Molakala S. Reddy

Structural Aspects of Salivary Glycoproteins

The protective functions of saliva are attributed, in part, to its serous and mucous glycoproteins. We have studied, as representative molecules, the proline-rich glycoprotein (PRG) from human parotid saliva and the high (MGI ) and low (MG2) molecular weight mucins from submandibular-sublingual saliva. PRG (38.9 kDa) contains 40% carbohydrate consisting of 6 triantennary N-linked units and a single peptide chain of 231 amino acids, 75% of which = PRO+GLY+GLN. PRGs secondary structure is comprised of 70% random coil (naked regions) and 30% β-turns (glycosylated domains). MGI (>103 kDa) contains 15% protein (several disulfide linked subunits), 78% carbohydrate (290 units of 4-16 residues), 7% sulfate, and small amounts of covalently linked fatty acids. MG2 (200-250 kDa) contains 30% protein (single peptide chain), 68% carbohydrate (170 units of 2-7 residues), and 2% sulfate. The major carbohydrate units of MG2 are: NeuAcα2, 3Galβ1, 3GalNAc, Galβ1, 3GalNAc, and Fucα1, 2Galβ1, 3GalNAc. MG1 contains hydrophobic domains, as evidenced by its ability to bind fluorescent hydrophobic probes; MG2 does not. Collectively, the biochemical and biophysical comparisons between MGI and MG2 indicate that these two mucins are structurally different. Several functional properties of MG1, MG2, and PRG have been examined, including their presence in two-hour in vivo enamel pellicle, binding to synthetic hydroxyapatite, lubricating properties, and interactions with oral streptococci. The data presented suggest that these glycoproteins may have multiple functions which are predicated, in part, on their carbohydrate units. The potential significance of the structure-function relationships of these glycoproteins to the oral ecology is discussed.

Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva

A high-molecular-weight mucin-glycoprotein (MG1) was isolated from human submandibular-sublingual saliva and was comprised of 14.9% protein, 29.0% N-acetylglucosamine, 9.4% N-acetylgalactosamine, 10.5% fucose, 24.2% galactose, 0.9% mannose, 4.0% N-acetylneuraminic acid, and 7.0% sulfate. Carbohydrate units were O-glycosidically linked and ranged in size from 4 to 16 residues. The biophysical properties of MG1 were compared to those of a smaller mucin (MG2) also isolated from submandibular-sublingual saliva. Fluorescence spectroscopy demonstrated that MG1 bound both 1-anilino-8-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPNA) in stable hydrophobic binding sites (melting temperature, 47 +/- 2 degrees C), whereas MG2 did not bind these hydrophobic probes. These hydrophobic domains occurred on nonglycosylated or naked portions of MG1 since Pronase treatment eliminated ANS binding. Reduction of disulfide bridges in MG1 increased the number of available hydrophobic binding sites. High ionic strength (0 to 2 M NaCl) had no effect on ligand binding, whereas lowering pH (9 to 2) increased ANS binding without affecting NPNA complexation. Circular dichroism (CD) data suggested that MG1s carbohydrate chains dominated its spectrum. In contrast, the peptide backbone dominated the CD spectrum of MG2. Collectively, the results of this study indicate that human submandibular-sublingual saliva contains two structurally distinct mucins.

Specificity of salivary-bacterial interactions: II. Evidence for a lectin on Streptococcussanguis with specificity for a NeuAcα2,3Ga1β1,3Ga1NAc sequence

Abstract Evidence is presented for the presence of a lectin on Streptococcus sanguis with specificity towards the major acidic oligosaccharide of human salivary mucin. Based upon hemagglutination inhibition studies, the strongest inhibitor was NeuAcα2,3Galβ1,3GalNAcol ⪢ NeuAcα2,3Galβ1,4Glc ⪢ NeuAc > Gal. Interactions were not heat sensitive or charge dependent, and were not affected by the presence of bacterial cell associated neuraminidase. The lectin could be extracted from Streptococcus sanguis with lithium 3,5-diiodosalicylate (LIS). Incubation of LIS extracts with carbohydrate ligands demonstrated that the specificity of binding was NeuAcα2,3Galβ1,3[ 3 H-]GalNAcol ⪢ Galβ1,3[ 3 H-]GalNAcol .

Purification of a low-molecular-weight, mucin-type glycoprotein from human submandibular-sublingual saliva.

A low-molecular-weight, monomeric, mucin-type glycoprotein (MG2) has been isolated from human submandibular-sublingual saliva. Initial purification involved sequential gel-filtration on Sephadex G-200 and Sepharose CL-2B, the latter in the presence of 6M urea. Fractions containing MG2 were next separated from contaminating secretory IgA by immunoaffinity chromatography or recycling through Sephadex G-200. Mucin fractions were 14C-labeled by reductive methylation, and then the final purification-step entailed recycling radiolabeled materials through Sephadex G-200. Radiolabeling aided in the assessment of purity, as judged by SDS-PAGE and ion-exchange chromatography. The carbohydrate portion accounted for 69.6% of the recovered weight and was composed of N-acetyl-glucosamine, N-acetylgalactosamine, galactose, fucose, and N-acetylneuraminic acid. Sulfate was also present. The protein comprised 30.4% of the recovered weight with threonine, serine, proline, and glycine accounting for 75.2% of the total amino acids. The oligosaccharides were alkali-labile, indicating an O-glycosyl linkage to the peptide. The mucin was weakly acidic and had an estimated mol. wt. of 200 000-250 000.

Clinical Science Adherence of Streptococcus sanguis to Salivary Mucin Bound to Glass

This study demonstrated that human submandibular-sublingual saliva (HSMSL) provided a better substrate than did whole saliva or parotid saliva for the binding of Streptococcus sanguis in a glass adherence assay. Additional evidence indicated that the lower molecular weight salivary mucin in HSMSL was involved in these interactions. Mucins sialic acid residues were found to play a major role in mediating the binding of certain strains of Streptococcus sanguis.

Oligosaccharide Structures of the Low-molecular-weight Salivary Mucin from a Normal Individual and one with Cystic Fibrosis

Studies were begun to compare the oligosaccharide structures of the low-molecular-weight mucin purified from the submandibular-sublingual saliva of a normal individual with that from one with cystic fibrosis. Following alkaline/borotritide cleavage, neutral and sialic acid-containing chains were purified by a combination of gel filtration, paper chromatography, and high-voltage paper electrophoresis. Oligosaccharides ranged in size from a disaccharide to a heptasaccharide. Approximately 80% of the oligosaccharides were GalB1, 3 GalNAc; Fucα1,2GalB1,3GalNAc; and NeuAcα2,3GalB1, 3GalNAc. The other structures were Fucα1,2GalB1,4(Fucα1,3)GlcNAcB1,6 (GalB1,3)GalNAc or GalB1,4(Fucα1,3)GlcNAcB1,6(Fucα1,2GalB1, 3)GalNAc; GalB1,4(Fucα1,3)GlcNAcB1,6(NeuAcα2,3GalB1,3)GalNAc; and Fucα1,2GalB1,4(Fucα1,3)GlcNAcB1,6 (NeuAcα2,3Gal-B1,3)GalNAc. There were no apparent qualitative differences in the neutral and sialic acid-containing units recovered from the normal and cystic fibrosis samples.

PppA, a Surface-Exposed Protein of Streptococcus pneumoniae, Elicits Cross-Reactive Antibodies That Reduce Colonization in a Murine Intranasal Immunization and Challenge Model

ABSTRACT The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an ∼20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.

Lubrication of selected salivary molecules and artificial salivas

The lubrication regime displayed by human salivas (parotid and submandibular-sublingual), purified salivary molecules (the mucins MG1 and MG2 and α-amylases), and selected artificial salivas (Oracare D, Saliva Substitute, and Orthana) was assessed in vitro using a friction-testing device. Thin-film (boundary) lubrication was observed for all of the salivary samples and two of the artificial salivas examined. Oracare D, a glycerol-based artificial saliva, was the exception since it lubricated by a thick-film (hydrodynamic) regime. On a molar basis, the best lubricants of the purified salivary molecules were MG1 > MG2 ≈ nonglycosylated α-amylases ≈ glycosylated α-amylases.

Structure of the carbohydrate chains of the proline-rich glycoprotein from human parotid saliva

Abstract The carbohydrate structure of a proline-rich glycoprotein from human parotid saliva is presented. Studies were performed on tritiated glycopeptides prepared by extensive pronase digestion of galactose oxidase/sodium borotritide treated glycoprotein. Structural characterization was carried out using a combination of periodate oxidation, methylation analysis, nitrous acid deamination, and glycosidase treatment. A triantennary asparagine-linked structure was found which contained peripheral Galβ1→4 (Fucα1→3) GlcNAc sequences.

Structural features of the human salivary mucin, MUC7.

Human salivary mucin (MUC7) is characterized by a single polypeptide chain of 357 aa. Detailed analysis of the derived MUC7 peptide sequence reveals five distinct regions or domains: (1) an N-terminal basic, histatin-like domain which has a leucine-zipper segment, (2) a moderately glycosylated domain, (3) six heavily glycosylated tandem repeats each consisting of 23 aa, (4) another heavily glycosylated MUC1- and MUC2-like domain, and (5) a C-terminal leucine-zipper segment. Chemical analysis and semi-empirical prediction algorithms for O-glycosylation suggested that 86/105 (83%) Ser/Thr residues were O-glycosylated with the majority located in the tandem repeats. The high (∼25%) proline content of MUC7 including 19 diproline segments suggested the presence of polyproline type structures. CD studies of natural and synthetic diproline-rich peptides and glycopeptides indicated that polyproline type structures do play a significant role in the conformational dynamics of MUC7. In addition, crystal structure analysis of a synthetic diproline segment (Boc-Ala-Pro-OBzl) revealed a polyproline type II extended structure. Collectively, the data indicate that the polyproline type II structure, dispersed throughout the tandem repeats, may impart a stiffening of the backbone and could act in consort with the glycosylated segments to keep MUC7 in a semi-rigid, rod shaped conformation resembling a ‘bottle-brush’ model.