Alkaline Borohydride Reduction Research Articles

Partial Characterization of the Epitope on Excretory-Secretory Products of Fasciola hepatica Recognized by Monoclonal Antibody ES78

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

Peripheral alpha-linked N-acetylglucosamine on the carbohydrate moiety of mucin derived from mammalian gastric gland mucous cells: epitope recognized by a newly characterized monoclonal antibody.

To obtain a tool to study the structural characterization and the detection of mucin derived from the gastric gland mucous cells, we developed a monoclonal antibody, designated HIK1083, against mucin purified from rat gastric mucosa. In an ELISA, HIK1083 reacted strongly with the mucin purified from a deep layer of the corpus and antrum but only slightly reacted with that obtained from the surface mucosal layer. The reaction of mucin and HIK1083 was inhibited by the oligosaccharides obtained by the alkaline borohydride reduction of antigenic mucin. Two purified oligosaccharide alditols reacting with the monoclonal antibody obtained from the antigenic mucin had one and two peripheral alpha-linked N-acetylglucosamine residues, respectively, according to the evidence from NMR spectroscopy. Moreover, among the commercially available p-nitrophenyl derivatives of monosaccharides, only p-nitrophenyl-N-acetyl-alpha-D-glucosaminide inhibited the reaction of this monoclonal antibody and the antigenic mucin in a concentration-dependent manner. These results, as well as the immunohistochemical observations, indicate that alpha-linked N-acetylglucosamine residues are specifically attached to the peripheral region of the carbohydrate moiety of the mucin synthesized in and secreted from the gastric-gland-type cells, and indicate that the monoclonal antibody HIK 1083 recognizes this structure.

Skeletal keratan sulphate chains isolated from bovine intervertebral disc may terminate in alpha(2----6)-linked N-acetylneuraminic acid.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (2-year-old animals) after digestion with chondroitin ABC lyase followed by digestion with diphenylcarbamoyl chloride-treated trypsin of A1D1 proteoglycans and gel-permeation chromatography on Sepharose CL-6B. The peptido-keratan sulphate fragments were subjected to alkaline borohydride reduction. The reduced chains were treated with keratanase in the presence of the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, and the digest was subjected to alkaline borohydride reduction. This produced oligosaccharides with galactitol at their reducing ends. This reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these was shown by 600 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-6Gal beta 1-4GlcNAc(6-SO4)beta 1-3Gal-ol The structure of this oligosaccharide shows that keratan sulphate chains from bovine intervertebral disc have non-reducing termini with N-acetylneuraminic acid linked alpha(2----6) as well as alpha(2----3) to an unsulphated galactose.

A non-reducing terminal fragment from tracheal cartilage keratan sulphate chains contains α(2-3)-linked N-acetylneuraminic acid

Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).

The Distinctive Pattern of Proteoglycan and Glycosaminoglycan Free Chain Synthesis by Cultured Human Epidermal Keratinocytes

The in vitro synthesis of proteoglycans and glycosaminoglycan free chains was studied in human epidermal keratinocytes. Preconfluent and confluent cultures established on 3T3 feeders were steady state labeled with [35S]-sulfate and [3H]-glucosamine after removal of the 3T3 cells. Products in nonionic detergent extracts of keratinocytes and in the medium were analyzed in the presence of protease inhibitors. Glycosaminoglycans as proteoglycans and as free chains were defined by susceptibility or resistance, respectively, to alkaline borohydride reduction. Products associated with the cells were approximately 30% proteoglycans and approximately 70% glycosaminoglycan free chains, whereas in the medium virtually all was proteoglycan. The heparan and chondroitin sulfate proteoglycans were small compared to those of many other cell types. Their Kav on Sepharose CL-4B was 0.56 (estimated 50 kDa), whereas the free chain Kav was 0.74 (estimated 12 kDa). Relative amounts of the sulfated products varied with confluence and differentiation; heparan and chondroitin sulfates were equally represented within the free chains and proteoglycans of the cells in preconfluent, proliferating cultures, whereas in postconfluent, differentiated cultures the major labeling was in the heparan sulfate products, consistent with our prior reports (J Invest Dermatol 88:215-9, 1987 and 91:492-8, 1988). The cellular localization of the products was probed with glycosaminoglycan degrading enzymes added to isotopically prelabeled cultures. The proteoglycans appeared to be located on the external surface of plasma membranes, whereas the glycosaminoglycan free chains resisted digestion and are either intracellular or membrane associated, but otherwise inaccessible. These data establish the distinctive pattern of low Mr proteoglycans and abundant cell-associated glycosaminoglycan free chains synthesized by keratinocytes.

Glycosaminoglycan Free Chains: External Plasma Membrane Components Distinct from The Membrane Proteoglycans

Heparan sulfate and chondroitin sulfate glycosaminoglycans of BALB/c 3T3 fibroblasts, metabolically labeled with [3H]glucosamine and [35S]sulfate precursors, are resolved by preparative Sepharose CL-4B chromatography into distinct products, the proteoglycans and the glycosaminoglycan free chains, the latter resistant to appreciable molecular weight shift upon alkaline borohydride reduction. The in situ localization of these cell layer molecules was probed with glycosaminoglycan degrading enzymes (lyases) of bacterial origin, which were used to digest isotopically prelabeled monolayer cultures prior to extraction with a nonionic detergent in the presence of protease inhibitors. Most of the total cellular complement of glycosaminoglycan free chains, in addition to the proteoglycans, proved accessible to the lyases under conditions which did not appreciably affect cell viability or morphology. Because these results were also obtained under low temperature (4 degrees C) conditions and in the presence of phenylarsine oxide, a sulfhydryl reagent that irreversibly inhibits endocytosis, the effects of the lyases are not dependent upon internalization by the cells. The cellular production and cell surface expression of the glycosaminoglycan free chains were not materially altered when lysosomal function was pharmacologically inhibited, confirming that the free chains are not intracellular intermediates in the lysosomal degradation pathways of proteoglycans. Contrary to the prevailing model, our observations establish that, at least in the cell line under study, glycosaminoglycan free chains are located on the external leaflet of the plasma membrane, as such suggesting that these products are biologically active components of cell surfaces.

Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage

Two discrete peptido-keratan sulphate fragments were isolated via chondroitinase ABC and trypsin digestion of a proteoglycan aggregate fraction prepared from bovine femoral head cartilage (six year old animals). The larger fragments (K(av) = 0.07, CL-6B) contained peptides substituted with several keratan sulphate (KS) chains from the KS-rich region of the proteoglycan and the smaller fragments (K(av) = 0.5, CL-6B) contained peptides with, perhaps, only one KS chain and the stubs of post-chondroitinase-treated chondroitin sulphate chains. The two peptido-KS samples and the KS chains derived from these by alkaline borohydride reduction were characterised by 13C-NMR spectroscopy. The two populations of KS chains were also examined by chromatography (Sephadex G-75), and keratanase digestion followed by chromatography on Bio-Gel P-10. From the results it was concluded that the KS chains from the two major trypsin-derived peptido-KS fragments had similar sulphation levels, distributions of hydrodynamic sizes and susceptibilities to keratanase.

Chlamydomonas agglutinin conjugated to agarose beads as an in vitro probe of adhesion

Flagellar sexual agglutinins are responsible for the primary recognition and adhesion events of mating in Chlamydomonas reinhardi which culminate in zygotic union of plus and minus gametes. Recent studies in this laboratory have shown the plus agglutinin to be an extremely large (>10 6 D) and asymmetric glycoprotein containing a high proportion of hydroxyproline and serine residues [14, 27, 28]. This paper reports an improved method for in vitro investigations of the adhesive nature of this molecule. Purified agglutinin is covalently attached to an insoluble (Affi-gel 15 agarose bead) support and shown to retain potent agglutination activity when presented to living minus gametes, which rapidly and extensively adhere to the coated bead surface by their flagella. The specificity of the response is documented by the lack of interaction of plus gametes with the immobilized plus agglutinin (IA +). Using this simple yet sensitive bioassay, we have subjected IA + beads to various enzymatic, chemical and physical treatments and assessed the effects on agglutinin activity. These studies reveal that Chlamydomonas plus agglutinin is sensitive to thermolysin or trypsin digestion, alkaline borohydride reduction, periodate oxidation, thiol reduction and heating at 65 °C, but unaffected by treatment with chymotrypsin, endo- or exoglycosidases, or incubation with isolated minus agglutinin. The implications of these results for agglutinin structure and possible functional interactions in initial recognition/adhesion events are discussed.

Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells.

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.

Immunochemical and chemical investigations of the structure of glycoprotein fragments obtained from epiglycanin, a glycoprotein at the surface of the TA3-Ha cancer cell

The structures of the carbohydrate chains present in fragments of a large-molecular-weight glycoprotein, epiglycanin, cleaved from the surface of viable TA3-Ha murine mammary carcinoma ascites cells and purified by gel filtration, were studied by immunochemical and chemical methods. Inhibitory activities for neuraminidase-treated and untreated glycoprotein material in the hemagglutination of NN-specific human erythrocytes by eight purified lectins were determined. Excellent inhibition was obtained in the Bauhinia purpurea, Arachis hypogaea, Iberis amara, and Wistaria floribunda systems, and weak inhibition against the Ricinus communis and Glycine max lectins. No activity against hemagglutination by the Phaseolus vulgaris and Phaseolus limensis lectins was observed. These results, when compared with those obtained by periodate oxidation, alkaline borohydride reduction, and partial methylation, suggest the possible presence of six different carbohydrate chains of 1 to 5 components in length, having as terminal groups N-acetylneuraminic acid, galactose, and 2-acetamido-2-deoxygalactose. All chains are attached to a single polypeptide chain by O-glycosyl bonds involving a 2-acetamido-2-deoxygalactose residue and a serine or threonine residue. It is suggested that the native molecule of epiglycanin of molecular weight 500,000 contains more than 500 carbohydrate chains attached to a single polypeptide chain of ≈ 1,300 amino acid units.