Ronald E. Loomis

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.

Surface properties of mussel adhesive protein component films.

Mussel adhesive protein (MAP) is the adhesive agent used by the blue sea mussel (Mytilus edulis) to attach the animal to various underwater surfaces. It is composed of 75-->85 repeating decameric units with the reported primary sequence NH2-A(1)-K(2)-P(3)-S(4)-Y(5)-Hyp(6)-Hyp(7)-T(8)-DOPA(9)-K(10)-COOH. This study identifies and compares the surface properties of the decameric unit, selected fragments and individual amino acid constituents with the complete MAP preparation. These molecular systems were examined: (a) in the solid state as thin films formed on germanium substrata using multiple-attenuated-internal-reflectance infrared (MAIR-IR) spectroscopy, ellipsometry and contact angle analysis; and (b) in the solution state using circular dichroism (CD) spectroscopy. Extensive molecular modelling of the decamer was performed making integral use of the experimentally derived data. These cumulative semi-empirical and empirical results suggest a conformation for the decamer that closely associates the L-DOPA and tyrosine residues with the solid substratum. This model provides the first representation of MAP derived from a rational integration of theoretical and experimental data. On the basis of this model, a possible explanation for the bioadhesive properties of MAP is suggested.

Isolation and characterization of a mucin-glycoprotein from rat submandibular glands

A blood group A+ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present.

Surface characterization of mussel adhesive protein films

Mussel adhesive protein (MAP) from the marine mussel Mytilus edulis is considered to be responsible for attaching it to surfaces in its native environment. The isolated and partially purified form of MAP, marketed as «Cell-Tak TM » (BioPolymers, Inc:, Farmington, CT), is currently being used as an in vitro aid for attachment of cells to culture vessels. The collective results reported here indicate that Cell-Tak TM binds strongly to clean germanium plates, serving as surrogate substrata for those found in many natural situations

Comparative biophysical study of adsorbed calf serum, fetal bovine serum and mussel adhesive protein films

Varying concentrations of different sera and adhesive agents are routinely used to increase cellular attachment to substrata. The surface-chemical effects of some of these surface-altering materials have been examined using ellipsometry, contact angle analysis and multiple-attenuated internal reflection infrared (MAIR-IR) spectroscopy. Specifically, 15% fetal bovine serum (FBS), Hams F-12 (containing 10% FBS + 1% penicillin/streptomycin), 10% calf serum and mussel adhesive protein (MAP) were allowed to adsorb on to similar and different surfaces and then compared. Each of these preparations is capable of altering the surface-chemical properties of substrata with varying resultant surface energies. It is therefore important to characterize serum in the proper concentrations on the substrata under consideration in order to understand the interfacial effects.

Conformational analysis of the cholecystokinin C-terminal octapeptide: a nuclear magnetic resonance and computer-simulation approach

The C-terminal octapeptide portion of cholecystokinin (CCK8) has well-defined biological properties which include action as a neurotransmitter and induction of gall-bladder contraction and pancreatic enzyme secretion. Many analogues of CCK8 have been prepared and tested for potency, making this an ideal model system in which to initiate evaluation of structure-function relationships. The present study uses high-resolution proton nuclear magnetic resonance (NMR) spectroscopy and energy minimization techniques to evaluate the solution (DMSO) and in vacuo conformation(s) of CCK8. The NMR results provide amide and C alpha H alpha chemical shift temperature dependencies and all phi dihedral angles and chi 1 rotamer populations. The energy minimization data located deep potential energy wells, for which all torsion angles are reported. Collectively, the data support models for CCK8 where the structures are characterized by a high degree of folding. These conformations are characterized by sharp turns, possibly stabilized by hydrogen-bonds. Taken together with pharmacologic data and somewhat similar folded structures implied from fragments of CCK8, it is suggested that both electrostatic and steric effects are needed for full biological potency.

Dynamics of a proline-rich glycoprotein from human parotid saliva: a 360-MHz proton nuclear magnetic resonance investigation

Abstract The 360-MHz proton n.m.r. spectra of a proline-rich glycoprotein from human parotid saliva (PRG) were obtained in order to analyse spin-lattice relaxation ( T 1 ) times and hydrogen-deuterium exchange rates. The ( T 1 ) values for the proline ring and bulk peptide backbone C α H α protons were found to be about 0.5 s. The HaD exchange data revealed that only four of the many N-H resonances remained after the first few minutes of exchange. Within 96 h at 12°C most of the remaining peak intensities were gone. These data imply a dynamic conformation with a majority of the backbone free of hydrogen-bonded structures.

Interactions of a human salivary proline-rich glycoprotein nonapeptide functional domain with model surfaces

The homeostasis of the tooths hydroxyapatite-like surface is primarily maintained by a proteinaceous film called the acquired enamel pellicle (AEP). Human saliva contains a family of proline-rich proteins (PRPs) which are among the first biomolecules to adsorb to the tooths surface, and consequently are deemed essential to AEP formation and function. One particular member of the PRPs, the proline-rich glycoprotein (PRG), has several areas of conserved sequence. One of these regions, G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9), or PRG9, occurs twice and has several proline residues occurring in different types of physico-chemical environments. An investigation into the static and dynamic adsorption processes of this peptide onto model surfaces (i.e. non-siliconized and siliconized germanium plates) was accomplished in this study. PRG9 was found to adsorb tightly to the non-siliconized germanium plates at near-monolayer thicknesses in both the static and the dynamic flow experiments. However, the peptide...