N. Laila Huq

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.

A Review of the Salivary Proteome and Peptidome and Saliva-derived Peptide Therapeutics

Saliva is a glandular secretion that is vital in the maintenance of healthy oral tissues. In this review we outline the high abundance salivary proteins, summarise the status of the salivary proteome and peptidome, the genetic origin and recognised functions of these proteins, the diseases associated with salivary disorders, and the emerging saliva-derived peptide therapeutics. Different proteomic approaches have reported the identification of over 1,300 proteins in saliva. However there are fewer than 100 high abundance proteins, identified by multiple methods including, two-dimensional polyacrylamide gel electrophoresis and HPLC combined with mass spectrometry. Analysis of the genes coding for the salivary proteins demonstrated a non-uniform chromosomal distribution with chromosome 4 having the largest proportion of genes expressed in salivary glands. Several diseases are associated with salivary disorders including Sjögren’s syndrome, Prader-Willi syndrome, dental caries and stress related disorders. Saliva as a diagnostic medium for various biochemical tests has provided a non-invasive and accessibility advantage over other more regularly tested body fluids such as blood and urine. To-date the emerging saliva-based therapeutics include artificial salivas and antimicrobial agents based on histatins and mucins.

A 1H-NMR study of the casein phosphopeptide αs1-casein(59–79)

Complete sequence-specific resonance assignments have been determined for a calcium phosphate sequestering, phosphoseryl-containing, tryptic peptide alpha s1-casein(59-79) containing the phosphorylated motif -SSSEE-. Spectra have been recorded in the presence of excess Ca2+ and at three different values of sample pH to characterize the changes in peptide conformation as calcium binds to the phosphorylated residues. The secondary structure of the peptide was characterized by sequential (i,i + 1), medium-range (i,i + 2/3/4), and long-range (i,i + 5) NOE connectivities, C alpha H chemical shifts, NH to C alpha H coupling constants and the observation of slowly exchanging amide protons. Two structured regions have been identified: residues P73 to V76 implicated in beta-turn conformations, and residues E61 to sigma 67 involved in a loop-type structure.

Porphyromonas gingivalis Cysteine Proteinase Inhibition by κ-Casein Peptides

ABSTRACT 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.

The Role of Multiphosphorylated Peptides in Mineralized Tissue Regeneration

Several multiple phosphoseryl-containing proteins have been identified in mineralising tissue and body fluids where they have been proposed to act as nucleators and/or regulators of biomineralisation. In particular, the unique features of the multiphosphorylated caseins from milk and phosphophoryn from dentine enable them to have a role in mineralised tissue regeneration. In this review, three examples of therapeutic applications using multiphosphorylated peptides are described.

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 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.

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 Ki 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.

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide αs1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With αs1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu63‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu70 was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to αs1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)75 with Ser75 in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)75‐Val‐Glu‐Glu also abolished inhibition of antibody binding to αs1‐casein (59–79) demonstrating that Ser(P)75 is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)66‐Ser(P)‐Ser(P)68 and in the sequence ‐Pro73‐Asn‐Ser(P)‐Val‐Glu77‐ are critical for antibody binding to αs1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright

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.