACS Paragon Plus Environment


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Substrates: 1 mM SDGRG, GRGDS, SLIGKV-NH2 and HCKFWW (Mass spectrometric analysis of the original GRGDS peptide sample alone (data not shown) indicated that the three significant unlabeled peaks in Panel B (top) were impurities present in the original sample while the new peak at m/z 331 in Panel B (bottom) could not be assigned); Gingipains: 64 nM; Cys: 5 mM; GlyGly: 0 or 10 mM. Enzymatic reactions were terminated after 2 h by acidification with 2 M acetic acid. Transpeptidated moieties are underlined. One of the two subunits in each intermolecular disulfide species is bracketed.

In the RgpB digest of GRGDS, the transpeptidation product GRGG (m/z 346) was also detected when GlyGly was included in the reaction (Fig 7B, Table 1) suggesting that transpeptidation may be insensitive to the total number of residues (up to 3) following the cleavage site. Cysteine transpeptidation was not detected for these two RgpB substrate peptides despite the presence of 5 mM cysteine. Similarly, Kgp hydrolysed SLIGK amide (SLIGKV-NH2) produced SLIGK (m/z 517) including its dimeric form (m/z 1034) as revealed by ESI-TOF (Fig 7C-D, Table 1). The Cys transpeptidation product, SLIGKC (m/z 620), was formed with and without GlyGly, with its dimeric form (m/z 1240) being also detected in the absence of GlyGly (Fig 7C-D, Table 1). The presence of GlyGly allowed the formation of the transpeptidation product SLIGKGG (m/z 631) including its dimeric form (m/z 1262) with decreasing production of both hydrolysis and Cys transpeptidation products (SLIGK and SLIGKC) according to their abundances in the ESI-TOF spectra (Fig 7C-D, Table 1). The identities of these three products were confirmed by Orbitrap LC-MS/MS (Fig 8A-C). Interestingly, ESI-TOF detected species at m/z 1016 and 1114 which were assigned to the transpeptidation products SLIGKSLIGK, and SLIGKSLIGKV-NH2 and the peak at m/z 1130 was matched to the double transpeptidation


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product, SLIGKSLIGKGG (Fig 7D, Table 1), demonstrating that Kgp catalysed transpeptidation can utilise larger acceptor molecules including the original SLIGKV-NH2 substrate and its hydrolysis product SLIGK and the transpeptidation product SLIGKGG. These transpeptidation products were also confirmed by Orbitrap LC-MS/MS, e.g. SLIGKSLIGKV-NH2 (Fig 8D). The dimeric form of the substrate SLIGKV-NH2 (m/z 1230) and the heterodimer (m/z 1148) formed between the hydrolysis product SLIGK and the transpeptidation product SLIGKGG were also detected (Fig 7D).



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Fig 8. Orbitrap LC-MS/MS identification of transpeptidated synthetic peptides. The digestion products analysed in Fig 7 (see Fig 7 legend) were also analysed by Orbitrap LCMS/MS. The precursor ions analysed by fragmentation include: (A) hydrolysis product m/z 517 SLIGK; (B)-(E) transpeptidation products m/z 620 SLIGKC, m/z 631 SLIGKGG, m/z 1114 SLIGKSLIGKV-NH2 and m/z 1273 HCKHCKFWW. Predicted sequences and


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fragmentation ion patterns are shown at the top of each spectrum. The ion types are labelled above the matched peak m/z.

Interpretation of the ESI-TOF spectrum of Kgp-digested HCKFWW was more complex due to both dimerization and disulfide bond formation (Fig 7E-F, Table 1). The substrate was detected mainly as doubly-charged dimer (m/z 906) and in monomer form with a cysteine disulfide by-product (HC(C)KFWW, m/z 1025) while the C-terminal hydrolysis product, FWW was detected as both monomer and dimer (m/z: 538 & 1075). The HCKFWW substrate was also found disulfide bonded to the HCK hydrolysis product (m/z 1290). Peaks at m/z 1405 and m/z 703 (2+) were only detected in the presence of GlyGly and corresponded to HCKGG disulfide bonded with HCKFWW while peaks at m/z 1273 and m/z 637 (2+) detected in both conditions were attributable to the transpeptidation product HCKHCKFWW with an intramolecular disulfide bond, which was confirmed with Orbitrap MS/MS (Fig 8E).

Trypsin digestion of selected substrates as control experiments Since the widely used serine protease trypsin was reported to have transpeptidation activity 12, similar experiments using this enzyme were performed. In contrast to the gingipain digests, ESI-TOF did not detect any transpeptidation species in the tryptic digests of the six substrates (Fig S3) except for a low abundance peak at m/z 631 representing the formation of SLIGKGG (Fig S3E). These results demonstrate that the observed transpeptidation activities of RgpB and rKgp were significantly greater than that of trypsin under the same conditions. Trypsin showed predominant hydrolysis activity in these control reactions with much stronger hydrolysis of GPKNA than of BApNA. Hydrolysis of GPKNA by trypsin proceeded essentially to completion (Fig S3B); while the abundance of the BApNA species remained



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relatively high after digestion (Fig S3A). Consistently, SLIGK amide and HCKFWW were largely consumed by trypsin (Fig S3E-H) while both SDGRG and GRGDS remained essentially undigested (Fig S3C-D).

GlyGly transpeptidation of human haemoglobin by gingipains To determine if the gingipains are able to act as transpeptidases on biologically relevant substrates, RgpB, rKgp, P. gingivalis whole cells with in situ native gingipains and control trypsin were tested against human haemoglobin (Hb). The reactions were performed over 24 hours and aliquots were removed at various time points and analysed by SDS-PAGE to assess the level of Hb degradation (Fig S4). Haemoglobin was largely resistant to digestion with RgpB, but could be digested with rKgp, trypsin and whole cells. The presence of GlyGly appeared to enhance digestion with rKgp or whole cells (Fig S4) consistent with the occurrence of transpeptidation. The samples that had been digested for 24 h were chosen for further analysis by orbitrap LC-MS/MS. A search of the data for peptides derived from the alpha and beta chains of haemoglobin cleaved at either Lys or Arg with and without GlyGly as a C-terminal modification resulted in the identification of 36, 18 and 24 GlyGly modified peptides for rKgp, RgpB and trypsin respectively (Table 3 and Table S3). RgpB and rKgp were each found to cleave and transpeptidate at both Lys and Arg residues (Table 3 and Table S3). The level of rKgp-catalysed transpeptidation relative to total cleavage (hydrolysis + transpeptidation) was spread over a very wide range from 0.2 -74% with a median value of 4.6%. There was no obvious size preference with both small and large peptides acting as suitable transpeptidation substrates, however, peptides found to have internal (uncleaved) Lys residues exhibited relatively higher transpeptidation levels. RgpB was less effective at cleaving Hb than rKgp. This is not unexpected as it has already been shown that Arg residues of Hb are not accessible


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until cleavage of exposed Lys residues has occurred 26. Furthermore, since there are few Arg residues in Hb (3 in the Hb α-chain, one of which is the C-terminal residue and 3 in the Hb βchain), few Arg cleavage products were amenable to LC-MS/MS identification. In addition to Arg-specific hydrolysis and GlyGly transpeptidation, RgpB also catalysed hydrolysis and transpeptidation at Lys residues, however, some of these peptides were only identified in hydrolysed form, which may have been due to the low signal intensities observed. The percentage of peptides transpeptidated relative to all hydrolysis and transpeptidation products ranged from 0.2 – 21% with a median of 4.1%, similar to rKgp. Trypsin also generated transpeptidation products but at a significantly lower level with a median transpeptidation level of only 0.4% (Table 3 and Table S3). In the presence of GlyGly, LC-MS/MS analysis of the 24 h Hb digest with P. gingivalis whole cells resulted in the identification of 35 transpeptidation products involving the Hb α- and β-chains for strain W50 and 33 for strain ATCC 33277 (Table 3 and Table S3). The level of transpeptidation was greatly increased relative to that observed with purified RgpB and rKgp with a median value of 44% and 34% respectively. Since gingipains are not the only cell surface proteinases of P. gingivalis, an MS/MS search was also conducted using “no enzyme” type specificity. With this search, 715 additional unique Hb peptides were identified for the W50 sample including 435 peptides without Arg or Lys at the C-terminus. Of these, only six were potential GlyGly transpeptidation products indicating a strong preference for transpeptidation at Arg and Lys residues consistent with the gingipains being largely responsible for the observed transpeptidation (data not shown). The very large number of non-tryptic peptides identified was due to the exponential increase in the number of potential peptides that can be produced due to non-specific cleavages and the presence of other endoproteases and exoproteases. When haemoglobin was digested with rKgp only 109 additional unique peptides were



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identified with the “no enzyme” type search but none were found to be transpeptidated with GlyGly (data not shown). There was no absolute specificity for transpeptidation observed since 24/27 of the Lys and Arg cleavage sites present in Hb were both involved in hydrolysis and transpeptidation (Table 3). The two Lys sites and one Arg site which were not initially detected as hydrolysed or transpeptidated were found to be both hydrolysed and transpeptidated when semi-tryptic peptides were searched for (data not shown). Hence all 27 sites were in fact transpeptidated.

Gingipain-catalysed rearrangement of haemoglobin peptides Given that haemoglobin proved to be an effective substrate for gingipain catalysed transpeptidation, and that peptides larger than GlyGly such as SLIGKV and HCKFWW (Fig 7) could act as acceptor molecules, we considered that during Hb digestion, the peptide products from cleavage may be able to serve as acceptors enabling transpeptidation. Since the concentration of Hb was only 6 µM whereas the concentration of GlyGly used was 10 mM, it was considered very doubtful that transpeptidation between Hb peptides would be observed. Nevertheless, the Hb samples digested for 24 h with rKgp, RgpB, W50 whole cells and ATCC 33277 whole cells without added GlyGly were subjected to orbitrap LC-MS/MS analysis. To enable identification, a custom sequence database was created that contained all possible combinations of Hb tryptic peptides joined together by transpeptidation. Surprisingly, rearranged Hb peptides were common. In the rKgp digest, 116 unique rearrangements were identified by MS/MS, while 56 and 68 were found for W50 and ATCC 33277 whole cells respectively (Table 4 and Table S4). Only four rearranged Hb peptides were found in the sample digested with RgpB, consistent with the low level of digestion achieved with this enzyme. The number of apparent acceptors used for the transpeptidation reactions was 33 and ranged from the single amino acid, Lys, to peptides as large as 29


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residues (Table 4). Out of the 28 possible peptides produced from full tryptic cleavage of Hb all except two were shown to function as acceptors, indicating that in general, most peptides are suitable acceptors for gingipain-catalysed transpeptidation. The most commonly detected acceptors were the N-terminal peptides VLSPADK, VLSPADKTNVK and VHLTPEEK followed by the small acceptors K and VK (Table 4). The detected acceptors were recorded as “apparent acceptors” since it is not known whether hydrolysis occurred after transpeptidation. For example, the detected product, VHLTPEEKVLSPADK may have been produced by hydrolysis of VHLTPEEKVLSPADKTNVK, making it uncertain whether the acceptor was VLSPADK, VLSPADKTNVK or an even longer peptide. When the haemoglobin samples digested in the presence of 10 mM GlyGly were also searched for rearranged Hb peptides, a smaller number of rearranged Hb sequences were detected (data not shown), suggesting the suppression of such transpeptidated Hb peptide rearrangement by the presence of an overwhelmingly high concentration of GlyGly as a competing acyl-acceptor.

Haemoglobin digestion is enhanced by the presence of pre-digested serum albumin It was noted above that Hb appeared to be digested faster in the presence of GlyGly (Fig S4), suggesting that transpeptidation with GlyGly was contributing significantly to the breakdown of Hb. To explore whether transpeptidation is likely to increase the digestion rate of proteins in vivo, we digested Hb in the presence of digested peptides of human serum albumin (HSA), an abundant protein in gingival crevicular fluid 37. Hb was digested by W50 whole cells over 24 h with and without HSA peptides and the extent of degradation was monitored by SDS-PAGE. From densitometric analysis of the full-length Hb band around 15 kDa, Hb was found to be digested faster in the presence of 2.5 mM HSA peptides as evidenced by the greater loss of the full-length Hb band at the 8 h and 24 h time points (Fig 9 and Table S5). LC-MS/MS analysis of the 24 h digest demonstrated the utilisation of HSA



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peptides as acceptor molecules against Hb. In total, 101 transpeptidation products were identified comprising 44 HSA-HSA products, 24 Hb-HSA products, 13 Hb-Hb products, 10 HSA-Hb products, 7 products involving Lys and HSA and the remaining three involving Lys and Hb (Table S6). Lys could have originated from either protein.

Fig 9. HSA peptides enhanced digestion of Hb by P. gingivalis W50 whole cells. Hb digests at 0, 8 and 24 h in the presence or absence of HSA peptides were analysed with SDS-PAGE. A solution of HSA peptides were produced by pre-digestion with trypsin and a total concentration of 0, 1 or 2.5 mM was included in the reactions. Hb: 6 µM; W50: 2.5 x 107 cells/mL; Cys: 5 mM.

DISCUSSION Although transpeptidation activity is not unknown for proteases, this is the first demonstration of a protease or transpeptidase that will cleave and non-specifically rearrange peptide sequences at a high level. While in theory, all proteases have this ability, none to our


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knowledge have been demonstrated to favour transpeptidation over hydrolysis with such a wide range of substrates and acyl acceptors. The recent crystal structures of gingpains RgpB and Kgp have revealed the molecular mechanisms for catalysis and substrate specificities 38-40. The Michaelis-Menten kinetics observed for gingipain proteolysis of chromogenic and fluorogenic substrates precluded any allosteric regulation 32, 35. Hence a molecule that increases proteolytic activity cannot be described as a positive effector. Furthermore, a hyperbolic relationship between the apparent rate of hydrolysis and increasing concentrations of a non-substrate molecule is indicative of an alternative acyl-acceptor molecule other than water participating in the enzymatic reaction. Typically, transpeptidases that utilise the Ping-Pong mechanism exhibit an increase in both kcat and Km as the concentration of acyl acceptor is increased with the kcat/Km ratio remaining relatively constant 41. In this study, we found this to be also the case for both RgpB and Kgp using GlyGly as the acyl acceptor, consistent with previously published data 35, which can now be interpreted to support a Ping-Pong mechanism for gingipain-catalysed transpeptidation reactions. Gingipain-catalysed transpeptidation reactions using, for example, the chromogenic substrates BApNA for RgpB and GPKNA for Kgp can be described as follows. Prior to hydrolysis, BApNA or GPKNA is located within the active site. The outcome of hydrolysis is the formation of two products from one substrate (Fig S6 Steps I-IV). Nucleophilic attack from the sulphur of the catalytic cysteine to the carbonyl of the substrate results in formation of the BA-enzyme or the GPK-enzyme intermediate with the release of C-terminal pnitroaniline concomitant with colour development (Fig S6 Steps I-II). In a subsequent step, a water molecule can react with the BA-enzyme/GPK-enzyme intermediate releasing hydrolysis products BA-OH or GPK-OH (Fig S6 Steps III and IV). Alternatively, as we have shown herein, peptides such as GlyGly can behave as acyl-acceptor molecules resulting in the



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formation of BAGG or GPKGG as a transpeptidation product (Fig S6 Steps V and VI). The GlyGly transpeptidation products maintain an internal Arg or Lys residue and hence remain as substrates for RgpB or Kgp. When the gingipain concentration was increased by 10-fold (Fig S1), the BApNA and GPKNA substrates were almost completely converted to hydrolysis products demonstrating that the presence of transpeptidation products was transient. When the reaction rate of RgpB and Kgp was measured as a function of GlyGly concentration, the reaction rate increased more than 4.6-fold and 8.6-fold for RgpB and rKgp respectively at 1 mM substrate and up to 200 mM GlyGly acceptor (Fig 1). Since the concentration of the water acceptor is 55 M, the gingipains appear to show a strong preference for a primary amine as acceptor, which is in agreement with the higher nucleophilicity of amine over water 42. The competition between GlyGly, Cys and water for the acceptor role was demonstrated by MS detection of the three alternative products as a function of GlyGly concentration. While the GlyGly transpeptidation product increased in abundance, both the Cys transpeptidation products and the hydrolysis products decreased (Fig 4). The ability of the gingipains to increase their reaction rate in the presence of an alternative acceptor molecule may have benefits in vivo. Since it was also observed that proteins such as haemoglobin (Fig S4), α1-achy 35, and inconsistently, azocasein and azocoll 31, 35

were degraded faster in the presence of GlyGly, the degradation of host proteins and

peptides in vivo will be more efficient when the concentration of peptide acceptors is high. In other words, the products of gingipain-catalysed protein digestion (cleaved peptides) will enhance the rate of continued protein digestion. This was confirmed in this study by demonstrating faster digestion of Hb in the presence of just 2.5 mM HSA peptides. During disease, P. gingivalis resides in inflamed periodontal pockets where they are exposed to the flow of both gingival crevicular fluid, inflammatory exudates and occasional bleeding 43, 44. The normal protein concentration in human serum is approximately 70 mg/mL, the digestion


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of which can potentially produce high concentrations of acceptor peptides. Assuming an average peptide mass of 1000 Da, 70 mg/mL of peptide corresponds to 70 mM. Since only a small proportion of the total protein would be digested at any one time, a low mM level of peptide acceptors within the local vicinity of the gingipains is a reasonable estimate. The transpeptidation activities of gingipains does not appear to have specificity for utilising peptide acyl-acceptors. SLIGK and HCKFWW from synthetic peptide substrates were also able to function as acceptors at only 1 mM producing transpeptidation products of substantial abundance (Fig 7, Table 1). The HCKFWW peptide in particular is rather bulky and includes four residues with large cyclic side chains. The ability of these two unrelated peptides to act as acceptors at a low concentration suggests that the gingipains may have very little specificity for peptide-based acceptors. It is interesting to note that these two peptides were able to dimerise (Table 1). This dimerization ability may have enhanced their ability to function as acceptors, as when the dimer binds to the Kgp active site and the acyl-enzyme intermediate is formed with the first subunit, the second subunit will be in close proximity and may be the favoured acceptor. In the case of gingipain digestion of haemoglobin, a wide range of various peptides as large as 29 amino acid residues were able to act as acyl-acceptors (Table 3). This lack of specificity for transpeptidation with respect to acyl-acceptor is consistent with specificity being pre-determined by the substrate binding pocket which prefers Arg (RgpB) and Lys (Kgp). Once the acyl-enzyme intermediate is formed, GlyGly or digested peptides from Hb or HSA would be expected to behave similarly to the water acceptor and attack all Lys-acyls (for Kgp) or all Arg-acyls (RgpB) equally (Fig S6). Despite this expectation, there was however, considerable differences in the level of transpeptidation observed for Hb relative to hydrolysis. In the case with GlyGly, the level ranged from 0.2-98% (Table 3). This very large range may be best explained by considering the lifetime of the transpeptidation product in the solution.



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Initially, from the discussed mechanism it would be expected that during a given digestion in excess GlyGly, the relative rates of hydrolysis and transpeptidation would be a constant. From what we have seen, the transpeptidation rate is much faster than the hydrolysis rate leading to an initial higher concentration of transpeptidation product. Over time however, the transpeptidation product will be removed by further hydrolysis. Together this suggests that the apparent level of transpeptidation of a particular site or peptide reflects the stability of that transpeptidation site or product against hydrolysis. In the case of a protein substrate like Hb, the transpeptidation level may also reflect the timing of transpeptidation. A site that is readily exposed to the gingipains and is a good substrate will be targeted early in the reaction and may continue to be targeted throughout the reaction, being both hydrolysed and transpeptidated until it is fully hydrolysed. Other sites in the protein maybe poorly exposed and only be available late in the reaction after other portions of the protein have been digested. Such sites will have less opportunity to be fully hydrolysed and may therefore exhibit a higher level of transpeptidation. The lifetime of transpeptidation products in vitro may therefore be governed by their ability to compete with the other substrates present in the reaction as well as the enzyme concentration. In vivo, however, the lifetime of transpeptidation products is further influenced by their uptake by bacteria, the presence of other proteinases and the flow of fluids such as gingival crevicular fluid that may remove transpeptidation products away from contact with gingipains and other proteinases. Probably due to the high structural similarity between RgpB and Kgp catalytic domains 38, 39, 45, the two gingipains exhibited cross activity in cleavage and transpeptidation of haemoglobin. Both rKgp and RgpB were found to hydrolyse and transpeptidate both Arg and Lys residues (Table 3). Cross activity of RgpB for Lys residues was shown previously and found to be under reversible redox control 46. It is unknown whether such redox regulation was a factor for the case of haemoglobin digestion.


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Possible conditions in vivo promoting transpeptidation include macromolecular crowding, proximity effects, and substrate specificity 47. This may explain why digestion of haemoglobin with whole P. gingivalis cells resulted in a higher percentage of GlyGly transpeptidation products relative to the purified gingipains. The adhesin domains of RgpA and Kgp which are complexed with the catalytic domains on the cell surface 48 may increase macromolecular crowding by binding to multiple substrates favouring transpeptidation. In addition, these adhesin domains may play a role in targeting and aligning the substrates for better transpeptidation. The prevalence of this transpeptidation capability for cell-surface located gingipains in vivo is yet to be explored. In vivo, P. gingivalis survives in a periodontal pocket in a milieu of available host and other bacterial proteins. The presence of peptides with rearranged amino acids or new peptides synthesised by ligation of non-contiguous peptides from host and bacterial proteins have not been investigated. These peptides would not be identified readily using current proteomic protocols that rely on matching peptides with known genomic sequences. In order to investigate the “transpeptidome”, new bioinformatic tools need to be developed that enable the identification of scrambled peptides. The impact and prevalence of transpeptidation is now emerging in the literature 3, 7. Within the cellular antigen presenting pathways, proteins are normally cleaved enabling the formation of contiguous antigens. However recent findings of non-contiguous antigens by transpeptidation have revealed a mechanism for the expansion of antigen diversification with implications in immunity that have yet to be elucidated 47. Although the gingipains are involved in events leading to the dysregulation of host-defensive inflammatory reactions, their involvement in transpeptidation has not been investigated. The generation of chimeric peptides in the extracellular microenvironment by the now disclosed transpeptidation activity of gingipains could potentially be involved in host immune dysregulation and auto-immune



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reactions 49. Previous studies have demonstrated the presence of auto-antibodies and autoimmune reactions in periodontitis lesions 50 and have further suggested an association between periodontitis and some systemic diseases including the autoimmune diseases rheumatoid arthritis and diabetes 51-53. Although the exact mechanisms of autoimmunity are still unclear, posttranslational modifications through extracellular proteolytic reactions to generate neoepitopes may have a role in pathogenesis 54. The transpeptidation activities of gingipains are reminiscent of the P. gingivalis sortase-like transpeptidase PorU, another cysteine protease of the gingipain family 55. PorU is a component of the type IX secretion system (T9SS) and functions to cleave the T9SS signal domain found at the C-terminus of substrate proteins including the gingipains. The cleavage of the signal is part of a transpeptidation reaction with the acceptor molecule being a serine residue that is linked to anionic lipopolysaccharide (A-LPS). Hence PorU conjugates the gingipains and other T9SS substrates to the cell surface 36, 56. PorU appears specific for proteins carrying the T9SS signal and preferentially cleaves at a moderately conserved site which includes a small residue (Gly, Ala or Ser) in the S1’ position. The presence of a small residue in this position may be related to its specificity toward the Ser-A-LPS moiety, while for the gingipains the apparent lack of specificity beyond Arg and Lys may reflect their accommodation of acceptor peptides with large N-terminal amino acids such as His in the HCKFWW peptide. In conclusion, we have demonstrated that the gingipain catalytic machinery is capable of transpeptidation. As the gingipains are virulence factors for the human pathogen P. gingivalis their transpeptidation activity therefore may be an important component of their role in host immune dysregulation and the link with autoimmune diseases.


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SUPPORTING INFORMATION

The following supporting information is available free of charge at ACS website https://protect-au.mimecast.com/s/5jr9C1WZKqhlnG1NuYEPGz?domain=pubs.acs.org. Fig S1. ESI-TOF analysis of transpeptidation of BApNA and GPKNA with a high concentration of RgpB and rKgp, respectively.

Fig S2. ESI-TOF spectra of the P. gingivalis whole cells digests of GPKNA and BApNA in the absence or presence of GlyGly.

Fig S3. ESI-TOF spectra of trypsin digests in the absence or presence of GlyGly.

Fig S4. Time course SDS-PAGE analysis of human haemoglobin digested by purified gingipains, P. gingivalis whole cells and control trypsin.

Fig S5. Primary sequences of haemoglobin subunits and human serum albumin.

Fig S6. Ping-Pong catalytic cycle of gingipain-mediated hydrolysis and transpeptidation modified from 20.

Table S1. Relative MS abundance of GPK, GPKC and GPKGG to the combined abundance (100%) from digestion of 1 mM GPKNA with various concentrations of rKgp at 10 mM GlyGly and 5 mM Cys.



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Table S2. Estimation of the extinction coefficient (ε214) of Kgp and RgpB hydrolysis and transpeptidation products from digestion of chromogenic substrates GPKNA and BApNA in the presence of GlyGly.

Table S3. Transpeptidation of Haemoglobin with GlyGly.

Table S4. Mascot Data for Haemoglobin digested with W50 Cells with no GlyGly.

Table S5. Densitometric analysis of cleavage levels of Hb digested with P. gingivalis W50 whole cells in the presence or absence of HSA peptide mix at 8 and 24 hours.

Table S6. Transpeptidation products during digestion of Hb with P. gingivalis W50 whole cells in the presence of pre-digested HSA

AUTHOR CONTRIBUTIONS. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ¶ These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS


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This work was supported by the Australian Government Department of Industry, Innovation and Science Grant ID 20080108. The authors thank Dr Shuai Nie, Dr Ching-Seng Ang and Dr Nicholas Williamson for the acquisition of Orbitrap LC-MS/MS data and their technical support through the Mass Spectrometry and Proteomics Facility at Bio21 Institute, The University of Melbourne, Australia.

ABBREVIATIONS GG or GlyGly: glycylglycine; rKgp: recombinant lysine specific gingipain; RgpB: arginine specific gingipain; GlyGlyGly: glycylglycylglycine; GlyAsn: glycylasparagine; L-BApNA; Nα-Benzoyl-L-arginine 4-nitroanilide; GPKNA: N-(p-tosyl)-Gly-Pro-Lys p-nitroanilide; HSA: human serum albumin; ABC: ammonium bicarbonate; T9SS: type IX secretion system; ALPS: anionic lipopolysaccharide; GpNA: γ-glutamyl-p-nitroanilide; BA: Nα-Benzoyl-Larginine; GPK: N-(p-tosyl)-Gly-Pro-Lys; Ts: Tosyl group; BZ: Benzamide; Gdn: Guanidine; FDR: false discovery rate.



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Table 1. ESI-TOF determined product species from the RgpB and rKgp digests of six different substrates. Digestions were performed in the presence of 5 mM Cys with or without inclusion of 10 mM GlyGly (20 mM for BApNA and GPKNA). Enzymes: 64 nM; substrates: 1 mM. Reactions were terminated by acidification before mass spectrometric analysis. All species are shown as monoisotopic masses with one proton (MH+) unless indicated otherwise. Enzyme

Substrate

Product Species

RgpB

BApNA

BApNA BA BAGG BAC SDGRG SDGR SDGRGG GRGDS GRGG GPKNA GPK GPKGG GPKC SLIGKV-NH2 SLIGKV-NH2 dimer SLIGK SLIGK dimer SLIGKSLIGK

SDGRG

GRGDS rKgp

GPKNA

SLIGKV-NH2

SLIGKSLIGKVNH2 SLIGKGG SLIGKGG dimer SLIGKSLIGKGG

Expected m/z 399.18 279.15 393.19 382.15 491.22 434.20 548.24 491.22 346.18 575.23 455.20 569.24 558.21 615.42 1229.84 517.33 1033.66 1015.64 (508.32) 1113.70 (557.35) 631.38 1261.75 1129.69

- GG Measured Abundance m/z 399.21 258670 279.16 4846 nd nd 382.18 30915 491.23 2878 434.21 359 nd nd 491.22 2410 nd nd 575.23 302967 455.20 215767 nd nd 558.21 114812 615.43 100049 1229.86 9930 517.35 37340 1033.68 1539 1015.67 6392 (508.34) (4390) 1113.76 4730 (557.39) (6294) nd nd nd nd nd nd

+ GG Measured Abundance m/z 399.20 79402 279.16 3242 393.21 4769 382.18 14897 491.22 2686 434.20 209 548.24 505 491.22 3121 346.18 1226 575.23 43106 455.20 142281 569.24 228298 558.21 38206 615.43 34875 1229.85 1117 517.35 13137 nd nd 1015.67 699 (508.34) (428) nd nd

631.39 1261.77 1129.71

39912 2632 1343 45

ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

HCKFWW

SLIGK+SLIGKGG dimer a SLIGKC SLIGKC dimer HCKFWW HCKFWW dimer (MH22+) FWW FWW dimer HC(C)KFWW (inter S-S) b HCKHCKFWW (intra S-S) c HCK(HCKFWW) (inter S-S) d HCKGG(HCKFWW) (Inter S-S) e

Page 46 of 51

1147.69

nd

nd

1147.72

664

620.34 1239.66 906.41 906.41

620.36 1239.70 nd 906.42

20601 977 nd 9606

620.36 nd nd 906.41

5923 nd nd 4877

538.24 1075.49 1025.41 (513.21) f 1272.56 (636.78) 1290.57 (645.79) 1404.61 (702.81)

538.26 1075.50 1025.43 (513.22) 1272.57 (636.79) 1290.58 (645.80) nd

28275 15966 39540 (24806) 2421 (4306) 1081 (3477) nd

538.25 1075.49 1025.42 (513.21) 1272.56 (636.79) 1290.57 (645.79) 1404.61 (702.81)

44572 26071 29204 (17407) 970 (1837) 569 (1464) 715 (3758)

a

SLIGK and SLIGKGG formed heterodimer

b

The Cys residue and the free Cys included in the reaction formed a disulfide bond intermolecularly.

c

HCK and HCKFWW formed a new peptide (transpeptidation product) with an intramolecular disulfide bond formed between the two Cys residues. d

HCK and HCKFWW were linked to each other by an intermolecular disulfide bond.

e

HCKGG and HCKFWW were linked to each other by a intermolecular disulfide bond.

f

The data in the brackets were from double charged species, [MH2]2+.

nd: not detected.

46


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Table 2. Comparison between transpeptidation and hydrolysis levels of GPKNA and BApNA with Kgp and RgpB, respectively, in the presence of GlyGly. Digested samples were analysed using reversed phase HPLC with in-situ UV detection. The peak areas were integrated from the peaks at 214 nm as shown in chromatograms in Fig. 5. Enzyme

Kgp

RgpB

a

GG (mM)

+GG a

Hyd a

Molar ratio

Amount (au) 0.69

Peak area (au) 3464

Amount (au) 0.33

+GG/ Hyd

10

Peak area (au) b 7927

120

13252

1.16

1875

0.18

6.4

200

15223

1.33

1653

0.16

8.3

10

3985

0.55

2593

0.42

1.3

120

9699

1.33

960

0.15

8.9

200

11551

1.59

846

0.14

11.4

2.1

+GG: GlyGly transpeptidation products GPKGG and BAGG; Hyd: hydrolysis products GPK and BA; b a.u.: arbitrary unit.

47


Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 48 of 51

Table 3. Orbitrap LC-MS/MS identified hydrolysis and transpeptidation products from human haemoglobin digested by gingipains in the presence of GlyGly. Enzymes: rKgp, RgpB and Trypsin (64 nM), P. ginivalis whole cells of strains W50 and ATCC 33277 (2.5 x107 cells/mL). The amino acid residue numbering is based on the sequences of haemoglobin alpha and beta subunits as in Fig S5. +GG (%): percentage of the mass abundance of GlyGly transpeptidation products within the hydrolysis and transpeptidation products. Hbα and Hbβ: haemoglobin subunits. Acyl donor Lys or Arg residues are coloured in red while uncleaved Lys or Arg residues are highlighted in cyan.

Protein

Peptide sequences identified from hydrolysis

+GG (%) rKgp RgpB W50 ATCC 33277

Hbα

1

1

VLSPADK7 VLSPADKTNVK11

1

VLSPADKTNVKAAWGK16

1 8

VLSPADKTNVKAAWGKVGAHAGEYGAEALER

31

16

TNVKAAWGK

12 17 17 32 41 41 41 62 93 91

31

AAWGKVGAHAGEYGAEALER

VGAHAGEYGAEALER

31 40

VGAHAGEYGAEALERMFLSFPTTK 40

MFLSFPTTK

56

TYFPHFDLSHGSAQVK

60

TYFPHFDLSHGSAQVKGHGK

61

TYFPHFDLSHGSAQVKGHGKK

90

VADALTNAVAHVDDMPNALSALSDLHAHK 99

VDPVNFK

99

LRVDPVNFK

100

127

LLSHCLLVTLAAHLPAEFTPAVHASLDK

Trypsin

27.1 10.7

0.7

98.0 18.9

97.8 19.4

14.0 0.5

25.4

1.7

82.7

78.0

-

23.8

18.8

46.7

52.8

-

41.4

-

77.5

74.8

-

23.1

10.6

60.0

43.3

5.3

1.8

7.6

28.6

34.3

0.2

1.7

-

-

-

-

0.2

7.5

8.0

4.2

0.3

1.8

12.8

22.1

8.8

0.3

41.1

-

46.3

41.2

-

31.1

-

80.8

75.0

-

4.7

-

48.7

16.1

3.8

0.5

-

33.6

20.0

0.4

4.6

-

31.8

15.4

0.2

3.4

-

46.5

-

48


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128

Hbβ

FLASVSTVLTSK139

0.5

0.2

11.6

5.9

0.3

VHLTPEEK8 1 VHLTPEEKSAVTALWGK17

2.7 14.4

0.4 -

27.1 58.2

22.4 52.6

0.3 -

1

24.0

10.7

85.1

-

-

4.0

-

64.9

60.5

0.4

74.3

21.2

50.0

60.3

36.1

12.4

2.5

69.3

44.7

0.2

4.1

3.1

43.6

40.0

0.4

4.5

2.7

26.7

21.1

0.2

12.1

18.3

22.7

19.1

6.8

22.4

-

24.2

25.3

-

14.8

-

18.1

16.7

-

1

9 9

VHLTPEEKSAVTALWGKVNVDEVGGEALGR30 17

SAVTALWGK

SAVTALWGKVNVDEVGGEALGR

18 31 41 41 60 62

VNVDEVGGEALGR LLVVYPWTQR

30

30

40 59

FFESFGDLSTPDAVMGNPK

61

FFESFGDLSTPDAVMGNPKVK

82

VKAHGKKVLGAFSDGLAHLDNLK 82

AHGKKVLGAFSDGLAHLDNLK

66

KVLGAFSDGLAHLDNLK

1.3

-

37.9

50.5

0.5

67

82

2.1

0.4

45.2

28.3

0.3

2.5

-

58.5

52.4

0.7

11.4

-

59.3

35.9

0.8

1.6

-

33.3

17.2

0.4

0.5

-

39.2

18.6

-

83 83 96

82

VLGAFSDGLAHLDNLK 95

GTFATLSELHCDK

GTFATLSELHCDKLHVDPENFR LHVDPENFR

105 121 133

104

104 120

LLGNVLVCVLAHHFGK 132

EFTPPVQAAYQK

0.6

5.2

40.0

32.1

0.4

144

0.4

0.6

26.5

25.0

0.2

Median

4.6

4.1

43.6

34.3

0.4

VVAGVANALAHK

49



Page 50 of 51

Table 4. Peptide acyl acceptors utilised in transpeptidation during gingipain digestion of haemoglobin without added GlyGly. gingipains: rKgp and RgpB (64 nM), P. gingivalis whole cells of W50 and ATCC 33277 strains (2.5 x 107 cells/mL); haemoglobin: 6 µM; digestion buffer: TC150. 24 h incubation before addition of actic acid to terminate the reactions. Samples were cleaned up with ZipTip C18 before subjected to Orbitrap LC-MS/MS analysis. The amino acid residue numbering is based on the sequences of haemoglobin alpha and beta subunits as in Fig S5.

No . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

Hb subuni t α β α α/β β α α β α α β β α β β α β α Β β α β β β α β β α α β β α α

Acyl acceptors (apparent) 1 VLSPADKTNVK11 1 VHLTPEEK8 1 VLSPADK7 K61/K66 60 VK61 32 MFLSFPTTK40 8 TNVK11 62 AHGK65 57 GHGK60 17 VGAHAGEYGAEALER31 18 VNVDEVGGEALGR30 133 VVAGVANALAHK144 12 AAWGK16 121 EFTPPVQAAYQK132 41 FFESFGDLSTPDAVMGNPK59 41 TYFPHFDLSHGSAQVK56 62 AHGKK66 91 LR92 67 VLGAFSDGLAHLDNLK82 41 FFESFGDLSTPDAVMGNPKVK61 128 FLASVSTVLTSK139 83 GTFATLSELHCDK95 31 LLVVYPWTQR40 9 SAVTALWGK17 93 VDPVNFK99 60 VKAHGK65 145 YH146 140 YR141 128 FLASVSTVLTSKYR141 96 LHVDPENFR104 9 SAVTALWGKVNVDEVGGEALGR30 8 TNVKAAWGK16 62 VADALTNAVAHVDDMPNALSALSDLHA HK90

rKg p 8 7 12 13 6 3 7 2 3 7 7 7 4 4 2 1 2 2 3 nd 1 2 2 2 2 1 1 2 nd 1 nd 1

Rgp B 1 nd nd nd nd 1 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 1 nd nd 1 nd nd nd

W5 0 15 11 8 3 3 4 2 2 1 nd nd nd 1 nd 1 2 1 1 nd nd 1 nd nd nd nd nd nd nd nd nd nd nd

1

nd

nd

ATC C tota 33277 l 13 37 13 31 10 30 5 21 5 14 4 12 nd 9 4 8 4 8 nd 7 nd 7 nd 7 1 6 1 5 2 5 2 5 nd 3 nd 3 nd 3 2 2 nd 2 nd 2 nd 2 nd 2 nd 2 nd 2 1 2 nd 2 nd 1 nd 1 1 1 nd 1 nd

1

nd: not detected.


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Porphyromonas gingivalis Gingipains Display Transpeptidation Activity

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