The Biochemistry of glycoproteins and proteoglycans

How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum.

The dramatic increase in sequence information in the form of expressed sequence tags (ESTs)1 and genomic sequence has created a ‘gene function gap’, with the identification of new genes faroutpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms2,3. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.

Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells

Reduced temperature prevents transfer of a membrane glycoprotein to the cell surface but does not prevent terminal glycosylation

An antibody preparation in which an N-glycosylation site of the Fc domain of the antibody is substituted with a biantennary oligosaccharide, characterised in that at least 20 % of the preparation comprises antibody molecules having an oligosaccharide that contains at least one galactose residue.

Preparation of glycosylated antibodies

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins.

A method for modifying eukaryotic and prokaryotic proteins to extend their in vivo circulatory lifetimes. In the preferred embodiment, enzymatic and/or chemical treatments are used to produce a modified protein carrying one or more covalently attached trisaccharide, sialic acid→galactose→N-acetylglucosamine→(SA→Gal.fwdarw.GlcNAc→), or tetrasaccharide (SA→Gal→GlcNAc→GlcNAc→) moieties. The method can be applied to any natural or recombinant protein possessing asparagine-linked oligosaccharides or to any non-glycosylated protein that can be chemically or enzymatically derivatized with the appropriate carbohydrate units. Following injection into an animal, the modified glycoproteins are protected from premature clearance by cells of the liver and reticulo-endothelial system which recognize and rapidly internalize circulating glycoproteins with carbohydrate chains containing terminal Gal, GlcNAc, fucose or mannose residues. The method can also be used to mask antigenic determinants on foreign proteins which would otherwise pro The United States Government has certain rights in this invention by virtue of National Institutes of Health grants No. CA26712, GN31318, and CA14051.

Synthetic method for enhancing glycoprotein stability

Enrichment of insertional mutants following retrovirus gene trap selection

Publisher Summary This chapter presents the procedures utilized by laboratories to radiolabel the dolichol-linked oligosaccharides and study their biosynthesis. Two general approaches are adopted. The chapter primarily summarizes the in vivo studies, where pulse-labeling is used to identify intermediates and determine precursor-product relationships. These studies reflect the true biosynthetic conditions and thereby allow an accurate determination of the kinetic interrelationships of the various oligosaccharide-lipid intermediates. In addition, the chapter concerns in vitro studies with isolated membrane preparations. The advantages of this approach lie in the ability to optimize label incorporated into the oligosaccharide-lipid intermediates, and the relative ease in manipulating incubation conditions to modify and test the biosynthetic machinery.

S. Catherine Hubbard

Papers

Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins.

Synthetic method for enhancing glycoprotein stability

Enrichment of insertional mutants following retrovirus gene trap selection

[35] N-asparagine-linked oligosaccharides: Biosynthesis of the lipid-linked oligosaccharides

The Lipid-Linked Oligosaccharide and Its Role in Glycoprotein Synthesis