title: Sequence TTKF ↓ QE defines the site of proteolytic cleavage in Mhp683 protein, a novel glycosaminoglycan and cilium adhesin of Mycoplasma hyopneumoniae.
abstract: 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 P45(683), P48(683), and P50(683). A peptide sequence (TTKF↓QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48(683) and P50(683), 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 P45(683), P48(683), and P50(683) reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1(683)-F5(683), spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1(683)-F5(683) also bound porcine epithelial cilia, and antisera to F2(683) and F5(683) significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45(683), P48(683), and P50(683) each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.
authors.affiliation: NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Camden 2567, New South Wales, Australia; School of Biological Sciences, University of Wollongong, Wollongong 2522, New South Wales, Australia.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Bogema
authors.affiliation: School of Molecular and Microbial Biosciences, University of Sydney, Sydney 2006, New South Wales, Australia.
authors.forename: Nichollas E
authors.initials: NE
authors.lastname: Scott
authors.affiliation: The ithree Institute, University of Technology, Sydney 2007, New South Wales, Australia.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Padula
authors.affiliation: The ithree Institute, University of Technology, Sydney 2007, New South Wales, Australia.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Tacchi
authors.affiliation: The ithree Institute, University of Technology, Sydney 2007, New South Wales, Australia.
authors.forename: Benjamin B A
authors.initials: BBA
authors.lastname: Raymond
authors.affiliation: NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Camden 2567, New South Wales, Australia.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Jenkins
authors.affiliation: School of Molecular and Microbial Biosciences, University of Sydney, Sydney 2006, New South Wales, Australia.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Cordwell
authors.affiliation: Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa 50011.
authors.forename: F Chris
authors.initials: FC
authors.lastname: Minion
authors.affiliation: School of Biological Sciences, University of Wollongong, Wollongong 2522, New South Wales, Australia; School of Chemistry and Molecular Biosciences and the Australian Infectious Diseases Research Centre, University of Queensland, Brisbane 4072, Queensland, Australia.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Walker
authors.affiliation: NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Camden 2567, New South Wales, Australia; The ithree Institute, University of Technology, Sydney 2007, New South Wales, Australia. Electronic address: steven.djordjevic@uts.edu.au.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Djordjevic
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001422:Bacterial Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D002923:Cilia / Q000378:metabolism / Q000382:microbiology; D006025:Glycosaminoglycans / Q000235:genetics / Q000378:metabolism*; D045705:Mycoplasma hyopneumoniae / Q000235:genetics / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000378:metabolism / Q000382:microbiology; D013552:Swine
chemical_list: D018829:Adhesins, Bacterial; D006025:Glycosaminoglycans