Akira Kobata

Structures of Sialylated O-Linked Oligosaccharides of Bovine Peripheral Nerve α-Dystroglycan THE ROLE OF A NOVEL O-MANNOSYL-TYPE OLIGOSACCHARIDE IN THE BINDING OF α-DYSTROGLYCAN WITH LAMININ

α-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein β-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve α-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve α-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the α-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaα2-3Galβ1-4GlcNAcβ1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the α-dystroglycan with laminin.

[17] Hydrazinolysis of asparagine-linked sugar chains to produce free oligosaccharides

Publisher Summary Several enzymic and chemical methods liberate asparagine-linked sugar chains as oligosaccharides. Among them, endo-β-N-acetylglucosaminidases have been used to elucidate the whole structures of highmannose type of sugar chains and all of the sugar chains of hen egg albumin, and to confirm the presence of hybrid-type asparagine-linked sugar chains. However, the substrate specificities of these enzymes have hampered their use for the study of complex-type asparaginelinked sugar chains, a most important series of this group. The method described in this chapter is the chemical procedure applicable to the study of asparagine-linked sugar chains without any limitations. The hydrazinolysis reaction is used for the isolation of whole asparagine-linked sugar chains as oligosaccharides by N-acetylation, instead of cleaving all glucosamine linkages by deamination. For determining an optimal condition to release oligosaccharides, the hydrazinolysis products of bovine IgG and of gltcoprotein-IV obtained from hen egg albumin are carefully examined.

[6] Analysis of oligosaccharides by gel filtration

Publisher Summary Development of various techniques to liberate sugar chains from glycoproteins, there is a need to fractionate oligosaccharides composed of 8-20 monosaccharides is increasing. Gel permeation chromatography is useful tool for this purpose, which separates oligosaccharides by their sizes and is limited in fractionating oligosaccharides as compared to paper chromatography. However, because of the occurrence of structural rules in oligosaccharides of natural origin, the method can be used more effectively than was previously believed. The evidence that N-acetylhexosamine, hexose, and methylpentose residues behave differently because of size also increases the usefulness of the method. Moreover, careful analysis of the mobilities of various oligosaccharides has disclosed that even isomeric oligosaccharides show slight differences in their mobilities on the column, and these differences can be correlated with the mobilities of particular disaccharide structures within each oligosaccharide.

Structure, biosynthesis and functions of glycoprotein glycans

Since the pioneering work on structure and function of heteroglycans compiled in the classical books edited by A. Gottschalk in 19721, there have been several promising developments in glycoconjugate research, as reviewed in this article. In Part 1, contributed by A. Kobata, current knowledge on heteroglycan structures is presented and representative examples taken from higher organisms are given. Part 2, written by J. F. G. Vliegenthart and J. P. Kamerling, covers the most important achievements in methodology: procedures to obtain pure glycans and to analyze their structures. Part 3, contributed by J. Paulson, is devoted to biosynthesis of glycans now describable as pathways since several of the glycosyltransferases have been isolated and analyzed for specificity. In Part 4, contributed by E. Buddecke, current knowledge on functional roles of glycans is presented. It will become apparent that the prerequisite for valid work either in biosynthetic or functional context depends on solid structural information. This is particularly true whenever glycosyltransferase reaction products are being analyzed, or glycans involved in biological functions are investigated. Although in past years, a great deal of important knowledge has been gathered by use of crude glycosidase or glycosyltransferase activities (a notable example is found in reference 2), one may now postulate that glycans implicated in biological reactions should be thoroughly analyzed. This review may familiarize ‘newcomers’ with the field of glycoconjugate research with special emphasis on glycoprotein glycans. Glycolipids are not included in this article as they have recently been reviewed by S. I. Hakomori3. The reader is also referred to several excellent monographs4,5 and the Proceedings of the Glycoconjugate Symposia held biannually6–8.

Asparagine-linked glycosylation of the scrapie and cellular prion proteins☆

Post-translational modification of the scrapie prion protein (PrP) is thought to account for the unusual features of this protein. Molecular cloning of a PrP cDNA identified two potential Asn-linked glycosylation sites. Both the scrapie (PrPSc) and cellular (PrPC) isoforms were susceptible to digestion by peptide N-glycosidase F (PNGase F) but resistant to endoglycosidase H as measured by migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PNGase F digestion of PrPC yielded two proteins of Mr26K and 28K; however, the 26-k species was only a minor component. In contrast, PNGase F digestion of PrPSc yielded equimolar amounts of two proteins of Mr26K and 28K. The significance of this altered stoichiometry between the 26- and 28-kDa deglycosylated forms of PrP during scrapie infection remains to be established. Both isoforms as well as PrP 27-30, which is produced by limited proteolysis of PrPSc, exhibited a reduced number of charge isomers after PNGase F digestion. The molecular weight of PrP 27-30 was reduced from 27K-30K by PNGase F digestion to 20K-22K while anhydrous hydrogen fluoride or trifluoromethanesulfonic acid treatment reduced the molecular weight to 19K-21K and 20K-22K, respectively. Denatured PrP 27-30 was radioiodinated and then assessed for its binding to lectin columns. PrP 27-30 was bound to wheat germ agglutinin (WGA) or lentil lectins and eluted with N-acetylglucosamine or alpha-methyl-mannoside, respectively. Digestion of PrP 27-30 with sialidase prevented its binding to WGA but enhanced its binding to Ricinus communis lectin. These findings argue that PrP 27-30 probably possesses Asn-linked, complex oligosaccharides with terminal sialic acids, penultimate galactoses, and fucose residues attached to the innermost N-acetyl-glucosamine. Whether differences in Asn-linked oligosaccharide structure between PrPC and PrPSc exist and are responsible for the distinct properties displayed by these two isoforms remain to be established.

Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours.

Most secretory and membrane‐bound proteins produced by mammalian cells contain covalently linked sugar chains. Alterations of the sugar chain structures of glycoproteins have been found to occur in various tumours. Because the sugar chains of glycoproteins are essential for the maintenance of the ordered social behaviour of differentiated cells in multicellular organisms, alterations to the sugar chains are the molecular basis of abnormal social behaviours in tumour cells, such as invasion into the surrounding tissues and metastasis. In this review, the structure and enzymatic basis of typical alterations of the N‐linked sugar chains, which are found in various tumours, are introduced. These data are useful for devising diagnostic methods and immunotherapies for the clinical treatment of tumours. Three β‐N‐acetylglucosaminyltransferases, GnT‐III, ‐IV and ‐V, play roles in the structural alteration of the complex‐type sugar chains in various tumours. In addition, transcriptional changes in various glycosyltransferases, together with the transporters of sugar nucleotides and sulfate, which are responsible for the formation of the outer chain moieties of complex‐type sugar chains, are the keys to inducing the alterations.

The substrate specificities of endo-β-N-acetylglucosaminidases CII and H

Abstract A minor glycopeptide was newly isolated from the exhaustive pronase digest of crystalline ovalbumin by Dowex-50w column chromatography, and its structure was determined as Manα1→3Manα1→6 (Manα1→3) Manβ1→4GlcNAcβ1→4GlcNAc→Asn. This glycopeptide (GP-VI) has the smallest carbohydrate unit among the ovalbumin glycopeptides so far reported, and is also the smallest glycopeptide of all which are susceptible to endo-β-N-acetylglucosaminidases CII and H. This finding, together with the already reported data of the action of both enzymes to glycopeptides of known structures, elucidates that the structural requirement of CII enzyme for its substrate is R→2Manα1→3 (R→6) Manα1→6 (R→2Manα1→3) (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn, in which R represents either hydrogen or sugars, and that of H enzyme is R→2Manα1→3 (R→6) Manα1→6 (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn.

Detection of O-mannosyl glycans in rabbit skeletal muscle α-dystroglycan

Abstract α-Dystroglycan, which is a cell surface component of dystroglycan complex, is known to bind laminin in basal lamina of muscle cells and Schwann cells. We found previously that a novel O-glycan, Siaα2-3Galβ1-4GlcNAcβ1-2Man, is the major oligosaccharide in bovine peripheral nerve α-dystroglycan, and that this structure might mediate the binding of laminin. In order to determine whether this structure is specific for peripheral nerve α-dystroglycan or present on different forms of α-dystroglycan, we analyzed the structures of the sialylated O-glycans of rabbit skeletal muscle α-dystroglycan. Their structures were elucidated to be a mixture of a core 1 O-glycan and the same O-mannosyl glycan that we found in bovine peripheral nerve. These results indicate that α-dystroglycan in different species and tissues share a common structure of its major O-linked acidic carbohydrate, suggesting its relevance to the basic functional role of α-dystroglycan.

Characterization of Dystroglycan‐Laminin Interaction in Peripheral Nerve

Abstract: Dystroglycan is encoded by a single gene and cleaved into two proteins, α‐ and β‐dystroglycan, by posttranslational processing. The 120‐kDa peripheral nerve isoform of α‐dystroglycan binds laminin‐2 comprised of the α2, β1, and γ1 chains. In congenital muscular dystrophy and dy mice deficient in laminin α2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan‐laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan‐laminin interaction in peripheral nerve. We demonstrate that (1) α‐dystroglycan is an extracellular peripheral membrane glycoprotein that links β‐dystroglycan in the Schwann cell outer membrane with laminin‐2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of α‐dystroglycan in the interaction with laminin.

Substrate specificity of diplococcal β-N-acetylhexosaminidase, a useful enzyme for the structural studies of complex type asparagine-linked sugar chains

Summary The substrate specificity of diplococcal β-N-acetylhexosaminidase was studied in detail by using oligosaccharides of known structure. The enzyme cannot cleave G1cNAcβ1→4Man and G1cNAcβ1→6Man linkages although it readily hydrolyzes G1cNAcβ1→2Man, G1cNAcβ1→3Gal and G1cNAcβ1→6Gal linkages. The G1cNAcβ1→2Man linkage in G1cNAcβ1→4(GlcNAcβ1→2)Man group is cleaved by the enzyme but the linkage in G1cNAcβ1→6(G1cNAcβ1→2)Man group is not, probably because of the steric effect of G1cNAcβ1→6Man residue on G1cNAcβ1→2Man linkage. Similar steric effect is also observed in the case of G1cNAcβ1→2Manα1→6(GlcNAcβ1→2Manα1→3) (G1cNAcβ1→4)Manβ1→4 G1cNAc0T. The enzyme can cleave only one of the two G1cNAcβ1→2Man linkages of the heptaitol and produces G1cNAcβ1→2Manα1→6(Manα1→3) (G1cNAcβ1→4)Manβ1→4G1cNAc0T. The substrate specificity of diplococcal β-N-acetylhexosaminidase can be used effectively for the structural studies of asparagine-linked sugar chains.