title: Cilium adhesin P216 (MHJ_0493) is a target of ectodomain shedding and aminopeptidase activity on the surface of Mycoplasma hyopneumoniae.
abstract: 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 (1072)T-N-F↓Q-E(1076) 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 (1072)T-N-F↓Q-E(1076) and (1344)I-T-F↓A-D-Y(1349) 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 LC-MS/MS analysis of biotinylated proteins and 2D SDS-PAGE. LC-MS/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.
authors.affiliation: The ithree institute and ‡Proteomics Core Facility, University of Technology Sydney , P.O. Box 123, Broadway, Sydney, NSW 2007, Australia.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Tacchi
authors.forename: Benjamin B A
authors.initials: BB
authors.lastname: Raymond
authors.forename: Veronica M
authors.initials: VM
authors.lastname: Jarocki
authors.forename: Iain J
authors.initials: IJ
authors.lastname: Berry
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Padula
authors.forename: Steven P
authors.initials: SP
authors.lastname: Djordjevic
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000378:metabolism*; D020816:Amino Acid Motifs; D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001422:Bacterial Adhesion; D001665:Binding Sites; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism; D006493:Heparin / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D045705:Mycoplasma hyopneumoniae / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011485:Protein Binding; D059748:Proteolysis; D034421:Sus scrofa; D053719:Tandem Mass Spectrometry
chemical_list: D018829:Adhesins, Bacterial; D008565:Membrane Proteins; D010446:Peptide Fragments; D006493:Heparin; D000626:Aminopeptidases