Anthony Herp

Current concepts of the structure and nature of mammalian salivary mucous glycoproteins

SummaryThe visco-elastic properties of salivary secretions are due to high molecular-weight glyco-proteins, known as mucins. Mucins are composed of numerous oligosaccharide side-chains O-glycosidically linked through 2-acetamido-2deoxy-α-d-galactose to the hydroxyl groups of seryl and threonyl residues of the protein core; on the average, every fourth amino acid residue is involved in such a bond. This work conveys their isolation and purification, compiles the compositional analysis of several mammalian submaxillary and sublingual mucins; defines the conditions of the alkaline β-elimination reaction, its mechanism and importance in structural studies of glycoproteins, and briefly discusses the influence of stimuli on mucous secretions, as well as biosynthesis, structural diversity, and physiological role of salivary mucous glycoproteins.

Depolymerization of Hyaluronic Acid by Autoxidants and Radiations

Hyaluronic acid, a linear polymer of alternating N-acetyl-D-glucosamine and D-glucuronic acid units with a molecular weight of 105 to 107, is known to be degraded by X-rays, ultraviolet radiation, and ultrasonic waves (1-6). This substance and other natural and synthetic polymers are also degraded by autoxidants such as ascorbic acid or ferrous or cuprous ions in the presence of oxygen (7), by mechanisms probably involving free radicals (5, 8, 9) similar to those involved in their degradation by radiations. This latter reaction has been described as the oxidativereductive depolymerization (ORD) reaction (10). Alexander et al. (8, 11) noted degradations of polymethacrylic acid by both X-rays and several autoxidants such as cysteine, but the present study is the first formal comparison of the effects of radiations and autoxidants on hyaluronic acid. The present work was carried out in order to compare the effects of radiation on hyaluronic acid with those produced by autoxidants under closely similar conditions. A semiquantitative comparison of the effects of X-rays, ,B-rays (P32, internally), and autoxidants is reported. The effect of visible light with riboflavin as sensitizer was also studied. For the ORD reaction, the results of a survey of possible autoxidants and antioxidants are included. Viscometric measurements were used as the criterion of the degree of degradation of hyaluronic acid.

Abnormalities of blood selenium and glutathione peroxidase activity in patients with acquired immunodeficiency syndrome and aids-related complex

Severe protein-calorie malnutrition is common in patients with AIDS and could contribute to the progressive deterioration characteristic of that disease. Selenium deficiency could also have a negative impact on immune function and other organ functions vital for recovery from infectious diseases. Therefore, to assess any role for selenium in AIDS, we determined plasma and erythrocyte selenium levels and glutathione peroxidase activity in 13 patients with AIDS compared to 8 patients with AIDS-related complex (ARC) and 14 healthy controls. Plasma selenium levels were significantly reduced in AIDS patients compared to controls (p<.0001) and to ARC (p<.02). Erythrocyte selenium levels in both AIDS and ARC were also reduced compared to controls (p<.02), but not to each other. Glutathione peroxidase activity in AIDS was 28.9±1.4 U/g Hb vs 38.4±6.9 in ARC (p=NS) and 52.3±1.7 in controls (p<.0001 vs AIDS;p<.02 vs ARC). When all groups were combined, there were significant correlations between total lymphocyte count and both plasma selenium (r=.53;p<.002) and erythrocyte glutathione peroxidase activity (r=.65;p<.0001). In addition, strong correlations were noted between plasma selenium and serum albumin (r=.68;p<.0001), plasma selenium and glutathione peroxidase (r=.77;p<.0001), and glutathione peroxidase and hematocrit (r=.66;p<.0001). In AIDS or ARC, no correlations between selenium with disease duration or weight loss were present. We conclude that, in comparison to normals, patients manifesting infection with human immunodeficiency virus have evidence of selenium deficiency as determined by diminished plasma and erythrocyte levels and glutathione peroxidase activity. These abnormalities are most marked in patients with AIDS, but are also present in patients with AIDS-related complex. Selenium deficiency has important implications for the progression and pathogenesis of clinical disease in AIDS.

Lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis

The lipid composition of tracheobronchial secretions from normal individuals and patients with cystic fibrosis was investigated. Lipids were extracted from he dialyzed and lyophilized samples, and fractionated on silicic acid columns into neutral lipids, glycolipids and phospholipids. The lipids contained each fraction were separated into individual components by thin-layer chromatography and quantified. The secretions of patients with cystic fibrosis and were found to contain about 30% more lipids than that of normal individuals and exhibited elevated levels of cholesterol, phospholipids and glycosphingolipids. The level of free fatty acids and glyceroglucolipids was higher in the normal secretions. The phospholipids of cystic fibrosis secretions exhibited higher content of sphingomyelin and phosphatidylserine, while the normal samples contained more lysophosphatidylcholine. The glycosphingolipids of both types of samples consisted mainly of glucosyl- and lactosylceramides. The major glyceroglucolipid of the normal tracheobronchial secretions was tetraglucosyl glyceroglucolipid, whereas hexa-and octaglucosyl glyceroglucolipids were the predominant compounds of the cystic fibrosis secretions.

β-elimination and reduction reactions and some applications of dimethylsulfoxide on submaxillary glycoproteins

Abstract It has been shown that dimethylsulfoxide is useful in separating protein contaminants and minor mucins from the submaxillary glycoproteins prepared by the method of Tettamanti, G. and Pigman, W. (1968) Arch. Biochem. Biophys., 124, 41–50. In addition, alkaline dimethylsulfoxide was found to greatly accelerate the β-elimination reaction of glycoproteins. As this procedure does not produce diffusible protein fragments, it should be of value in studies of the protein core and for the investigation of those protein-carbohydrate linkages that are resistant to treatment by alkali. Optimal conditions for carrying out the β-elimination and reduction reactions of bovine submaxillary glycoprotein in aqueous alkaline NaBH4 systems have been described. Under such conditions, the conversion of 2-aminopropenoic and 2-amino-2-butenoic acid residues into alanine and α-aminobutyric acid, respectively, is complete. This reduction procedure is of a broad application and can be used for the reduction of the phenylhydrazone derivatives of α-keto acids to the corresponding amino acids. A comparison of our procedure with that described by Simpson, L. D., Hranisavljevic, J. and Davidson, E. A. (1972) Biochemistry, 11, 1849–1856, shows that the method outlined here is superior both in efficiency and for quantitative recovery of unsaturated hydroxyamino acids.

Biochemistry and Lectin Binding Properties of Mammalian Salivary Mucous Glycoproteins

The molecules responsible for the highly viscous properties of mucus are secretory glycoproteins referred to as mucins. Salivary mucins are characterized by a high sugar to protein ratio and are of a broad range of molecular weight from 7 x 10(4) to millions. With a few exceptions, they contain up to 30% of hexosamine (galactosamine and glucosamine), 8-33% of sialic acid, trace to 15% of galactose or fucose and little or no mannose. The size of carbohydrate side chains of these glycoproteins ranges from one to about fifteen units of sugar. These carbohydrate side chains are usually O-glycosidically linked through N-acetylgalactosamine to a peptidyl serine or threonine. In some instances, ester sulfate groups, mainly on N-acetylglucosamine, are also a structural feature. In many of these glycoproteins, the saccharide sequence is the same as that which determines the specificity of blood groups. Carbohydrate sequence analysis shows that salivary mucins exhibit considerable polydispersity, great diversity and remarkable structural flexibility not only among animal species but also within the same mucin molecule. Based on their lectin-binding ability, they can be used for purification of lectins, and lectins coupled to resin may be useful for the isolation of mucin-type glycoproteins. The epithelial mucous secretions modulate oral microbial flora; many secretory components serve as lectin-receptors for the attachment of microbes. The judicious use of lectins with widely differing binding characteristics has already been valuable in the in situ localization of salivary glycoproteins, in elucidating structural details, recording sugar density within a given tissue section, and defining host-parasite interactions. It is hoped that their use, together with monoclonal antibody (158) and tissue culture techniques (159, 160) will further clarify the roles of individual secretory mucous glycoproteins in health and disease.

A guide for carbohydrate specificities of lectins.

Lectins are carbohydrate-binding proteins of nonimmune origin, which have been widely used in the fields of cell biology, biochemistry and histochemistry to isolate and/or to characterize cell surface carbohydrates(1–3). Lectins require configurational and structural complementarity of sugars for interaction to occur. All lectin molecules have two or more carbohydrate binding sites, a property essential to their ability to agglutinate cells or to precipitate complex carbohydrates(1, 2, 4). Until the early seventies, the carbohydrate specificities of lectins were mainly determined by the abilities of monosaccharides or their glycosides to inhibit lectin-induced haemagglutination(1, 4). Makela(5), in the late fifties, divided lectin-reactive monosaccharides into four classes, based on their configuration at C-3 and C-4 of the pyranose form, as shown in Fig. 1. Lectins that bind to Makela’s group II sugars are Gal-specific whereas those reacting with sugars of group III are Man and/or Glc-specific lectins. Fucose-binding lectins are specific for group I sugars. Lectins binding sugars belonging to group IV have not yet been reported.

Subunit structure and dissociation of Callinectes sapidus hemocyanin

The hemocyanin of the blue crab, Callinectes sapidus has two major components sedimenting with approximate sedimentation coefficients of 17 S and 25 S. Molecular weight data based on light scattering and sedimentation equilibrium measurements at pH 7.8 suggest that the two components have molecular weights of approximately 450 000 and 900 000 in the presence of stabilizing Ca2+. In the absence of Ca2+, the molecular weights are found to be about 5% lower, suggesting some dissociation of the hemocyanin components. Circular dichroism and optical rotatory dispersion measurements in the far-ultraviolet region gave nearly identical spectra for the two components. Based on the reference parameters of Chen et al. (Chen, Y.H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131 and Chen, Y.H., Yang, J.T. and Chan, K.H. (1974) Biochemistry 13, 3340--3359), estimates of 16--20% alpha-helix, 40--60% beta-structure, and 30--40% random organization were obtained for the two hemocyanin components. Exposure to 6 M Gdn HCl gave light scattering molecular weights of approx. 68 000 and 77 000, which is close to one-sixth of the molecular weight of the 17 S component. These results support the view that the two components of C. sapidus hemocyanin share the hexameric and dodecameric organization common to arthropod hemocyanins. The salts of the Hofmeister series and the ureas are found to dissociate the dodecameric component with the former exhibiting the usual order of effectiveness of NaCl, NaBr, NaI, and NaClO4 dissociation, while the ureas show an inverse order of decreasing effectiveness in going from urea to methyl-, ethyl- and propylurea. This suggests that polar and ionic interactions are relatively more important than hydrophobic interactions for the stabilization of the dodecameric form of C. sapidus hemocyanin. The dissociation behavior of the 17 S hexameric species by GdnHCl in the 0--1.5 M concentration region (where essentially no denaturation occurs), based on light scattering molecular-weight measurements, is satisfactorily accounted for by equations describing the dissociation of hexamers to monomers.

Depolymerization of polysaccharides through the generation of free radicals at a platinum surface: a novel procedure for the controlled production of free-radical oxidations☆

Abstract Electrical cells were devised to investigate aspects of the free-radical degradation of alginic and hyaluronic acids by l -ascorbic acid. By partitioning autoxidant and polysaccharide in separate electrode compartments, it was possible to induce depolymerization of the carbohydrate. Contaminating metals in the phosphate buffer were shown to serve as electron carriers in the absence of an addition of such mediators. More efficient depolymerization resulted when an external source of dc voltage was used in place of the electrode potential generated by the autoxidant. Addition of Fe(III) as the EDTA complex accentuated the degradation. Ferrous ion was detected at the cathode of the cell; hydrogen peroxide was observed to form at low concentrations, and to disappear at high concentrations from the solution. Depolymerization of the sugar acids is thought to result from the cyclic reduction and autoxidation of iron ions. Depolymerization, normally followed by increase in the specific fluidity of the polymer solution, was accompanied by generation of reducing properties.

Studies on the isolation and composition of human ocular mucin

A method for the isolation and purification of human ocular mucin from the brief saline extract of human ocular mucus is reported. Initial purification of ocular mucin was achieved by sequential chromatography of the saline-soluble mucus extract from an individual donors mucus pool on columns of Sephadex G-50 and Sepharose CL-4B. A portion of such mucin isolate was subjected to quantitative analysis of the O-seryl (threonyl)-N-acetylgalactosaminyl linkage, characteristic of mucins, by alkaline beta-elimination and tritiated borohydride reduction. Following Bio-Gel P-2 filtration, the mucin isolate whose cleaved oligosaccharides contained tritiated galactosaminitol greater than 0.5 microCi mg-1, a value that represents at least 64% of that observed for bovine and ovine submaxillary reference mucins, was considered to be mucin-rich. These isolates were subjected to further purification on Sephacryl S-500 and DEAE-Trisacryl M column chromatographies. The purified mucin had a minimum molecular weight of 120 kDa. It consisted of 25-30% protein and 54-55% carbohydrate. Its amino acid and carbohydrate compositions are characteristic of a mucin structure. The purity of the mucin was verified by SDS-gradient PAGE. Upon isoelectric focusing, polydispersity/microheterogeneity were exhibited in the pI range 5.0-6.6.