A novel Ser O-glucuronidation in acidic proline-rich proteins identified by tandem mass spectrometry.

Human acidic proline-rich salivary protein PRP-1 and its C-terminally truncated form PRP-3 were analyzed by electrospray tandem mass spectrometry. Post-translational modifications were detected and characterized. A pyroglutamic acid residue was demonstrated at the N-terminus, Ser-8 and Ser-22 were shown to be phosphorylated and an O-linked glucuronic acid conjugation was identified. The latter modification was located to Ser-17 and found to be present in approximately 40% of the polypeptides.

Gln-Gly cleavage: correlation between collision-induced dissociation and biological degradation.

Tryptic digestion of the 150-residue human acidic salivary proline-rich protein 1 (PRP-1) generated eight peptides, two of which corresponded to the N-terminal 30-residue segment. In each of the other six tryptic peptides, a consensus repeat with the structure PQGPPQQGG was present. A facile Gln-Gly cleavage between the second and the third residues of the repeat was observed during collision-induced dissociation experiments. We postulate possible mechanisms to account for this reactivity, involving attack on the peptidyl carbonyl group by the Gln sidechain. Significantly, the Gln-Gly cleavage has been shown to be biologically important in the bacterial degradation of PRPs in saliva, generating bacteria-binding Pro-Gln C-termini. We suggest a link between the gas-phase chemistry and the biochemical degradation of these molecules.

Secretory immunoglobulin A heavy chain presents Galbeta1-3GalNAc binding structures for Actinomyces naeslundii genospecies 1.

Adherence of Actinomyces naeslundii ATCC 12104 to hydroxyapatite beads coated with protein fractions of parotid saliva, obtained by gel filtration on S-200 HR columns, showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to high-molecular-weight proteins (Strömberg et al., 1992). The present study investigates the nature of these high-molecular-weight binding proteins and determines their specific ability to mediate adherence to representative strains of Actinomyces species. Strain ATCC 12104 bound specifically in a lactose-inhibitable manner to the heavy chain of secretory immunoglobulin A (S-IgA), contained within a high-molecular-weight parotid protein fraction separated on SDS-PAGE and transferred to a solid membrane support. Lactose-inhibitable binding to the heavy chain of S-IgA from human colostrum was also demonstrated. Peanut agglutinin bound to the heavy chain of parotid and colostrum S-IgAs contained on solid support membranes, confirming the presence of Galbeta1-3GalNAc residues on these molecules. Both salivary and colostrum S-IgA aggregated with strain ATCC 12104 in a GalNAcbeta1-3Galalpha-O-ethyl-inhibitable fashion. Further separation of high-molecular-weight salivary proteins on S-500 HR columns showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to both mucin- and S-IgA-containing fractions. The presence of S-IgA in salivary pellicles formed in vivo on teeth was demonstrated by Western blot analysis of pellicle extracts with anti-IgA antibodies. Among strains representing A. naeslundii genospecies 1 and 2 and A. odontolyticus, only those of genospecies 1 with a particular adherence profile showed efficient GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to S-IgA. Thus, oligosaccharides on S-IgA may promote bacterial aggregation (or adherence) and provide a mechanism by which S-IgA can interact with bacteria without prior immunological challenge.

Agglutinin and acidic proline-rich protein receptor patterns may modulate bacterial adherence and colonization on tooth surfaces.

Bacterial binding to salivary proteins may in part account for individual differences in the colonization of tooth surfaces. High-molecular-weight glycoproteins, agglutinins, mediate S. mutans adherence, whereas acidic proline-rich proteins mediate adherence of other early-colonizing streptococci and Actinomyces. The aim of the present study was to examine the composition of adherence-related salivary proteins and dental plaque micro-organisms in three individuals with a low, moderate, and high capacity to mediate S. mutans adherence. The S. mutans (strain Ingbritt) binding activity resided with a 300-kDa agglutinin which was six-fold more prevalent in the high S. mutans binding saliva compared with the low one. Binding to all three salivas was completely blocked by a monoclonal anti-agglutinin antibody. The moderate S. mutans binding saliva was found to contain adherence-inhibiting components. Furthermore, the low and moderate S. mutans binding salivas mediated binding of A. naeslundii strain LY7 to a greater extent than the saliva with high S. mutans binding. The A. naeslundii binding activity resided with the acidic proline-rich proteins (APRPs) and paralleled the relative content of 106- and 150-residue APRPs. Low A. naeslundii binding coincided with an almost two-fold higher ratio of 106/150 APRPs compared with the high A. naeslundii binding saliva. During conventional gel filtration, a degradation of the acidic, basic, and glycosylated proline-rich proteins was evident in the saliva with high S. mutans and low A. naeslundii binding. This saliva donor had a comparably high rate of dental plaque formation, high counts of S. mutans, and low counts of other streptococci and Actinomyces.

Function of the rat salivary glands after exposure to inorganic mercury.

In spite of many studies on the toxicity of mercury, very little is known about the effects of mercury on the function of exocrine glands. In the present paper selected functions of Sprague-Dawley rat salivary glands were studied after the exposure of the animals to inorganic mercury at two different doses; 3.25 mg/kg body weight given during 25 days and 7.0 mg/kg body weight given during 27 days. The function of the salivary glands was estimated by saliva secretion rate, secretion of electrolytes, proteins and biosynthesis of glycoproteins. The function was compared between mercury exposed rats and age and sex matched control rats that were given injections with equal volumes of 0.154 mol/l NaCl on the same time schedule. In the present study we report that no significant effect on saliva secretion rate, concentrations of salivary constituents or biosynthesis of glycoproteins in the salivary glands could be observed in rats as a result of mercury exposure at two levels that gave 30 or 60 times higher serum mercury concentrations than in the majority of the Swedish population.

Interactions between peroxide and salivary glycoprotein: protection by peroxidase.

The effect of ascorbic acid oxidation and of peroxide on salivary glycoproteins has been studied. No signs of depolymerization of the glycoproteins were observed as a result of treatment of whole saliva or the isolated glycoprotein fraction from human saliva with an ascorbic acid, hydrogen peroxide and cupric ion combination. Sialic acid was analyzed with two methods which gave different chromogens of the acid. The conclusion could be drawn from the results that the effect of ascorbic acid oxidation was a decarboxylation of sialic acid in salivary glycoproteins. This also destroys the bacteria-agglutinating activity of saliva. Full protection of the agglutinin activity is given by salivary peroxidase. It is suggested that one role of peroxidases in saliva is to protect biologically active proteins from the action of oxygen or its radicals.

Peroxidase activity and thiocyanate accumulation in salivary glands.

Salivary glands with high, low, or no peroxidase activity do not differ in [S14CN-] after the i.v. injection of KS14CN, nor do the glands differ from blood and muscle in [S14CN-]. The content of SCN- in a salivary gland does not mirror the gland's participation in the peroxidase-mediated antimicrobial mechanism.

Quantitative characterization of the binding of histamine by heparin.

Tissue histamine is stored in mast cell granules, presumably as a histamine-heparin complex. Heparin is a polyelectrolyte, with a fraction of its anionic charge neutralized by condensed counterions. The interaction of heparin with histamine in aqueous solution was quantitatively characterized by 1H nuclear magnetic resonance (NMR) spectroscopy. Binding constants were determined from chemical shift-pH titration data for the C2H proton of the imidazolium ring for a wide range of histamine, heparin, and Na+ concentrations. The results indicate a binding stoichiometry of 1 histamine per heparin disaccharide repeat unit. The binding is electrostatic, as indicated by the strong dependence of the binding constant on Na+ concentration. From an analysis of the binding constants using the counterion condensation theory of polyelectrolytes, it was determined that the binding of H2A2+ results in displacement of 1.72 Na+ ions from the counterion condensation volume of heparin and that H2A2+ makes 2 ionic interactions with heparin. The displacement of Na+ from the counterion condensation volume of heparin by H2A2+ was also studied by 23Na NMR. From 23Na spin-lattice relaxation time data, it was determined directly that 1.78 Na+ ions are displaced per H2A2+ bound by heparin. The results are discussed in terms of the ion exchange process which takes place when histamine is released by mast cells.

Adhesion of Candida albicans, but not Candida krusei, to salivary statherin and mimicking host molecules.

The aim of the present study was to identify salivary molecules affecting adhesion of Candida albicans and Candida krusei to salivary pellicles and epithelial cells. Strains of C. albicans (GDH18, GDH3339, CA1957, ATCC 28366 and ATCC 10321), but not C. krusei (strains ATCC 14243 and Ck9), bound to saliva-coated hydroxyapatite and buccal epithelial cells. Parotid saliva fractions containing statherin, glycosylated proline-rich proteins (PRP) and as yet unidentified components mediated adhesion of strain GDH18; Fuc alpha 1-2Gal beta 1-4Glc partly inhibited the adhesion to those fractions not containing statherin. Pure statherin, but not PRP-1, mediated dose-dependent adhesion of C. albicans strain GDH18 to hydroxyapatite beads. Candida isolates (GDH18, GDH3339 and CA1957) bound somewhat more avidly to statherin/saliva relative to ATCC strains 28366 and 10321, while the opposite was true for adhesion to buccal epithelial cells. Adhesion of C. albicans strain GDH18 to saliva-coated hydroxyapatite and buccal epithelial cells was completely (93%) and partly (43%) blocked by statherin-specific immunoglobulin G (IgG) antibodies, respectively. Control IgG antibodies did not block Candida adhesion. Blockage of Candida adhesion to epithelial cells also occurred with Fuc alpha 1-2Gal beta 1-4Glc (49%) and N-acetylglucosamine (38%), while statherin specific IgG antibodies in combination with Fuc alpha 1-2Gal beta 1-4Glc almost completely eliminated Candida adhesion (79%). In addition, statherin in solution blocked the adhesion of strain GDH18 to epithelial cells by inducing aggregation of Candida cells.

Analysis of the interaction of water with the manganese cluster of photosystem II using isotopically labeled water.

The association of water with the Mn of the water oxidizing complex was investigated using H2(17)O- and 2H2O-reconstituted lyophilized photosystem II particles. The pulsed electron paramagnetic resonance (EPR) technique of electron spin echo envelope modulation (ESEEM) was used to investigate the interaction of the magnetic 2H and 17O nuclei with the paramagnetic S2 state of the Mn complex and other photosystem II components. ESEEM offers a much more specific and sensitive detection of this type of interaction than continuous wave (CW) EPR. Unlike earlier reports using CW EPR, these experiments did not detect any interaction of water with the multiline EPR signal from the S2 state of the Mn complex. No signals indicating specific interaction of either H or O with the multiline signal were detected. Signals due to 2H and 17O were detected only at the Larmour frequency, indicating nonspecific "distant ENDOR" effects. A weak interaction with 17O was detected both in S1, when the Mn is EPR silent, and in S2, but only on the high-field side of g = 2. This interaction may be with the Rieske iron-sulfur center in the cytochrome b6f complex. The results were the same whether the multiline signal was generated by 200 K illumination of dark-frozen samples, or by room temperature illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Illumination at room temperature in the presence of an electron acceptor to allow multiple turnovers of the system with cycling of the S states did not result in the appearance of any new interactions. These results appear to exclude close (less than 6 A) binding of water to the Mn center giving rise to the multiline signal, and also to exclude mechanisms in which water oxidation involves the breaking and re-formation of the mu-oxo bridges of the Mn complex. They cannot, however, exclude models in which water binding to the manganese complex and direct oxidation by the manganese complex occur in the higher S states, or are catalyzed by one bis(mu-oxo) Mn dimer while oxidizing equivalents are accumulated in the S2 state by a second bis(mu-oxo) Mn dimer.

DNA-synthesizing cells in the blood in coeliac disease and inflammatory bowel disease.

The level of 3H-labelled thymidine ([3H]TdR) incorporation of blood cells of patients with coeliac disease was shown in two separate studies to be significantly lower than that of a normal control group. In the first study the 'background' DNA synthesis in unstimulated cultures containing a standard number of blood lymphocytes was measured on days 4, 5 and 6. In the second study a standard volume of freshly drawn whole blood was added to culture medium and the [3H]TdR incorporation measured over the first 24 hr and from 48 to 72 hr. In all cases the [3H]TdR incorporation of cells of coeliac patients on a normal or a gluten-free diet was lower than that of the control group. It is suggested that sequestration of DNA-synthesizing cells in the mucosa of the small bowel may partly explain these results. In whole-blood cultures from patients with inflammatory bowel disease in remission [3H]TdR incorporation over the first 24 hr was raised in Crohn's disease but normal in ulcerative colitis.

Isolation and characterization of bovine gingival proteoglycans versican and decorin.

1. We have isolated, chemically and immunologically characterized versican and decorin from bovine gingiva. 2. Versican was of large molecular weight and the molecular size of the core protein was estimated to be greater than 200 kDa. 3. The glycosaminoglycan chains were susceptible to chondroitinase ABC and N-linked oligosaccharides were present on the protein core of the molecule. 4. Immunological studies provided evidence that a hyaluronic acid binding region was present in the core protein of versican. 5. The overall structure was similar to that of versican isolated from bovine sclera. 6. Decorin had a molecular weight of 102 kDa and its glycosaminoglycan chain was completely digested by specific glycosidases. 7. The partially deglycosylated core protein had a molecular weight of 55 kDa and N-linked oligosaccharides were present on the molecule.

Possible release of an ArgGlyArgProGln pentapeptide with innate immunity properties from acidic proline-rich proteins by proteolytic activity in commensal streptococcus and actinomyces species.

This study suggests degradation of salivary acidic proline-rich proteins (PRPs) into potential innate-immunity-like peptides by oral Streptococcus and Actinomyces species. PRP degradation paralleled cleavage of Pro-containing substrates. PRP degradation by S. gordonii strain SK12 instantly released a Pyr(1)-Pro(104)Pro(105) and a Gly(111)-Pro(149)Gln(150) peptide together with a presumed Arg(106)Gly(107)Arg(108)Pro(109)Gln(110) pentapeptide. The synthetic Arg(106)Gly(107)Arg(108)Pro(109)Gln(110) peptide desorbed bound bacteria and counteracted sucrose-induced decrease of dental plaque pH in vitro.

Influence of diuretics on urinary general base catalytic activity and cyclophosphamide-induced bladder toxicity.

The influence of diuretics on the induction of bladder toxicity by cyclophosphamide was investigated in rats. Following ip administration, about 3.5% of the cyclophosphamide was excreted as 4-hydroxycyclophosphamide. This amount was found to be compatible with the view that the urotoxic effects of cyclophosphamide are caused by the acrolein generated in the urine from 4-hydroxycyclophosphamide, the primary metabolite of cyclophosphamide. In situ, acrolein was more potent than 4-hydroperoxycyclophosphamide with regard to producing an increase in bladder weight; phosphoramide mustard was essentially without urotoxic activity. The urotoxic potency of 4-hydroperoxycyclophosphamide, but not that of acrolein, increased as the pH and/or the phosphate concentration of the infusion medium increased. This was as expected in view of the knowledge that release of acrolein from 4-hydroxycyclophosphamide or 4-hydroperoxycyclophosphamide is facilitated by the presence of general base catalysts, eg, phosphate and bicarbonate, and that the rate at which this reaction proceeds in the presence of these catalysts increases as their concentration and the pH increases. In vivo, diuretics that acidified the urine, eg, ammonium chloride and furosemide, prevented the increase in bladder weight ordinarily elicited by the dose of cyclophosphamide used in these experiments. In contrast, a diuretic, acetazolamide, that markedly increased urinary bicarbonate concentration and alkalinized the urine, did not. None of the diuretics altered the systemic metabolism and urinary excretion of cyclophosphamide nor did they alter the systemic action, as judged by spleen weight, of cyclophosphamide. These observations demonstrate that the pH of the urine and the urinary concentration of general base catalysts greatly influence the urotoxic potential of oxazaphosphorines such as cyclophosphamide. They indicate that while the use of acidifying diuretics is likely to be beneficial in minimizing oxazaphosphorine-induced bladder toxicity, the use of alkalinizing diuretics may not be helpful.

Probing the active site of human manganese superoxide dismutase: the role of glutamine 143.

Structural and biochemical characterization of the nonliganding residue glutamine 143 near the manganese of human Mn superoxide dismutase (hMnSOD), a homotetramer of 22 kDa, reveals a functional role for this residue. In the wild-type protein, the side-chain amide group of Gln 143 is about 5 A from the metal and is hydrogen-bonded to Tyr 34, which is a second prominent side chain adjacent to the metal. We have prepared the site-specific mutant of hMnSOD with the conservative replacement of Gln 143 --> Asn (Q143N). The crystal structure of Q143N shows that the side-chain amide nitrogen of residue 143 is 1.7 A more distant from the manganese than in the wild-type enzyme. The Tyr 34 side-chain hydroxyl in Q143N is also moved to become 0.6 A more distant from the metal due to an additional water molecule. Differential scanning calorimetry showed that Q143N is slightly more stable than the wild-type enzyme with Tm for the main unfolding transition increased by 2 degrees C to 90.7 degrees C. Pulse radiolysis and stopped-flow spectrophotometry reveal that unlike wild-type hMnSOD, which is strongly inhibited by peroxide, Q143N MnSOD exhibits no product inhibition even at concentrations of O2. - in the millimolar range, and its catalysis follows Michaelis kinetics with no evidence of cooperativity. However, the overall catalytic activity of this mutant was decreased 2-3 orders of magnitude compared with the wild-type MnSOD, which can account for its lack of product inhibition. Q143N MnSOD lacked the visible absorption spectrum typical of wild-type Mn(III)SOD. Also, unlike the wild-type Mn(III)SOD, which is electron paramagnetic resonance (EPR) silent, Q143N MnSOD has a complex EPR spectrum with many resonances in the region below 2250 G. We conclude that the Gln 143 --> Asn mutation has increased the reduction potential of manganese to stabilize Mn(II), indicating that Gln 143 has a substantial role in maintaining a reduction potential favorable for the oxidation and reduction cycles in the catalytic disproportionation of superoxide. A solvent hydrogen isotope effect near 2 for kcat in catalysis by Q143N hMnSOD indicates rate-contributing proton transfers to form product hydroperoxide anion or hydrogen peroxide. The data demonstrate a prominent role for Gln 143 in maintaining the microenvironment of the manganese and in efficient catalysis of superoxide dismutation to oxygen and hydrogen peroxide.