Protein cleavage is a ubiquitous post-translational modification of proteins involved in a variety of biological processes such as cell signalling, protein activation, localisation and degradation. Protein cleavage has also been linked to virulence in a variety of infectious diseases [1,2,3]; however very few high-throughput, global analyses of cleaved proteins have been performed on pathogenic bacteria. Therefore experimental investigation is needed to determine the extent and functional roles of protein processing in the production of mature proteins.
Mycoplasma hyopneumoniae is a swine pathogen that targets the ciliated epithelial lining of the respiratory tract. We have described extensive proteolytic processing of members of two adhesin families known as the P97 and P102 adhesin families that bind extracellular matrix including glycosaminoglycans, fibronectin and the fibrinolytic protein, plasminogen [4,5,6]. Our aim is to determine whether proteolytic cleavage targets other cell-surface proteins. To achieve this, a high-throughput, proteome-wide survey of functional, cleaved proteins is needed to determine sites of protein cleavage.
Using protein dimethyl labelling and ‘shotgun’ mass spectrometry, the N-terminal sequences of mature M. hyopneumoniae proteins were obtained, identifying true protein start sites and the exact location of post-translational protein cleavage. This data was combined with cell-surface studies, protein affinity chromatography and mass mapping by SDS-PAGE to confirm the intact masses of identified proteins.
Analysis of our proteome-wide, N-terminal sequence data confirmed multiple cleavage sites in the P97 and P102 adhesin families. In addition, subsets of cytosolic proteins that also moonlight on the cell surface were targets of cleavage events. Homologues of these ‘moonlighting’ proteins have also been described as virulence factors in other pathogenic bacteria. The sequence motifs associated with these cleavage events retain remarkable similarity, suggesting that similar proteases process these proteins on the cell surface, contributing to the diversity of host/pathogen interactions.