Bioinformatic investigation of the cost management strategies of five oral microbes.

Some amino acids are more energetically costly to synthesize de novo, therefore many microbes have evolved to regulate the metabolic expenditure of the cell and reduce the energy burden of extracellular unrecyclable proteins. Several oral bacterial species take up amino acids and peptides obtained from proteolysis of host proteins and hence do not rely only on de novo synthesis. The aim of this study was to investigate if five oral bacterial species implement cost management strategies to reduce the energy burden of extracellular unrecyclable proteins. Since the relative de novo amino acid synthesis costs are proportional to the masses of the amino acids, the energy costs of producing proteins were assessed by calculating the mean amino acid mass for each protein. For Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia and Streptococcus sanguinis, the outer membrane/extracellular proteins are made up of a much larger percentage of lower average mass amino acids whereas cytoplasmic proteins are made up of a larger proportion of higher average mass amino acid residues. These results are consistent with the five oral bacterial species employing energy-saving mechanisms in the production of extracellular unrecyclable proteins. Interestingly, the P. gingivalis and S. sanguinis genomes exhibited significantly lower predicted mean amino acid masses compared with those of the genomes of the other three species, suggesting that this may provide them with an energy advantage with respect to protein biosynthetic cost.

New approaches to enhanced remineralization of tooth enamel.

Dental caries is a highly prevalent diet-related disease and is a major public health problem. A goal of modern dentistry is to manage non-cavitated caries lesions non-invasively through remineralization in an attempt to prevent disease progression and improve aesthetics, strength, and function. Remineralization is defined as the process whereby calcium and phosphate ions are supplied from a source external to the tooth to promote ion deposition into crystal voids in demineralized enamel, to produce net mineral gain. Recently, a range of novel calcium-phosphate-based remineralization delivery systems has been developed for clinical application. These delivery systems include crystalline, unstabilized amorphous, or stabilized amorphous formulations of calcium phosphate. These systems are reviewed, and the technology with the most scientific evidence to support its clinical use is the remineralizing system utilizing casein phosphopeptides to stabilize and deliver bioavailable calcium, phosphate, and fluoride ions. The recent clinical evidence for this technology is presented and the mechanism of action discussed. Biomimetic approaches to stabilization of bioavailable calcium, phosphate, and fluoride ions and the localization of these ions to non-cavitated caries lesions for controlled remineralization show promise for the non-invasive management of dental caries.

Cysteine protease inhibitors: from evolutionary relationships to modern chemotherapeutic design for the treatment of infectious diseases.

Cysteine proteases are one of the largest groups of proteases and are involved in many important biological functions in all kingdoms of life. They are virulence factors of a range of eukaryotic, bacterial and viral pathogens and are involved in host invasion, pathogen replication and disruption of the host immune response. Their activity is regulated by a range of protease inhibitors. This review discusses the various families of cysteine protease inhibitors, their different modes of inhibition and their evolutionary relationships. These inhibitors as well as the recent discovery of propeptide and propeptide-like inhibitors provide insights into the structures that are important for particular inhibitory mechanisms, thus forming the foundation for the design of future therapeutics.

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.

Protein dynamics of bovine dentin phosphophoryn.

Bovine dentin 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 dentin. The elucidation of the structure-function relationship of phosphophoryn has been a challenge because of its high-molecular weight, high negative charge, repetitive sequence, and lability. We have used the dynamic behavior of the (1)H NMR signal at 600 MHz to provide insight into the molecular dynamics of phosphophoryn. Our results indicate that phosphophoryn is a molecule of uniformly high mobility, thus belonging to a recently identified class of intrinsically disordered proteins that are characterized by sequences of low complexity and rich in polar and charged residues. The significance of our results is that phosphophoryn, because of its uniform nature has the potential to be replaced by biomimetic synthetic peptide analogs that together with amorphous calcium phosphate may lead to the development of novel, nontoxic, apatite-based dental restorative materials.

NMR studies of a novel calcium, phosphate and fluoride delivery vehicle-alpha(S1)-casein(59-79) by stabilized amorphous calcium fluoride phosphate nanocomplexes.

The repair of early tooth enamel lesions has been recently demonstrated by tryptic phosphopeptides derived from milk caseins that associate with amorphous calcium phosphate (ACP) forming stable complexes. These casein phosphopeptides (CPP), containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, form calcium phosphate delivery vehicles that retard enamel demineralization and promote remineralization. Recently, we have shown that these peptides also stabilize calcium fluoride phosphate as soluble complexes. These complexes designated CPP-ACFP, have the potential to provide superior clinical efficacy in preventing dental caries and treating and repairing early stages of disease. In an approach to determine the ultrastructure of the casein phosphopeptide-amorphous calcium fluoride phosphate complexes, we have studied the structure of the predominant peptide alpha(S1)-CN(59-79) bound to ACFP using nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. The alpha(S1)-CN(59-79) peptide stabilized calcium fluoride phosphate as amorphous nanocomplexes with a hydrodynamic radius of 2.12+/-0.26 nm. The nanocomplexes exhibited stoichiometry of one peptide to 15 calcium, nine phosphate and three fluoride ions. Sequence-specific resonance assignments were determined for the peptide alpha(S1)-CN(59-79) complexed to the ACFP. The secondary structure of the peptide alpha(S1)-CN(59-79) was characterized by sequential (i, i+1), medium-range (i, i+2) nOes and H alpha chemical shifts. The spectral data were compared with that of the peptide alpha(S1)-CN(59-79) bound to calcium ions, revealing that the structurally significant secondary NH and alpha-chemical shifts were similar.

Cation-dependent structural features of beta-casein-(1-25).

Complete sequence-specific, proton-resonance assignments have been determined for the calcium phosphate-stabilizing tryptic peptide beta-casein-(1-25) containing the phosphorylated sequence motif Ser(P)(17)-Ser(P)-Ser(P)-Glu-Glu(21). Spectra of the peptide have been recorded, in separate experiments, in the presence of excess ammonium ions, sodium ions and calcium ions, and of the dephosphorylated peptide in the presence of excess sodium ions. We observed significant changes to chemical shifts for backbone and side-chain resonances that were dependent upon the nature of the cation present. Medium-range nuclear Overhauser effect (nOe) enhancements, characteristic of small structured regions in the peptide, were observed and also found to be cation dependent. The secondary structure of the peptide was characterized by sequential and medium-range (i, i+2/3/4, which denotes an interaction between residue i and residue i+2, i+3 or i+4 in the peptide) nOe connectivities, and Halpha chemical shifts. Four structured regions were identified in the calcium-bound peptide: residues Arg(1) to Glu(4) were involved in a loop-type structure, and residues Val(8) to Glu(11), Ser(P)(17) to Glu(20) and Glu(21) to Thr(24) were implicated in beta-turn conformations. Comparison of the patterns of medium-range nOe connectivities in beta-casein-(1-25) with those in alpha(S1)-casein-(59-79) suggest that the two peptides have distinctly different conformations in the presence of calcium ions, despite having a high degree of sequential and functional similarity.

N-terminal sequence analysis of bovine dentin phosphophoryn after conversion of phosphoseryl to S-propylcysteinyl residues.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser[P]) residues, is synthesized by odontoblasts and is believed to be involved in matrix-mediated biomineralization of dentin. We have purified BDP, using selective precipitation and ion exchange chromatography, from an EDTA soluble dentin extract and converted the Ser(P) residues to S-propylcysteinyl residues that are stable to Edman degradation, facilitating the determination of the amino acid sequence of the N-terminal 38 residues. After the initial Asp-Ser(P)-Pro-Asn-Ser(P)-Ser(P)-Asp-Glu-Ser(P)-Asn-Gly-, the sequence contained the repeated motifs Asp-Ser(P) and Asp-Ser(P)-Ser(P). Purified BDP migrated as a single band on gradient SDS-PAGE with an apparent molecular weight of 156 kDa. This value was consistent with the molecular weight of the dephosphorylated protein of 105 kDa determined by means of MALDI mass spectrometry.

Epitope analysis of the multiphosphorylated peptide alpha s1-casein (59-79).

The multiphosphorylated tryptic peptide alpha(s1)-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 alpha(s1)-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 alpha(s1)-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 x(s1)-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 x(s1)-casein(59-79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic.

A 1H-NMR study of the casein phosphopeptide alpha s1-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.

Complete amino acid sequence and comparative molecular modelling of HPr from Streptococcus mutans Ingbritt.

The heat-stable phosphocarrier protein (HPr) of Streptococcus mutans was extracted from whole cells using sodium lauroylsarcosinate/EDTA and purified to homogeneity by a single-step, ion-exchange chromatographic procedure. The complete amino acid sequence of the protein was determined from peptides generated by trypsin, alpha-chymotrypsin, endoproteinase Glu-C, and cyanogen bromide treatment. The HPr from S. mutans contains 86 or 87 amino acyl residues, depending on removal of the N-terminal Met and the protein shows high sequence homology with HPr from other Gram-positive bacteria. The predicted tertiary structure of the S. mutans HPr, from model building by homology, is an open-faced beta-sandwich consisting of two alpha-helices and a four-stranded antiparallel beta-sheet.

Partial amino acid sequence of osteocalcin from an extinct species of ratite bird.

Osteocalcin the major gamma carboxyglutamic acid containing protein of vertebrate bone has been purified from the bones of a specimen of Pachyornis elephantopus, a species of the extinct class of New Zealand ratite birds, the moas. The sequence of the N-terminal region of moa osteocalcin was determined using gas phase N-terminal sequencing. The N-terminal sequences of the ostrich and rhea osteocalcins were also determined. Alignment of the N-terminal sequence of osteocalcin from the extinct moa against the osteocalcins of the extant ostrich, rhea and emu reveals the homology amongst the ratite species is greater than the homology with the chicken osteocalcin.

Recovery from steroid-induced osteoporosis.

The reversibility of steroid-induced osteoporosis, a major complication of Cushing syndrome and long-term use of exogenous corticosteroids, is not well documented. We measured the bone mineral density of lumbar vertebras and the femoral neck by dual-photon absorptiometry and determined the biochemical variables of bone turnover in two patients successfully treated for Cushing syndrome who were followed for the next 24 months. Our data are the first to show marked increases in bone density (up to 20%) during the recovery period. The accompanying biochemical changes, particularly the marked increase in serum osteocalcin levels, confirm that enhanced bone formation occurred during the recovery phase. These findings suggest that steroid-induced osteoporosis can be reversed at least in young persons.

The amino acid sequence of Emu osteocalcin: gas phase sequencing of Gla-containing proteins.

Osteocalcin (OC), the major gamma carboxyglutamic acid (Gla)-containing protein of vertebrate bone, has been isolated from bones of the emu (Dromaius novaehollandae) and the primary structure determined by a combination of gas phase N-terminal sequencing of the intact molecule and a proteolytic fragment, and carboxypeptidase Y C-terminal sequencing. Gla residues were located by counting tritium radioactivity in fractions from the N-terminal sequencing of the tritiated/thermally decarboxylated molecule. Emu OC consists of 48 amino acid residues containing 3 Gla residues, and a single disulphide bond. The C-terminal 29 residues are identical to those of the human and sheep OC sequences. Alignment of the N-terminal sequence against those of other OCs reveals greater sequence homology with chicken OC than with mammalian OCs.

Methionine oxidation in human growth hormone and human chorionic somatomammotropin. Effects on receptor binding and biological activities.

The oxidation of the methionine residues of human growth hormone (hGH) and human chorionic somatomammotropin (hCS) to methionine sulfoxide by hydrogen peroxide has been studied. The kinetics of oxidation of individual methionine residues has been measured by reverse-phase high pressure liquid chromatography tryptic peptide mapping. Met-170 is completely resistant to oxidation in both hormones. The other 3 methionine residues in hCS (Met-64, Met-96, and Met-179) have markedly different reaction rates. Oxidation of the methionine residues does not appear to cause gross conformational changes in either hGH or hCS, as judged by CD and 1H NMR spectroscopy. Oxidation of Met-14 and Met-125 in hGH has little effect on affinity of the hormone for lactogenic receptors or on its potency in the Nb2 rat lymphoma in vitro bioassay for lactogenic hormones. The oxidation of Met-64 and/or Met-179 in hCS reduces profoundly both its affinity for lactogenic receptors and its in vitro biological potency. It is inferred by induction that residues 64 and/or 179 are critical for the binding of both hGH and hCS to lactogenic receptors and the expression of lactogenic biological activity.

Low serum osteocalcin levels in glucocorticoid-treated asthmatics.

Serum osteocalcin (OC) levels were measured in 19 asthmatic patients receiving long term glucocorticoid therapy and in age- and sex-matched asthmatic patients not receiving this treatment. In the glucocorticoid-treated patients, the mean OC level was approximately 50% less than that in the control group (P less than 0.001), and there was a direct correlation between serum OC and 1,25-dihydroxyvitamin D [1,25-(OH)2D; r = 0.71; P less than 0.001]. Multiple regression analysis in a total of 39 glucocorticoid-treated patients indicated that OC correlated directly to 1,25-(OH)2D and inversely to glucocorticoid dose. There was no correlation between OC and 1,25-(OH)2D in the control group and no significant difference in mean serum 1,25-(OH)2D between the steroid-treated asthmatic patients and the asthmatic control patients. The effect of a 4-day course of oral 1,25-(OH)2D on serum OC was studied in six patients with glucocorticoid excess and six normal subjects. There was a similar percent increase in OC levels in both groups, though the basal concentrations and absolute increases were substantially less in the steroid-treated group. It is likely that the depression of serum OC in glucocorticoid-treated patients results from the reduction in the rate of bone formation induced by these hormones.

Immunochemical detection and characterisation of osteocalcin from moa bone.

Osteocalcin (the 6,000 dalton Mr gamma-carboxyglutamate-containing protein of bone) has been detected in acid extracts of bones of the extinct class of New Zealand ratite birds, the moas, using a radioimmunoassay for sheep osteocalcin. The immunoreactive osteocalcin of the extracts of two of these bones (the fibulae from two specimens of Pachyornis elephantopus found in South Island swamps) has been fractionated by gel filtration chromatography and reversed-phase high performance liquid chromatography, and behaves in a manner characteristic of osteocalcin from modern bones. Carbon-14 dating of bones and gizzard contents found in association with these specimens indicates approximate ages of 3,600 and 7,400 years respectively.

The amino acid sequences of goat, pig and wallaby osteocalcins.

The amino acid sequences of osteocalcins from cortical long bones of goat, pig and wallaby have been determined by manual microsequencing methods. The location of the gamma-carboxyglutamic acid residues were identified by N-terminal degradation of peptides in which these residues had been tritium-labelled in the gamma-H position and subsequently thermally decarboxylated to glutamic acid residues. Glutamic-17 is not gamma-carboxylated in wallaby osteocalcin, unlike other osteocalcins so far sequenced (except human). This may be related to sequence changes in the flanking residues. Proline-9 is fully hydroxylated in wallaby osteocalcin, despite the sequence substitution Ala-8----Phe-8.