Structural characterization of the isoenzymatic forms of human myeloperoxidase.

Myeloperoxidase from human neutrophils was isolated by ion-exchange and gel-filtration chromatography and shown by SDS-polyacrylamide gel electrophoresis to be comprised of alpha and beta subunits with apparent Mr values of 58,000 and 15,000, respectively. The apparent Mr of the native protein was 130,000-140,000, indicating that the holoenzyme has the quaternary structure alpha 2 beta 2. Automated Edman degradation of the separated alpha and beta subunits showed that the amino-terminal sequences were different from one another and demonstrated no sequence microheterogeneity. Comparison of these sequences with those in the National Biomedical Research Foundation data bank of protein sequences revealed that the subunits of human myeloperoxidase were not homologous to any known protein. Myeloperoxidase purified from HL-60 cells grown in culture demonstrated the same alpha 2 beta 2 subunit structure. Three isoenzymes of myeloperoxidase, prepared by gradient elution from a CM-Sepharose column, underwent quantitative analysis. No structural basis for the different elution pattern of the myeloperoxidase isoenzymes was discerned by amino-acid analysis, N-terminal sequence, polyacrylamide gel electrophoresis, or digestion with neuraminidase or enzymes known to cleave N-linked heterosaccharides. The structural basis for the myeloperoxidase isoenzymes of human neutrophils, each possessing equivalent activity, is not apparent from these studies.

The amino acid sequence of human beta-microseminoprotein.

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.

The Amount of MUC5B mucin in cervical mucus peaks at midcycle.

The physical character and amount of mucus secreted by the endocervix changes dramatically during the menstrual cycle to facilitate sperm migration at the time of midcycle ovulation. Mucins are highly glycosylated, high-molecular-weight proteins, which are the major structural components of the protective mucus gel covering all wet-surfaced epithelia, including that of the endocervix. We have previously demonstrated that the endocervical epithelium expresses messenger RNA (mRNA) of three of the large gel-forming mucins, designated MUC5AC, MUC5B, and MUC6, with mRNA of MUC5B predominating. Because mucin protein levels may be regulated posttranscriptionally, measurement of MUC5B protein levels with cycle are needed for correlation to mRNA levels. Measurement of specific mucin gene products within mucus secretions has been limited by availability of specific, well-characterized antibodies and by volume requirements of the isolation protocols for mucins, which include CsCl density centrifugation and fraction isolation. To measure MUC5B protein within the cervical mucus through the hormone cycle, we developed a polyclonal antibody specific to the mucin. The antibody, designated no. 799, is to a synthetic peptide mimicking a 19-amino-acid segment of an intercysteine-rich region within the D4 domain in the 3' region of the MUC5B protein. It recognizes native as well as denatured MUC5B on immunoblot, is preadsorbable with its peptide, and binds to apical secretory vesicles of epithelia expressing MUC5B. We used the MUC5B antibody along with a cervical mucin standard cervical mucin isolate in enzyme-linked immunosorbent assay to determine the relative amount of MUC5B mucin in samples of human cervical mucus taken through the menstrual cycle. We demonstrate a peak of MUC5B mucin in human cervical mucus collected at midcycle, compared with mucus from early or late in the cycle. This peak in MUC5B content coincides with the change in mucus character that occurs at midcycle, suggesting that this large mucin species may be important to sperm transit to the uterus.

Human serum alpha 2HS-glycoprotein modulates in vitro bone resorption.

We previously isolated a family of bone-resorbing proteins from human cancer ascites fluid and established that the three purified bone-resorbing proteins were chemically and immunochemically related to each other and to alpha-2HS glycoprotein (alpha 2HS). After this finding we purified the normal human serum counterpart of these ascites proteins and studied its effects on bone resorption. The bone-resorbing properties of normal human serum alpha 2HS were examined in vitro over a wide dose range. This normal human serum glycoprotein had a biphasic effect on 45Ca2+ release from bone. More specifically, this protein stimulated bone resorption at the lower concentrations tested, with a maximum effect [treated over control ratio of 2.5 +/- 0.30 (+/- SE); P less than 0.01] at 40 micrograms/mL. In contrast, at doses above 40 micrograms/mL, a sharp decline in calcium mobilization occurred, with a return to baseline occurring above 80 micrograms/mL. These results suggest that serum alpha 2HS may participate in the regulation of bone metabolism in vivo.

Molecular mapping of statherin- and histatin-binding domains in human salivary mucin MG1 (MUC5B) by the yeast two-hybrid system.

MGI is a high-molecular-weight mucin secreted by mucous acinar cells in human submandibular and sublingual glands. We have recently shown that the tracheobronchial mucin MUC5B is a major component of MG1. MUC5B is organized into cysteine-rich N- and C-terminal regions that flank a central tandem-repeat region containing cysteine-rich subdomains and imperfect 29-residue tandem repeats. In earlier work, we have shown that this mucin selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins in salivary secretions, and the aim of this study was to identify specific binding domains within MUC5B using the yeast two-hybrid system. Interactions of cysteine-rich domains in the tandem-repeat region (Cys1-Cys4) and C-terminal region (Cys8a, Cys8b, Cys8c) of MUC5B with statherin and histatins were investigated. These studies indicated that histatin 1 selectively bound to Cysl and Cys2, whereas statherin and histatin 1, 3, and 5 selectively bound to Cys8a. Analysis of the primary sequences of the identified binding domains suggests that these domains most probably can fold into globular-like structures in the native mucin. A ProDom blast search revealed that sequences in Cys1, Cys2, and Cys8a exhibit similarity to domains in evolutionarily diverse extracellular proteins known to participate in a wide variety of protein-protein interactions.

Pro-inflammatory cytokines up-regulate MUC1 gene expression in oral epithelial cells.

The membrane-bound mucin MUC1 is expressed ubiquitously on epithelial surfaces and is thought to provide protection from bacterial and chemical injury. The present study was undertaken to determine whether MUC1 was expressed in cultured oral epithelial cells and whether expression is modulated by pro-inflammatory mediators released as part of the host response to infection by oral pathogens. Northern and Western blotting experiments showed that KB cells express MUC1 mRNA and protein. When cells were treated with interleukins (IL-1beta, IL-6), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma), or combinations of these, real-time PCR demonstrated that MUC1 mRNA increased 1.4- to 3.2-fold. Interestingly, a significant increase in levels of MUC1 protein was also observed. While no effect was observed when KB cells were incubated with LPS from Porphyromonas gingivalis, infection of KB monolayers with this oral pathogen caused a 2.85-fold increase in MUC1 transcript levels. These results suggest that increased MUC1 synthesis may be a key element in the host response to infection with oral pathogens.

Molecular basis of salivary proline-rich protein and peptide synthesis: cell-free translations and processing of human and macaque statherin mRNAs and partial amino acid sequence of their signal peptides.

Acidic proline-rich phosphoproteins and phosphopeptides are abundant components of parotid and submandibular salivary secretions in man and in the subhuman primate, Macaca fascicularis. The major acidic proline-rich proteins and the proline-rich phosphopeptide, statherin, of man and macaques have been shown to be potent inhibitors of calcium phosphate precipitation and are thought to function in the oral environment by maintaining saliva supersaturated with respect to calcium phosphate salts. Little is known about the biosynthesis of these proline-rich phosphoproteins and peptides, and the aim of the present work was to determine the structural relationship between statherin precursors and native human and macaque statherin. RNA was isolated from human submandibular gland, and poly(A+) mRNA was selected by affinity chromatography on oligo(dT) cellulose and translated in a reticulocyte lysate. Electrophoretic analysis of the translation products revealed that this mRNA directed the synthesis of a large number of polypeptides with Mrs ranging from 5000 to 70,000. Immunoprecipitates, prepared with an antiserum directed against human statherin, contained a single component with a Mr of 7800, approximately 2000 daltons larger than native statherin. Radiosequencing of the in vitro precursor of statherin in immunoprecipitates demonstrated the presence of a 19-residue signal peptide. These results suggest that statherin is derived from a unique structural gene, and does not result from proteolytic processing of a large polyprotein precursor.

Structural relationship between human salivary histatins.

Histatins are a group of electrophoretically distinct histidine-rich polypeptides with microbicidal activity found in human parotid and submandibular gland secretions. Recently, we have shown that histatins 1, 3, and 5 are homologous proteins that consist of 38, 32, and 24 amino acid residues, respectively, and that these polypeptides kill the pathogenic yeast, Candida albicans. We now describe the isolation and structural characterization of histatins 2, 4, 6, and 7-12, the remaining members of this group of polypeptides. Histatin 2 was found to be identical to the carboxyl terminal 26 residues of histatin 1; histatin 4 was found to be identical to the carboxyl terminal 20 residues of histatin 3; and histatin 6 was found to be identical to histatin 5, but contained an additional carboxyl terminal arginine residue. The amino acid sequences of histatins 7-12 formally correspond to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively, of histatin 3, but could also arise proteolytically from histatin 5 or 6. These results establish, for the first time, the complete structural relationships between all members of this group of microbicidal proteins in human parotid saliva. The relationship of histatins to one another is discussed in the context of their genetic origin, biosynthesis and secretion into the oral cavity, and potential as reagents in anti-candidal studies.

Immunoquantification of human salivary mucins MG1 and MG2 in stimulated whole saliva: factors influencing mucin levels.

While more and more is known about the structure and function of human salivary mucins, there is relatively little information on quantification of these glycoconjugates in whole saliva and on factors influencing their secretion. The goal of the present work was to develop capture ELISAs that would allow for rapid, inexpensive, and reliable measurement of the salivary mucins MG1 and MG2, and to use these immunological procedures to investigate the significance of age, gender, flow rate, and protein concentration on mucin levels in whole saliva. Previously, we described a rabbit polyclonal antibody against MG1 (Troxler et al., 1995) and a rabbit polyclonal peptide antibody against an epitope in the N-terminal region of MG2 (Liu et al., 1999) which were used to develop the capture ELISAs. We verified the accuracy and specificity of these assays by showing correct measurement of known quantities of purified MG1 or MG2 added to whole saliva and lack of cross-reactivity between mucins and heterologous antisera on Western blots or in ELISAs. Whole saliva was collected from 60 subjects under conditions of masticatory stimulation, flow rates were recorded, and mucin concentrations were determined. The results showed that the mean concentration of MG1 and MG2 was 23.3 +/- 14.6 mg% and 13.3 +/- 11.6 mg%, respectively, and that mucins constitute approximately 16% of the total protein in whole saliva. No significant correlations were found between mucin levels and age or flow rate; however, a significant correlation was found between MG2 levels and total protein concentration. Furthermore, there were statistically significant gender differences in flow rate and MG1 levels, but not in MG2 levels. The availability of these immunoassays for quantification of MG1 and MG2 will help to elucidate the role of mucin in oral health and disease.

MG2 and lactoferrin form a heterotypic complex in salivary secretions.

Protein-protein interactions are necessary for homeostasis to be maintained and for biological systems to be integrated. Heterotypic complexes occur in saliva, and a complex between MG2 and SIgA has been suggested to promote microbial clearance from the oral cavity. In this study, we used a peptide display library to investigate previously unrecognized heterotypic complexes involving MG2 and other proteins. The library was panned with MG2 12 times, and analyses of clones identified the sequence Ala-Leu-Leu-Cys-, which occurs in salivary lactoferrin. Blotting experiments confirmed that MG2 and lactoferrin form a heterotypic complex in vitro and in vivo. Periodate treatment of MG2 did not affect the interaction. A synthetic lactoferrin peptide containing the motif Ala-Leu-Leu-Cys-blocked the interaction between MG2 and lactoferrin, confirming the specificity of the interaction identified by panning. This complex may enhance the properties of these salivary components in the oral environment.

The effects of duration and intensity of stimulation on total protein and mucin concentrations in resting and stimulated whole saliva.

The present investigation has characterized the influence of the duration and intensity of stimulation on the secretion pattern of total protein and salivary mucins MG1 and MG2 in whole saliva. Resting and stimulated whole saliva was collected from six healthy subjects on 2 consecutive days. Whole saliva was collected for 2 five-minute intervals under resting conditions followed by collection under masticatory stimulation induced by the chewing of parafilm (1 g) at 10 or 60 strokes/min for 15 min. Flow rates were different under the 2 levels of stimulation. The concentration of total protein was different in resting and stimulated whole saliva but was not affected by the duration or intensity of stimulation. Analysis of mucin concentrations determined by capture ELISAs revealed that the pattern of MG1 secretion was similar to that of total protein. The pattern of MG2 secretion was unique in that no differences were observed in the concentration of this mucin under resting and stimulated conditions. This study shows that the pattern of protein secretion in whole saliva does not reflect the combined pattern observed for protein secretion in parotid and submandibular/sublingual glands, and that the secretion patterns of MG1 and MG2 in whole saliva are quite different from one another.

Molecular cloning of human submandibular histatins.

Histatins are a group of histidine-rich polypeptides found in human parotid and submandibular gland secretions. These polypeptides are microbiocidal, possibly involved in maintaining the acquired enamel pellicle, and enhance the glycolytic activity of certain oral micro-organisms. Histatins 1, 3 and 5 are homologous proteins with 38, 32 and 24 amino acid residues, respectively; the cDNAs coding for histatins 1 and 3 have now been isolated and sequenced. The cDNA sequences were highly homologous but contained differences throughout their length, indicating that they arise from different genes that may be derived from a common ancestral gene. Northern blots were hybridized to a series of oligonucleotide probes, designed on the basis of histatin cDNA sequences, and these positively identified mRNAs for histatins 1 and 3. In addition, there was a third mRNA, which hybridized to several histatin oligonucleotide probes, suggesting that histatin 5 might be derived from a distinct mRNA and not by proteolytic processing of histatin 3. A Northern blot of macaque parotid gland total RNA also showed three histatin mRNAs, indicating that similar histatins exist in a non-human primate.

Heterogeneity of high-molecular-weight human salivary mucins.

The existence of high-molecular-weight glycoproteins in saliva and salivary secretions has been recognized for nearly 30 years. These proteins, called mucins, are essential for oral health and perform many diverse functions in the oral cavity. Mucins have been intensively studied, and much has been learned about their biochemical properties and their interactions with oral micro-organisms and other salivary proteins. In the past several years, the major high-molecular-weight mucin in salivary secretions has been identified as MUC5B, one of a family of 11 human mucin gene products expressed in tissue-specific patterns in the gastrointestinal, respiratory, and reproductive tracts. MUC5B is one of four gel-forming mucins which exist as multimeric proteins with molecular weights greater than 20-40 million daltons. The heavily glycosylated mucin multimers form viscous layers which protect underlying epithelial surfaces from microbial, mechanical, and chemical assault. Another class of mucin molecules, the membrane-bound mucins, is structurally and functionally distinct from the gel-forming mucins. These proteins do not form multimers and can exist as both secreted and membrane-bound forms, with the latter anchored to epithelial cell membranes through a short membrane-spanning domain. In the present work, we show that two of the membrane-bound mucins, MUC1 and MUC4, are expressed in all major human salivary glands as well as in buccal epithelial cells. While the functions of these mucins in the oral environment are not understood, it is possible that they form a structural framework on the cell surface which not only is cytoprotective, but also may serve as a scaffold upon which MUC5B, and possibly other salivary proteins, assemble.

Primary structure of allophycocyanin from the unicellular rhodophyte, Cyanidium caldarium. The complete amino acid sequences of the alpha and beta subunits.

The complete amino acid sequences of the alpha and beta subunits of allophycocyanin from the unicellular rhodophyte, Cyanidium caldarium, were determined by automated Edman degradation of the proteins and peptides derived from them by chemical and enzymatic cleavages. The sequence of the alpha subunit was determined from the sequences of tryptic, endoproteinase lysine-C, and cyanogen bromide peptides and carboxypeptidase A and Y digestion of the protein. The sequence of the beta subunit was determined from the sequences of tryptic, endoproteinase lysine-C, Staphylococcus aureus V8 protease, and cyanogen bromide peptides and in addition, a peptide derived from acid cleavage of an aspartyl-prolyl bond. The carboxyl-terminal sequence of the protein was determined by digestion with carboxypeptidase A. The alpha subunit contains 160 amino acids, one phycocyanobilin chromophore attached at residue 80 by a cysteinyl-thioether linkage, and the Mr calculated from the sequence is 18,160. The beta subunit contains 161 amino acids, one phycocyanobilin chromophore attached at residue 81 by a cysteinyl-thioether linkage, and the Mr calculated from the sequence is 18,125. The amino acid sequences of the alpha and beta subunits of allophycocyanin from C. caldarium are the first complete amino acid sequences of an allophycocyanin from a eukaryotic red alga. A matrix comparison of the alpha and beta subunits of C. caldarium allophycocyanin and phycocyanin (Offner, G.D., Brown-Mason, A.S., Ehrhardt, M. M., and Troxler, R. F. (1981) J. Biol. Chem. 256, 12167-12175; Troxler, R. F., Ehrhardt, M. M., Brown-Mason, A. S., and Offner, G. D. (1981) J. Biol. Chem. 256, 12176-12184) shows homology ranging from 26 to 39%. Comparison of the sequences of alpha and beta subunits of C. caldarium allophycocyanin with the sequences of the corresponding subunits of allophycocyanin from two prokaryotic cyanobacteria (Sidler, W., Gysi, J., Isker, E., and Zuber, H. (1981) Hoppe-Seyler's Z. Physiol. Chem. 362, 611-628; DeLange, R. J., Williams, L. C. and Glazer, A. N. (1981) J. Biol. Chem. 256, 9558-9566) shows homology ranging from 81 to 85%. The significance of this with respect to phycobiliprotein structure and function is discussed.

Characterization of a fatty acid-binding protein from rat heart.

A fatty acid-binding protein has been isolated from rat heart and purified by gel filtration chromatography on Sephadex G-75 and anion-exchange chromatography on DE52. The circular dichroic spectrum of this protein was not affected by protein concentration, suggesting that it does not aggregate into multimers. Computer analyses of the circular dichroic spectrum predicted that rat heart fatty acid-binding protein contains approximately 22% alpha-helix, 45% beta-form and 33% unordered structure. Immunological studies showed that the fatty acid-binding proteins from rat heart and rat liver are immunochemically unrelated. The amino acid composition and partial amino acid sequence of the heart protein indicated that it is structurally related to, but distinct from, other fatty acid-binding proteins from liver, intestine, and 3T3 adipocytes. Using a binding assay which measures the transfer of fatty acids between donor liposomes and protein (Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984) J. Biol. Chem. 259, 13395-13401), it was shown that both rat heart and liver fatty acid-binding proteins bind 2 mol of oleic acid or palmitic acid/mol of protein. The structural and functional relationship of rat heart fatty acid-binding protein to fatty acid-binding proteins from other tissues is discussed.

Heme regulates expression of phycobiliprotein photogenes in the unicellular rhodophyte, Cyanidium caldarium.

Allophycocyanin and phycocyanin in the red alga (Cyanidium caldarium) are chloroplast-encoded, light-harvesting accessory pigments composed of alpha and beta subunit polypeptides (17-19 kDa) to which 1 or more residues of the heme-derived bile pigment chromophore phycocyanobilin are attached by cysteinyl thioether linkages (Offner, G.D., and Troxler, R.F. (1983) J. Biol. Chem. 258, 9931-9940). Western blot experiments utilizing phycobiliprotein antisera revealed that immunoreactive allophycocyanin and phycocyanin apoproteins were absent in cells grown in the dark and present in cells exposed to light. Northern blot experiments using genomic DNA hybridization probes indicated that phycobiliprotein mRNAs were absent in the dark, whereas cells exposed to light contained two allophycocyanin mRNA transcripts, 1.4 and 1.6 kilobases in length, and one phycocyanin mRNA transcript, 3.0 kilobases in length, providing evidence that phycobiliproteins are encoded in photogenes which are only transcriptionally active in the light. Northern and Western analyses demonstrated that cells incubated in the dark with the heme precursor delta-aminolevulinate contained allophycocyanin and phycocyanin mRNAs and apoproteins, indistinguishable in size, number, and quantity from those made in the light. Cells incubated in the dark with delta-aminolevulinate, protoporphyrin IX, or heme, but not biliverdin or phycocyanobilin, synthesized allophycocyanin and phycocyanin alpha and beta apoproteins, suggesting a role for heme in the control phycobiliprotein gene expression. Cells incubated with heme in the dark produced allophycocyanin and phycocyanin mRNA transcripts, but did not produce mRNAs for four other photogenes coding for a P-700 reaction center protein, a 32-kDa herbicide-binding protein, and the large and small subunits of ribulose-bisphosphate carboxylase. These results show, for the first time, that heme is a regulatory factor specifically involved in transcriptional regulation of chloroplast genes for phycobiliproteins.

Phycobiliprotein synthesis in the unicellular rhodophyte, Cyanidium caldarium. Cell-free translation of the mRNAs for the alpha and beta subunit polypeptides of phycocyanin.

Phycobiliproteins are a class of abundant light-harvesting proteins assembled in complex multimeric aggregates located on thylakoid membranes of red algae and cyanobacteria. This study was undertaken to investigate the molecular basis of phycobiliprotein synthesis in the unicellular red alga, Cyanidium caldarium. RNA was isolated and separated into poly(A)-enriched and poly(A)-deficient fractions by oligo(dT)-cellulose chromatography. Both fractions stimulated incorporation of radiolabeled amino acids into protein in a reticulocyte lysate translation system. The alpha and beta subunit polypeptides of phycocyanin were among the products of poly(A)-deficient (but not poly(A)-enriched) RNA directed translation reactions on the basis of Mr and immunological cross-reactivity with immune serum prepared against native phycocyanin. After chromatography of post-oligo(dT)-cellulose poly(A)-deficient RNA on poly(U) agarose, the messenger RNAs for the alpha and beta subunit polypeptides of phycocyanin were again recovered in the poly(A)-deficient fraction, confirming that these messenger RNAs did not appear to be polyadenylated. Automated radiosequencing of in vitro synthesized alpha and beta subunit polypeptides of phycocyanin labeled with either [35S]methionine, [3H]isoleucine, or [3H]phenylalanine revealed partial amino acid sequences which were the same as the NH2-terminal sequences of native phycocyanin subunits. This demonstrates that a "transit peptide", such as that found in several proteins made in the cytosol and transported into chloroplasts, is not present on the subunits of phycocyanin.

Identification of a 130-kilodalton human biliary concanavalin A binding protein as aminopeptidase N.

BACKGROUND/AIMS: Human gallbladder bile contains a group of nonmucin glycoproteins that binds to the lectin concanavalin A (con A) and has been reported to promote cholesterol monohydrate crystal nucleation, an event preceding the formation of gallstones. Several of these proteins, including a 130-kilodalton protein, have been isolated and shown to promote nucleation in vitro. The aim of this study was to identify this and other major biliary con A binding glycoproteins. METHODS: Gallbladder bile was chromatographed on con A agarose, and the eluted proteins were electrophoresed, blotted, and subjected to amino-terminal sequence analysis. RESULTS: The major con A binding proteins were identified as aminopeptidase N (a 130-kilodalton protein), alpha 2 macroglobulin, hemopexin, immunoglobulin heavy chains, and the beta chain of haptoglobin. After further purification, aminopeptidase N was found to be enzymatically active and to promote cholesterol crystallization at its approximate physiological concentration in bile. CONCLUSIONS: It is likely that aminopeptidase N is the previously characterized 130-kilodalton biliary crystallization promoting protein. Aminopeptidase N is probably released from the biliary canalicular membrane by the detergent activity of bile salts and may be one factor that promotes cholesterol crystallization in the gallbladder.

Phosphoproteins in the parotid saliva from the subhuman primate Macaca fascicularis. Isolation and characterization of a proline-rich phosphoglycoprotein and the complete covalent structure of a proline-rich phosphopeptide.

Parotid saliva from the cynomolgus monkey (Macaca fascicularis) and from pooled human collections displayed the same groups of proteins when fractionated by anion exchange and gel filtration chromatography. We have isolated and characterized a proline-rich phosphoglycoprotein (MPRP) and a proline-rich phosphopeptide (M-statherin) from macaque parotid saliva. MPRP has an apparent molecular weight of 16,900 and displays an unusual chemical composition. It is enriched in proline, glycine, and acidic amino acids, but lacks cysteine, methionine, and tyrosine. MPRP contains 25% (w/w) carbohydrate with 7.0 mol of neutral hexoses, 5.3 mol of galactosamine, 5.9 mol of sialic acid, and 3 mol of phosphorus/mol of protein. M-statherin is a 42-residue phosphopeptide with a high proline, glutamic acid, and tyrosine content, but which lacks threonine, valine, cysteine, methionine, isoleucine, and histidine. The complete covalent structure of M-statherin (Mr = 5,368) is: NH2-Asp-Pse-Pse-Glu-Glu-Lys-Phe-Leu-Arg-Arg-Leu-Arg-Arg-Phe-Asp-Glu-Gly-Arg-Tyr- -Gly-Pro-Tyr-Gln-Pro-Phe-Ala-Pro-Gln-Pro-Leu-Tyr-Pro-Gln-Pro-Tyr-Gln-Pro-Tyr-Gln-Pro-Gln-Tyr-COOH This is the first complete amino acid sequence of a component in the salivary secretion of a subhuman primate. Phosphoserine occurs at residues 2 and 3. All 13 acidic and basic amino acids are located in the NH2-terminal half of the molecule. The carboxyl-terminal half of the molecule is hydrophobic where the tripeptide Tyr-Gln-Pro is repeated three times, the dipeptide Gln-Pro occurs twice, and the tripeptides Tyr-Gly-Pro, Phe-Ala-Pro, and Leu-Tyr-Pro occur once. Evaluation of secondary structure by the Chou-Fasman method predicts an alpha helix in the NH2-terminal half (residue 4-16) and a beta pleated sheet in the carboxyl-terminal half (residues 22-26; 38-42) of the molecule. Both MPRP and M-statherin inhibit spontaneous and seeded precipitation from solutions supersaturated with respect to calcium phosphate salts. This suggests that these macaque compounds may function by maintaining saliva supersaturated with respect to calcium phosphate salts, a necessary requirement for stabilization of hydroxyapatite in the surface layers of teeth.U