Jarocki, VM, Raymond, BBA, Tacchi, JL, Padula, MP & Djordjevic, SP 2019, 'Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y.', Scientific reports, vol. 9, no. 1.View/Download from: Publisher's site
Mycoplasma hyopneumoniae is an economically-devastating and geographically-widespread pathogen that colonises ciliated epithelium, and destroys mucociliary function. M. hyopneumoniae devotes ~5% of its reduced genome to encode members of the P97 and P102 adhesin families that are critical for colonising epithelial cilia, but mechanisms to impair mucociliary clearance and manipulate host immune response to induce a chronic infectious state have remained elusive. Here we identified two surface exposed M. hyopneumoniae proteases, a putative Xaa-Pro aminopeptidase (MHJ_0659; PepP) and a putative oligoendopeptidase F (MHJ_0522; PepF), using immunofluorescence microscopy and two orthogonal proteomic methodologies. MHJ_0659 and MHJ_0522 were purified as polyhistidine fusion proteins and shown, using a novel MALDI-TOF MS assay, to degrade four pro-inflammatory peptides that regulate lung homeostasis; bradykinin (BK), substance P (SP), neurokinin A (NKA) and neuropeptide Y (NPY). These findings provide insight into the mechanisms used by M. hyopneumoniae to influence ciliary beat frequency, impair mucociliary clearance, and initiate a chronic infectious disease state in swine, features that are a hallmark of disease caused by this pathogen.
Jarocki, VM, Steele, JR, Widjaja, M, Tacchi, JL, Padula, MP & Djordjevic, SP 2019, 'Formylated N-terminal methionine is absent from the Mycoplasma hyopneumoniae proteome: Implications for translation initiation.', International journal of medical microbiology : IJMM, vol. 309, no. 5, pp. 288-298.View/Download from: Publisher's site
N-terminal methionine excision (NME) is a proteolytic pathway that cleaves the N-termini of proteins, a process that influences where proteins localise in the cell and their turnover rates. In bacteria, protein biosynthesis is initiated by formylated methionine start tRNA (fMet-tRNAfMet). The formyl group is attached by formyltransferase (FMT) and is subsequently removed by peptide deformylase (PDF) in most but not all proteins. Methionine aminopeptidase then cleaves deformylated methionine to complete the process. Components of NME, particularly PDF, are promising therapeutic targets for bacterial pathogens. In Mycoplasma hyopneumoniae, a genome-reduced, major respiratory pathogen of swine, pdf and fmt are absent from its genome. Our bioinformatic analysis uncovered additional enzymes involved in formylated N-terminal methionine (fnMet) processing missing in fourteen mycoplasma species, including M. hyopneumoniae but not in Mycoplasma pneumoniae, a major respiratory pathogen of humans. Consistent with our bioinformatic studies, an analysis of in-house tryptic peptide libraries confirmed the absence of fnMet in M. hyopneumoniae proteins but, as expected fnMet peptides were detected in the proteome of M. pneumoniae. Additionally, computational molecular modelling of M. hyopneumoniae translation initiation factors reveal structural and sequence differences in areas known to interact with fMet-tRNAfMet. Our data suggests that some mycoplasmas have evolved a translation process that does not require fnMet.
Berry, IJ, Jarocki, VM, Tacchi, JL, Raymond, BBA, Widjaja, M, Padula, MP & Djordjevic, SP 2017, 'N-terminomics identifies widespread endoproteolysis and novel methionine excision in a genome-reduced bacterial pathogen.', Scientific Reports, vol. 7, no. 1, pp. 1-17.View/Download from: Publisher's site
Proteolytic processing alters protein function. Here we present the first systems-wide analysis of endoproteolysis in the genome-reduced pathogen Mycoplasma hyopneumoniae. 669 N-terminal peptides from 164 proteins were identified, demonstrating that functionally diverse proteins are processed, more than half of which 75 (53%) were accessible on the cell surface. Multiple cleavage sites were characterised, but cleavage with arginine in P1 predominated. Putative functions for a subset of cleaved fragments were assigned by affinity chromatography using heparin, actin, plasminogen and fibronectin as bait. Binding affinity was correlated with the number of cleavages in a protein, indicating that novel binding motifs are exposed, and protein disorder increases, after a cleavage event. Glyceraldehyde 3-phosphate dehydrogenase was used as a model protein to demonstrate this. We define the rules governing methionine excision, show that several aminopeptidases are involved, and propose that through processing, genome-reduced organisms can expand protein function.
Widjaja, M, Harvey, KL, Hagemann, L, Berry, IJ, Jarocki, V, Raymond, BBA, Tacchi, JL, Gründel, A, Steele, JR, Padula, MP, Charles, IG, Dumke, R & Djordjevic, SP 2017, 'Elongation factor Tu is a multifunctional and processed moonlighting protein.', Scientific Reports, vol. 7, no. 1, pp. 1-17.View/Download from: Publisher's site
Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline. Recombinant MpnEf-Tu bound strongly to a diverse range of host molecules, and when bound to plasminogen, was able to convert plasminogen to plasmin in the presence of plasminogen activators. Fragments of Ef-Tu retain binding capabilities to host proteins. Bioinformatics and structural modelling studies indicate that the accumulation of positively charged amino acids in short linear motifs (SLiMs), and protein processing promote multifunctional behaviour. Codon bias engendered by an A + T rich genome may influence how positively-charged residues accumulate in SLiMs.
Tacchi, JL, Raymond, BBA, Haynes, PA, Berry, IJ, Widjaja, M, Bogema, DR, Woolley, LK, Jenkins, C, Minion, FC, Padula, MP & Djordjevic, SP 2016, 'Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae', OPEN BIOLOGY, vol. 6, no. 2.View/Download from: Publisher's site
Jarocki, VM, Santos, J, Tacchi, JL, Raymond, BBA, Deutscher, AT, Jenkins, C, Padula, MP & Djordjevic, SP 2015, 'MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae', Open Biology, vol. 5, no. 1, pp. 1-13.View/Download from: Publisher's site
Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae.
Jarocki, VM, Tacchi, JL & Djordjevic, SP 2015, 'Non-proteolytic functions of microbial proteases increases pathological complexity', PROTEOMICS, vol. 15, no. 5-6, pp. 1075-1088.View/Download from: Publisher's site
Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.
Raymond, BBA, Jenkins, C, Seymour, LM, Tacchi, JL, Widjaja, M, Jarocki, VM, Deutscher, AT, Turnbull, L, Whitchurch, CB, Padula, MP & Djordjevic, SP 2015, 'Proteolytic processing of the cilium adhesin MHJ_0194 (P123(J)) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules', CELLULAR MICROBIOLOGY, vol. 17, no. 3, pp. 425-444.View/Download from: Publisher's site
Pendarvis, K, Padula, MP, Tacchi, JL, Petersen, AC, Djordjevic, SP, Burgess, SC & Minion, F 2014, 'Proteogenomic mapping of Mycoplasma hyopneumoniae virulent strain 232', BMC Genomics, vol. 15, no. 1, pp. 1-8.View/Download from: Publisher's site
Pokharel, D, Padula, MP, Lu, JF, Tacchi, JL, Luk, F, Djordjevic, SP & Bebawy, M 2014, 'Proteome analysis of multidrug-resistant, breast cancer-derived microparticles.', Journal of Extracellular Vesicles, vol. 3, no. 1, pp. 1-10.View/Download from: Publisher's site
Cancer multidrug resistance (MDR) occurs when cancer cells evade the cytotoxic actions of chemotherapeutics through the active efflux of drugs from within the cells. Our group have previously demonstrated that multidrug-resistant breast cancer cells spontaneously shed microparticles (MPs) and that these MPs can transfer resistance to drug-responsive cells and confer MDR on those cells in as little as 4 h. Furthermore, we also showed that, unlike MPs derived from leukaemia cells, breast cancer-derived MPs display a tissue selectivity in the transfer of P-glycoprotein (P-gp), transferring the resistance protein only to malignant breast cells. This study aims to define the proteome of breast cancer-derived MPs in order to understand the differences in protein profiles between those shed from drug-resistant versus drug-sensitive breast cancer cells. In doing so, we detail the protein cargo required for the intercellular transfer of MDR to drug-sensitive recipient cells and the factors governing the transfer selectivity to malignant breast cells. We describe the first proteomic analysis of MPs derived from human breast cancer cells using SDS PAGE and liquid chromatography-tandem mass spectrometry (LC/MS/MS), in which we identify 120 unique proteins found only in drug-resistant, breast cancer-derived MPs. Our results demonstrate that the MP-mediated transfer of P-gp to recipient cells occurs alongside CD44; the Ezrin, Radixin and Moesin protein family (ERM); and cytoskeleton motor proteins within the MP cargo.
Tacchi, JL, Raymond, BB, Jarocki, VM, Berry, IJ, Padula, M & Djordjevic, SP 2014, 'Cilium Adhesin P216 (MHJ_0493) Is a Target of Ectodomain Shedding and Aminopeptidase Activity on the Surface of Mycoplasma hyopneumoniae', Journal of Proteome Research, vol. Epub.View/Download from: Publisher's site
MHJ_0493 (P216) is a highly expressed cilium adhesin in Mycoplasma hyopneumoniae. P216 undergoes cleavage at position 1074 in the S/T-X-F?-X-D/E-like motif 1072TN-F?Q-E1076 generating N-terminal and C-terminal fragments of 120 kDa (P120) and 85 kDa (P85) on the surface of M. hyopneumoniae. Here we show that several S/T-X-F?X-D/E-like motifs exist in P216 but only 1072TN-F?Q-E1076 and 1344I-T-F?A-D-Y1349 were determined to be bona fide processing sites by identifying semitryptic peptides consistent with cleavage at the phenylalanine residue. The location of S/T-X-F?-X-D/E-like motifs within or abutting regions of protein disorder greater than 40 consecutive amino acids is consistent with our hypothesis that site access influences the cleavage efficiency. Approximately 20 cleavage fragments of P216 were identified on the surface of M. hyopneumoniae by LCMS/MS analysis of biotinylated proteins and 2D SDS-PAGE. LCMS/MS analysis of semitryptic peptides within P216 identified novel cleavage sites. Moreover, detection of a series of overlapping semitryptic peptides that differed by the loss a single amino acid at their N-terminus is consistent with aminopeptidase activity on the surface of M. hyopneumoniae. P120 and P85 and their cleavage fragments bind heparin and cell-surface proteins derived from porcine epithelial-like cells, indicating that P216 cleavage fragments retain the ability to bind glycosaminoglycans.
Hutchinson, AT, Malik, A, Berkahn, MB, Agostino, M, To, J, Tacchi, JL, Djordjevic, SP, Turnbull, L, Whitchurch, CB, Edmundson, AB, Jones, PM, Raison, RL & Ramsland, PA 2013, 'Formation of Assemblies on Cell Membranes by Secreted Proteins: Molecular Studies of Free Lambda Light Chain Aggregates Found on the Surface of Myeloma Cells.', Biochemical Journal, vol. 454, no. 3, pp. 479-489.View/Download from: Publisher's site
We have described the presence of cell membrane-associated ? free immunoglobulin light chains (FLC) on the surface of myeloma cells. Notably, the anti-?FLC mAb, MDX-1097, is being assessed in clinical trials as a therapy for ? light chain isotype multiple myeloma. Despite the clinical potential of anti-FLC mAbs, there have been limited studies on characterizing membrane-associated FLCs at a molecular level. Furthermore, it is not known if ?FLCs can associate with cell membranes of myeloma cells. In this study, we describe the presence of ?FLCs on the surface of myeloma cells. We found that cell surface-associated ?FLC are bound directly to the membrane and in an aggregated form. Subsequently, membrane interaction studies revealed that ?FLCs interact with saturated zwitterionic lipids such as phosphatidylcholine and phosphatidylethanolamine, and using automated docking, we characterize a potential recognition site for these lipids. Atomic force microscopy confirmed that membrane-associated ?FLCs are aggregated. Given our findings, we propose a model whereby individual FLCs show modest affinity for zwitterionic lipids, with aggregation stabilizing the interaction due to multivalency. Notably, this is the first study to image FLCs bound to phospholipids and provides important insights into the possible mechanisms of membrane association by this unique myeloma surface antigen.
Raymond, B, Tacchi, JL, Jarocki, VM, Minion, F, Padula, M & Djordjevic, SP 2013, 'P159 from Mycoplasma hyopneumoniae binds porcine cilia and heparin and is cleaved in a manner akin to ectodomain shedding', Journal of Proteome Research, vol. 12, no. 12, pp. 5891-5903.View/Download from: Publisher's site
Mycoplasma hyopneumoniae colonizes the ciliated epithelial lining of the upper respiratory tract of swine and results in chronic infection. Previously, we have observed that members of P97 and P102 paralog families of cilium adhesins undergo endoproteolytic processing on the surface of M. hyopneumoniae. We show that P159 (MHJ_0494), an epithelial cell adhesin unrelated to P97 and P102 paralog families, is a cilium adhesin that undergoes dominant cleavage events at S/T-X-F?X-D/E-like motifs located at positions 233F?Q234 and 981F?Q982, generating P27, P110, and P52. An unrelated cleavage site 738L-K-V?G-A-A743 in P110 shows sequence identity with a cleavage site (L-N-V?A-V-S) identified in the P97 paralog, Mhp385, and generates 76 (P76) and 35 kDa (P35) fragments. LCMS/MS analysis of biotinylated surface proteins identified six peptides with a biotin moiety on their N-terminus indicating novel, low abundance neo-N-termini. LCMS/MS of proteins separated by 2D-PAGE, 2D immunoblotting using monospecific antiserum raised against recombinant fragments spanning P159 (F1P159-F4P159), and proteins that bound to heparin-agarose were all used to map P159 cleavage fragments. P159 is the first cilium adhesin not belonging to the P97/P102 paralog families and is extensively processed in a manner akin to ectodomain shedding in eukaryotes.
Robinson, MW, Buchtmann, KA, Jenkins, C, Tacchi, JL, Raymond, BBA, To, J, Chowdhury, PR, Woolley, LK, Labbate, M, Turnbull, L, Whitchurch, CB, Padula, MP & Djordjevic, SP 2013, 'MHJ_0125 is an M42 glutamyl aminopeptidase that moonlights as a multifunctional adhesin on the surface of Mycoplasma hyopneumoniae', OPEN BIOLOGY, vol. 3.View/Download from: Publisher's site
Bogema, DR, Deutscher, AT, Woolley, LK, Seymour, LM, Raymond, BB, Tacchi, JL, Padula, M, Dixon, NE, Minion, F, Jenkins, C, Walker, MJ & Djordjevic, SP 2012, 'Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography', mBio, vol. 3, no. 2, pp. 1-11.View/Download from: Publisher's site
Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF down arrow QQ(677), consistent with a cleavage motif resembling S/T-X-F down arrow X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography.
Deutscher, AT, Tacchi, JL, Minion, F, Padula, M, Crossett, B, Bogema, DR, Jenkins, C, Kuit, TA, Walker, MJ & Djordjevic, SP 2012, 'Mycoplasma hyopneumoniae surface proteins Mhp385 and Mhp384 bind host cilia and glycosaminoglycans and are endoproteolytically processed by proteases that recognize different cleavage motifs', Journal of Proteome Research, vol. 11, no. 3, pp. 1924-1936.View/Download from: Publisher's site
P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of Mycoplasma hyopneumoniae that bind glycosaminoglycans, plasminogen, and fibronectin and perform essential roles in colonization of ciliated epithelia. We show that the P102 paralogue Mhp384 is efficiently cleaved at an S/T-X-F down arrow X-D/E-like site, creating P60(384) and P50(384). The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115(385)) and 88 kDa (P88(385)) and 27 kDa (P27(385)) cleavage fragments identified by LC-MS/MS. This is the first time a preprotein belonging to the P97 and P102 paralogue families has been identified by mass spectrometry. The semitryptic peptide (752)IQFELEPISLNV(763) denotes the C-terminus of P88(385) and defines the novel cleavage site L-761-N-V down arrow A-V-S-766 in Mhp385. P115(385), P88(385), P27(385), P60(384), and P50(384) were shown to reside extracellularly, though it is unknown how the fragments remain attached to the cell surface. Heparin- and cilium-binding sites were identified within P60(384), P50(384), and P88(385). No primary function was attributed to P27(385); however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (alpha 3 chain). P97 and P102 paralogue families are adhesins targeted by several proteases with different cleavage efficiencies, and this process generates combinatorial complexity on the surface of M. hyopneumoniae.
Seymour, LM, Jenkins, C, Deutscher, AT, Raymond, BB, Padula, M, Tacchi, JL, Bogema, DR, Eamens, GJ, Woolley, LK, Dixon, NE, Walker, MJ & Djordjevic, SP 2012, 'Mhp182 (P102) binds fibronectin and contributes to the recruitment of plasmin(ogen) to the Mycoplasma hyopneumoniae cell surface', Cellular Microbiology, vol. 14, no. 1, pp. 81-94.View/Download from: Publisher's site
Mycoplasma hyopneumoniae is a major, economically damaging respiratory pathogen. Although M. hyopneumoniae cells bind plasminogen, the identification of plasminogen-binding surface proteins and the biological ramifications of acquiring plasminogen requires further investigation. mhp182 encodes a highly expressed 102 kDa protein (P102) that undergoes proteolytic processing to generate surface-located N-terminal 60 kDa (P60) and C-terminal 42 kDa (P42) proteins of unknown function. We show that recombinant P102 (rP102) binds plasminogen at physiologically relevant concentrations (KD similar to 76 nM) increasing the susceptibility of plasmin(ogen) to activation by tissue-specific plasminogen activator (tPA). Recombinant proteins constructed to mimic P60 (rP60) and P42 (rP42) also bound plasminogen at physiologically significant levels. M. hyopneumoniae surface-bound plasminogen was activated by tPA and is able to degrade fibrinogen, demonstrating the biological functionality of M. hyopneumoniae-bound plasmin(ogen) upon activation. Plasmin(ogen) was readily detected in porcine ciliated airways and plasmin levels were consistently higher in bronchoalveolar lavage fluid from M. hyopneumoniae-infected animals. Additionally, rP102 and rP42 bind fibronectin with KDs of 26 and 33 nM respectively and recombinant P102 proteins promote adherence to porcine kidney epithelial-like cells. The multifunctional binding ability of P102 and activation of M. hyopneumoniae-sequestered plasmin(ogen) by an exogenous activator suggests P102 plays an important role in virulence.
Bogema, DR, Scott, NE, Padula, M, Tacchi, JL, Raymond, B, Jenkins, C, Cordwell, SJ, Minion, F, Walker, MJ & Djordjevic, SP 2011, 'Sequence TTKF | QE defines the site of proteolytic cleavage in Mhp683, a novel glycosaminoglycan and cilium adhesin of Mycoplasma hyopneumoniae', Journal Of Biological Chemistry, vol. 286, no. 48, pp. 41217-41229.View/Download from: Publisher's site
Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45683, P48683, and P50683. A peptide sequence (TTKF?QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48683 and P50683, determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45683, P48683, and P50683 reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1683F5683, spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1683F5683 also bound porcine epithelial cilia, and antisera to F2683 and F5683 significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45683, P48683, and P50683 each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.
Berry, IJ, Tacchi, JL, Jarocki, VM, Raymond, BBA, Padula, MP & Djordjevic, SP 2014, 'The Significance of Post-Translational Proteolysis in the Model Pathogen, Mycoplasma hyopneumoniae.', 2nd "Proteomics & Beyond" Symposium, Proteomics & Beyond Symposium, Australian Proteomics Analysis Facility.
Proteolytic cleavage is one of the most ubiquitous post-translational modifications to proteins, responsible for protein signalling, activation, localisation and ultimately degradation. Due to a variety of experimental limitations this important physiological process has been largely understudied, particularly in prokaryotes and archaea. In order determine the scale and functional roles of protein processing in bacteria and to explore the underlying mechanisms in the production of mature proteins, a high-throughput systems wide approach is needed..
The primary focus of this project is to sequence the N-terminal sequences of mature protein products of prokaryotic pathogens. Using this data, we can identify true protein start sites and any downstream post-translational processing. The genome-reduced agriculturally-important pathogen Mycoplasma hyopneumoniae, was selected as a model organism for these studies due to the large body of literature demonstrating the extensive proteolytic processing of many highly expressed surface proteins of this organism, which are critical for pathogenesis.
Using a high-throughput methodology called Terminal Amine Isotopic Labelling of Substrates (TAILS), the data obtained from the analysis of M. hyopneumoniae has provided evidence confirming true start sites of protein translation and complementary data pinpointing the precise sites of proteolytic cleavage. Our data indicates that an unprecedented number of cell surface proteins are targets for posttranslational processing.