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Porphyromonas gingivalis is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arg-gingipains (RgpA and RgpB) are major virulence factors, and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalyzed transpeptidation. Purified gingipains and P. gingivalis whole cells were used to digest selected substrates including human hemoglobin in the presence or absence of peptide acceptors. Mass spectrometric analysis of the substrates digested with gingipains in the presence of GlyGly showed that transpeptidation outcompeted hydrolysis, whereas the trypsin-digested controls exhibited predominantly hydrolysis activity. The transpeptidation levels increased with increasing concentration of GlyGly. Purified gingipains and whole cells exhibited extensive transpeptidation activities on human hemoglobin. All hemoglobin cleavage sites were found to be suitable for GlyGly transpeptidation, and this transpeptidation enhanced hemoglobin digestion. The transpeptidation products were often more abundant than the corresponding hydrolysis products. In the absence of GlyGly, hemoglobin peptides produced during digestion were utilized as acceptors leading to the detection of up to 116 different transpeptidation products in a single reaction. P. gingivalis cells were able to digest hemoglobin faster when acceptor peptides derived from human serum albumin were included in the reaction, suggesting that gingipain-catalyzed transpeptidation may be relevant for substrates encountered in vivo. The transpeptidation of host proteins in vivo may potentially lead to the breakdown of immunological tolerance, culminating in autoimmune reactions.

Tryptic digestion of the calcium-sensitive caseins yields casein phosphopeptides (CPP) that contain clusters of phosphorylated seryl residues. The CPP stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the CPP with calcium phosphate using a variety of nuclear magnetic resonance (NMR) techniques. Translational diffusion measurements indicated that the β-CN(1-25)-ACP nanocomplex has a hydrodynamic radius of 1.526 ± 0.044 nm at pH 6.0, which increases to 1.923 ± 0.082 nm at pH 9.0. (1)H NMR spectra were well resolved, and (3)JH(N)-H(α) measurements ranged from a low of 5.5 Hz to a high of 8.1 Hz. Total correlation spectroscopy and nuclear Overhauser effect spectroscopy spectra were acquired and sequentially assigned. Experiments described in this paper have allowed the development of a structural model of the β-CN(1-25)-amorphous calcium phosphate nanocomplex.