Keith J. Cross

The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-II reverse turn.

Many short synthetic peptides have now been shown to induce antibodies reactive with their cognate sequences in the intact folded protein1–8. Aside from the usefulness of such antibodies as site-specific reagents, the frequency with which this recognition occurs has raised several theoretical issues, the central one being that of how an antibody to a short synthetic peptide, which represents one of the most disordered states of a site in a protein, can react with the more ordered version of the same sequence in the folded protein. This apparent paradox can be resolved if the target site on the protein approaches disorder or if the peptide in solution or on a carrier adopts, with significant frequency, a conformation compatible with that of the cognate site in the protein. Various studies already suggest that antigenic sites in proteins correspond to regions of high atomic mobility1,9–15. We now show, using high-field nuclear magnetic resonance (NMR) spectroscopy, that a nonapeptide selected by several monoclonal antibodies as the immunodominant site of a 36-amino-acid immunogen (residues 75–110 of influenza virus haemagglutinin16,17) adopts a highly populated type-II reverse-turn conformation in water. This suggests that in this case the antibodies have selected a sequence possessing a conformational preference. Apart from helping us to understand immunological recognition, anti-peptide antibodies may provide reagents of sufficient precision for an immunological approach to the problem of protein folding18–23.

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.

Kappacin, a Novel Antibacterial Peptide from Bovine Milk

ABSTRACT Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk κ-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans andPorphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated κ-casein (residues 106 to 169) [κ-casein(106–169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)149κ-casein-A(138–158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)149κ-casein-A(138–158) and its nonphosphorylated counterpart κ-casein-A(138–158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)149 κ-casein-A(138–158) displayed growth-inhibitory activity against S. mutans(MIC, 59 μg/ml [26 μM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

Enamel Subsurface Lesion Remineralisation with Casein Phosphopeptide Stabilised Solutions of Calcium, Phosphate and Fluoride

Casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) and amorphous calcium fluoride phosphate (CPP-ACFP) solutions have been shown to remineralise enamel subsurface lesions. The aim of this study was to determine the effect of ion composition of CPP-ACP and CPP-ACFP solutions on enamel subsurface lesion remineralisation in vitro. CPP-bound and free calcium, phosphate and fluoride ion concentrations in the solutions were determined after ultrafiltration. The ion activities of the free ion species present were calculated using an iterative computational program. The mineral deposited in the subsurface lesions was analysed using transverse microradiography and electron microprobe. CPP was found to stabilise high concentrations of calcium, phosphate and fluoride ions at all pH values (7.0–4.5). Remineralisation of the subsurface lesions was observed at all pH values tested with a maximum at pH 5.5. The CPP-ACFP solutions produced greater remineralisation than the CPP-ACP solutions at pH 5.5 and below. The mineral formed in the subsurface lesions was consistent with hydroxyapatite and fluorapatite for remineralisation with CPP-ACP and CPP-ACFP, respectively. The activity gradient of the neutral ion pair CaHPO4⁰ into the lesion was significantly correlated with remineralisation and together with HF⁰ were identified as important species for diffusion.

Casein Phosphopeptides in Oral Health - Chemistry and Clinical Applications

The casein phosphopeptides (CPP) are derived from the milk protein casein by tryptic digestion. The CPP, containing the sequence -Pse-Pse-Pse-Glu-Glu- where Pse is a phosphoseryl residue, stabilize calcium and phosphate ions in aqueous solution and make these essential nutrients bioavailable. Under alkaline conditions the calcium phosphate is present as an alkaline amorphous phase complexed by the CPP, referred to as casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). The CPP-ACP complexes readily incorporate fluoride ions forming casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP). A mechanism is discussed which provides a rationale for the ability of the CPP-ACP to remineralize carious lesions in dental enamel. Clinical applications of the CPP-ACP as agents in the treatment of dental caries and other hypomineralized conditions are reviewed. It is concluded that the CPP are a safe and novel carrier for calcium, phosphate and hydroxide (fluoride) ions to promote enamel remineralization with application in oral care products, dental professional products and foodstuffs.

Characterization of a Novel Outer Membrane Hemin-Binding Protein of Porphyromonas gingivalis

Porphyromonas gingivalis is a gram-negative, anaerobic coccobacillus that has been implicated as a major etiological agent in the development of chronic periodontitis. In this paper, we report the characterization of a protein, IhtB (iron heme transport; formerly designated Pga30), that is an outer membrane hemin-binding protein potentially involved in iron assimilation by P. gingivalis. IhtB was localized to the cell surface of P. gingivalis by Western blot analysis of a Sarkosyl-insoluble outer membrane preparation and by immunocytochemical staining of whole cells using IhtB peptide-specific antisera. The protein, released from the cell surface, was shown to bind to hemin using hemin-agarose. The growth of heme-limited, but not heme-replete, P. gingivalis cells was inhibited by preincubation with IhtB peptide-specific antisera. The ihtB gene was located between an open reading frame encoding a putative TonB-linked outer membrane receptor and three open reading frames that have sequence similarity to ATP binding cassette transport system operons in other bacteria. Analysis of the deduced amino acid sequence of IhtB showed significant similarity to the Salmonella typhimurium protein CbiK, a cobalt chelatase that is structurally related to the ATP-independent family of ferrochelatases. Molecular modeling indicated that the IhtB amino acid sequence could be threaded onto the CbiK fold with the IhtB structural model containing the active-site residues critical for chelatase activity. These results suggest that IhtB is a peripheral outer membrane chelatase that may remove iron from heme prior to uptake by P. gingivalis.

The outer membrane protein LptO is essential for the O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C‐terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron‐dense surface layer. Both mutants showed co‐accumulation of A‐LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra‐ and penta‐acylated forms of mono‐phosphorylated lipid A in the wild‐type and porT mutant, while only the penta‐acylated forms of mono‐phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O‐deacylation of mono‐phosphorylated lipid A. Increased levels of non‐phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing mono‐phosphorylated tetra‐acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a β‐barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co‐ordinated secretion of A‐LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.

Divalent Metal Cations Increase the Activity of the Antimicrobial Peptide Kappacin

ABSTRACT Kappacin, nonglycosylated κ-casein(106-169), is a novel antimicrobial peptide produced from κ-casein found in bovine milk. There are two major genetic forms of kappacin, A and B, and using synthetic peptides corresponding to the active region, κ-casein(138-158), of these forms, we have shown that the Asp148 to Ala148 substitution is responsible for the lesser antibacterial activity of κ-casein-B(106-169). Kappacin was shown to have membranolytic action at concentrations above 30 μM at acidic pH when tested against artificial liposomes. There was little membranolytic activity at neutral pH, which is consistent with the lack of antibacterial activity of kappacin against Streptococcus mutans at this pH. Kappacin specifically bound two zinc or calcium ions per mol, and this binding enhanced antibacterial activity at neutral pH. Nuclear magnetic resonance analysis indicated that a κ-casein-A(138-158) synthetic peptide undergoes a conformational change in the presence of the membrane solvent trifluoroethanol and excess divalent metal ions. This change in conformation is presumably responsible for the increase in antibacterial activity of kappacin detected in the presence of excess zinc or calcium ions at neutral pH. When tested against the oral bacterial pathogen S. mutans cultured as a biofilm in a constant-depth film fermentor, a preparation of 10 g/liter kappacin and 20 mM ZnCl2 reduced bacterial viability by 3 log10 and suppressed recovery of viability. In contrast 20 mM ZnCl2 alone reduced bacterial viability by ≈1 log10 followed by rapid recovery. In conclusion, kappacin has a membranolytic, antibacterial effect that is enhanced by the presence of divalent cations.

Effect of Addition of Citric Acid and Casein Phosphopeptide-Amorphous Calcium Phosphate to a Sugar-Free Chewing Gum on Enamel Remineralization in situ

Casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) has been shown to remineralize enamel subsurface lesions in situ. The aim of this study was to investigate the effects of CPP-ACP in a fruit-flavoured sugar-free chewing gum containing citric acid on enamel remineralization, and acid resistance of the remineralized enamel, using an in situ remineralization model. The study utilized a double-blind, randomized, crossover design with three treatments: (i) sugar-free gum (2 pellets) containing 20 mg citric acid and 18.8 mg CPP-ACP, (ii) sugar-free gum containing 20 mg citric acid alone, (iii) sugar-free gum not containing CPP-ACP or citric acid. Ten subjects were instructed to wear removable palatal appliances, with 4 half-slab insets of human enamel containing demineralized subsurface lesions and to chew gum (2 pellets) for 20 min 4 times per day for 14 days. At the completion of each treatment the enamel half-slabs were removed and half of the remineralized lesion treated with demineralization buffer for 16 h in vitro. The enamel slabs (remineralized, acid-challenged and control) were then embedded, sectioned and subjected to microradiography to determine the level of remineralization. Chewing with gum containing citric acid and CPP-ACP resulted in significantly higher remineralization (13.0 ± 2.2%) than chewing with either gum containing no CPP-ACP or citric acid (9.4 ± 1.2%) or gum containing citric acid alone (2.6 ± 1.3%). The acid challenge of the remineralized lesions showed that the level of mineral after acid challenge was significantly greater for the lesions exposed to the gum containing CPP-ACP.

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.