<i>Mycoplasma hyopneumoniae</i> Surface Proteins Mhp385 and Mhp384 Bind Host Cilia and Glycosaminoglycans and Are Endoproteolytically Processed by Proteases That Recognize Different Cleavage Motifs

P97 and P102 paralogues occur as endoproteolytic cleavage fragments on the surface of <i>Mycoplasma hyopneumoniae</i> 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↓X-D/E-like site, creating P60₃₈₄ and P50₃₈₄. The P97 paralogue Mhp385 is inefficiently cleaved, with tryptic peptides from a 115 kDa protein (P115₃₈₅) and 88 kDa (P88₃₈₅) and 27 kDa (P27₃₈₅) 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 ⁷⁵²IQFELEPISLNV⁷⁶³ denotes the C-terminus of P88₃₈₅ and defines the novel cleavage site ⁷⁶¹L-N-V↓A-V-S⁷⁶⁶ in Mhp385. P115₃₈₅, P88₃₈₅, P27₃₈₅, P60₃₈₄, and P50₃₈₄ 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 P60₃₈₄, P50₃₈₄, and P88₃₈₅. No primary function was attributed to P27₃₈₅; however, this molecule contains four tandem R1 repeats with similarity to porcine collagen type VI (α3 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 <i>M. hyopneumoniae