Duraikkannu Loganathan

A new method for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates☆

A new method is described for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates. The reducing ends of various mono-, di-, tri-, and tetra-saccharides were conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonate (a fluorescent and negatively charged compound) by reductive amination using sodium cyanoborohydride. The sugar conjugates were purified by preparative gradient polyacrylamide gel electrophoresis followed by a newly developed technique involving their semi-dry transfer to positively charged nylon membranes and elution with sodium chloride. The structures of a monosaccharide- and trisaccharide-conjugate were established by f.a.b.-m.s. and 2D n.m.r. Seven linear oligosaccharide-fluorescent conjugates were treated sequentially with exoglycosidases and with endoglycosidases. Analysis of the products by gel electrophoresis provided sequence information. These methods may be useful for sequencing oligosaccharides that are chemically or enzymically (endoglycosidase) released from glycoproteins, glycolipids, and proteoglycans.

Heparin, Heparinoids and Heparin Oligosaccharides: Structure and Biological Activities

Heparin is a polydisperse, highly sulfated polysaccharide which has been in widespread clinical use over the past half century. This chapter describes our current understanding of heparin’s structure and its biosynthesis. In addition to heparin’s usefullness as an anticoagulant, it has a wide range of additional activities. These include its antiathero-sclerotic activity, complement inhibitory activity, angiogenic activity, and other additional, recently discovered activities. The rationale for developing biomimetic polymers to replace the natural product, heparin, is discussed. Synthetic polymers, semi-synthetic sulfated polysaccharides, fully synthetic heparin oligosaccharides, fractionated heparins, low molecular weight heparins, and enzymatically prepared heparin oligosaccharides all have been used as heparin substitutes. This chapter examines heparin’s structure-activity relationships with a focus on the potential utility of these biomimetic polymers as new therapeutic agents.

Carbohydrate analysis of glycoproteins A review

Many of the products prepared by biotechnological approaches, including recombinant genetic engineering, cell tissue culture, and monoclonal technologies, are glycoproteins. As little as five years ago, glycosylation was believed to play no significant role in the function of glycoproteins. Recent large scale testing of glycoprotein-based pharmaceuticals has indicated that both the extent and type of glycosylation can play a central role in glycoprotein activity. Although methods for compositional and sequence analysis of proteins and nucleic acids are generally available, similar methods have yet to be developed for carbohydrate oligomers and polymers. This review focuses on new, developing methods for the analysis and sequencing of the carbohydrate portion of glycoproteins. Included are: (1) the release of oligosaccharides and hydrolysis of carbohydrate chains using enzymatic and chemical methods; (2) fractionation by LPLC, electrophoresis, HPLC, and lectin affinity chromatography; (3) detection through the preparation of derivatives or by new electrochemical methods; (4) analysis by spectroscopic methods, including MS and high-field NMR; and (5) their sequencing through the use of multiple, well-integrated techniques. The ultimate goal of the analytical approaches discussed is to firmly establish structure and, thus, permit the study of structure-function relationships and eventually to allow the intelligent application of carbohydrate remodeling techniques in the preparation of new glycoproteins.

Structure and Metabolism of Glycosaminoglycans

This chapter examines new methods for the structural analysis of glycosaminoglycans. Recent advances in instrumentation and the availability of specific enzymes that act on these acidic polysaccharides has brought the sequencing of these complex molecules within reach. An understanding of structure is important in defining the true biological role of endogenous glycosaminoglycans and proteoglycans. A second benefit of improved analytical capabilities is that it provides a better understanding of the metabolism of these complex biopolymers.