Production and properties of adhesin-free gingipain proteinase RgpA.

The Arg-specific gingipains of Porphyromonas gingivalis RgpA and RgpB have 97% identical sequences in their catalytic domains yet their propeptides are only 76% identical. RgpA isolates as a proteinase-adhesin complex (HRgpA) which hinders direct kinetic comparison of RgpAcat as a monomer with monomeric RgpB. We tested modifications of rgpA identifying a variant that enabled us to isolate histidine-tagged monomeric RgpA (rRgpAH). Kinetic comparisons between rRgpAH and RgpB used benzoyl-L-Arg-4-nitroanilide with and without cysteine and glycylglycine acceptor molecules. With no glycylglycine, values of Km, Vmax, kcat and kcat/Km for each enzyme were similar, but with glycylglycine Km decreased, Vmax increased and kcat increased ~ twofold for RgpB but ~ sixfold for rRgpAH. The kcat/Km for rRgpAH was unchanged whereas that of RgpB more than halved. Recombinant RgpA propeptide inhibited rRgpAH and RgpB with Ki 13 nM and 15 nM Ki respectively slightly more effectively than RgpB propeptide which inhibited rRgpAH and RgpB with Ki 22 nM and 29 nM respectively (p < 0.0001); a result that may be attributable to the divergent propeptide sequences. Overall, the data for rRgpAH reflected observations previously made by others using HRgpA, indicating rRgpAH fidelity and confirming the first production and isolation of functional affinity tagged RgpA.

Porphyromonas gingivalis laboratory strains and clinical isolates exhibit different distribution of cell surface and secreted gingipains.

Background: The cell-surface cysteine proteinases RgpA, RgpB (Arg-gingipain), and Kgp (Lys-gingipain) are major virulence factors of P. gingivalis, a keystone pathogen in the development of destructive periodontal disease. The gingipains function as proteinases and transpeptidases utilising small peptides such as glycylglycine as acceptor molecules. However, the characteristics of the gingipains from most P. gingivalis strains have not been determined. Methods: We determined the phenotypes of a panel of P. gingivalis laboratory strains and global clinical isolates with respect to growth on blood agar plus whole-cell and vesicle-free culture supernatant (VFSN) Arg- and Lys-specific proteinase activities. Results: The P. gingivalis isolates exhibited different growth characteristics and hydrolysis of haemoglobin in solid media. Whole-cell Arg-gingipain Vmax varied 5.8-fold and the whole cell Lys-gingipain Vmax varied 2.1-fold across the strains. Furthermore, the P. gingivalis strains showed more than 107-fold variance in soluble Arg-gingipain activity in VFSN and more than 371-fold variance in soluble Lys-gingipain activity in VFSN. Glycylglycine and cysteine stimulated Arg- and Lys-specific cleavage activities of all strains. The stimulation by cysteine was in addition to its redox effect consistent with both glycylglycine and cysteine promoting transpeptidation. Conclusion: The global P. gingivalis clinical isolates exhibit different Arg- and Lys­gingipain activities with substantial variability in the level of soluble proteinases released into the environment.

Porphyromonas gingivalis Gingipains Display Transpeptidation Activity.

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.

Casein Phosphopeptide-Amorphous Calcium Phosphate Reduces Streptococcus mutans Biofilm Development on Glass Ionomer Cement and Disrupts Established Biofilms.

Glass ionomer cements (GIC) are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm) formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge.

Casein Phosphopeptide-Amorphous Calcium Phosphate Nanocomplexes: A Structural Model.

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 ß-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 ß-CN(1-25)-amorphous calcium phosphate nanocomplex.

Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health.

The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis.

Propeptide-mediated inhibition of cognate gingipain proteinases.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K(i) values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.

Porphyromonas gingivalis cysteine proteinase inhibition by kappa-casein peptides.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study κ-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associated with P. gingivalis whole cells, purified RgpA-Kgp proteinase-adhesin complexes, and purified RgpB proteinase. The peptide κ-casein(109-137) exhibited synergism with Zn(II) against both Arg- and Lys-specific proteinases. The active region for inhibition was identified as κ-casein(117-137) using synthetic peptides. Kinetic studies revealed that κ-casein(109-137) inhibits in an uncompetitive manner. A molecular model based on the uncompetitive action and its synergistic ability with Zn(II) was developed to explain the mechanism of inhibition. Preincubation of P. gingivalis with κ-casein(109-137) significantly reduced lesion development in a murine model of infection.

Association of bovine dentine phosphophoryn with collagen fragments.

Bovine dentine phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser(P)) residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentine. Phosphophoryn was purified from bovine dentine using EDTA extraction, Ca(2+) precipitation, anion exchange and size exclusion chromatography. The purified protein migrated on SDS-PAGGE as a single band. The protein was dephosphorylated using a chelex alkaline dialysis procedure, repurified using anion exchange and size exclusion chromatography and then subjected to cleavage with trypsin. The digest was subjected to reversed-phase HPLC and analysed by Q-TOF mass spectrometry. The only non-trypsin peptides that could be identified were two collagen Type I alpha2 peptides whose sequence was determined by fragmentation analysis. The association of collagen fragments with highly purified phosphophoryn suggests that the EDTA extraction method yields BDP that is strongly bound to collagen fragments. This association now helps explain discrepancies in molecular weight and amino acid composition data for various phosphophoryn preparations compared with the same data calculated from the C-terminal extension of mouse, rat and human dentine sialophosphoprotein (DSPP) gene products. Analysis of the mutation pattern of the clinical disorder Osteogenesis Imperfecta within the region enclosed by the identified collagen fragments reveals that phosphophoryn associates with a segment of collagen that is crucial for structure and/or function.

A review of protein structure and gene organisation for proteins associated with mineralised tissue and calcium phosphate stabilisation encoded on human chromosome 4.

Several proteins associated with mineralised tissue (teeth and bone) or involved in calcium phosphate stabilisation in the body fluids, milk and saliva have been mapped to the q arm of human chromosome 4. These include the dentine/bone proteins dentine sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP1), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein, osteopontin (OPN), enamelin, ameloblastin, milk caseins, salivary statherin, and proline-rich proteins. The proposed function of those that are multiphosphorylated is: (i) the stabilisation of calcium phosphate in solution (e.g. casein, statherin) preventing spontaneous precipitation and seeded-crystal growth or (ii) promoting biomineralisation (e.g. the phosphophoryn domain of DSPP), where the protein described as a template macromolecule, is proposed to act as a nucleator/promoter of crystal growth. The genes of these proteins have been subjected to conserved chromosomal synteny during mammalian evolution. The multiphosphorylated proteins statherin, caseins, phosphophoryn, BSP and OPN have been characterised as intrinsically disordered. The codon usage patterns for the amino acid serine reveal a bias for AGC and AGT codons within the human genes dspp, dmp1 and bsp, mouse dspp and dmp1 but not significantly for statherin or caseins. This pattern was also observed in the gene encoding hen phosvitin that also contains stretches of multiphosphorylated serines and in the dmp1 gene sequences of mammalian, reptilian and avian classes. In conclusion, these intrinsically disordered multiphosphorylated proteins are the translation products of genes displaying examples of codon usage bias, internal repeats and conserved chromosomal synteny within the mammalian class.

Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes.

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides 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 casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

Nascent helix in the multiphosphorylated peptide alphaS2-casein(2-20).

Sequence-specific nuclear magnetic resonance (NMR) assignments have been determined for the peptide alphaS2-CN(2-20) containing the multiphosphorylated motif-8Ser(P)-Ser(P)-Ser(P)-Glu-Glu12- in the presence of molar excess Ca2+. The secondary structure of the peptide was characterized by sequential (i,i + 1), medium-range (i,i + 2/3/4) nOes and H alpha chemical shifts. Molecular modelling of the peptide based on these constraints suggests a nascent helix for residues Ser(P)9 to Glu12. The spectral data for alphaS2-CN(2-20) were compared with those of other casein phosphopeptides beta-CN(1-25) and alphaS1-CN(59-79) that also contain the multiphosphorylated motif. This comparison revealed a similar pattern of secondary amide chemical shifts in the multiphosphorylated motif. However, the patterns of medium-range nOe connectivities in the three peptides suggests they have distinctly different conformations in the presence of Ca2+ despite having a high degree of sequential similarity.