Several clones of Chinese hamster ovary cells resistant to the cytotoxicity of the phytohemagglutinin from Phaseolus vulgaris show decreased binding o 125I-labeled phytohemagglutinin and contain decreased levels of a UDP-N-acetylglucosamine-glycoprotein N-acetylglucosaminyltransferase (EC 2.4.1.51; UDP-2-acetamido-2-deoxy-D-glucose:glycoprotein 2-acetamido-2-deoxy-D-glucosyltransferase) activity when compared to wild-type cells. The decrease in transferase activity varies from 45% to 96%, depending on the exogenous acceptor used in the enzyme assay. No differences between lectin-resistant and wild-type cells were noted for several other glycosyltransferases. The absence of a particular N-acetylglucosaminyltransferase in the lectin-resistant cells apparently results in defective glycosylation of lectin-binding glycoproteins on the cell surface. A phytohemagglutinin-resistant clone which shows decreased binding of llsIlabeled phytohemagglutinin but does not exhibit the enzyme deficiency has also been isolated.
Many mammalian and avian complex carbohydrates (glycoproteins and glycolipids) have highly branched oligosaccharides. Although the function of complex carbohydrates is not known, there is evidence to suggest that oligosaccharide branching may be an important factor in the process by which cells recognize one another and their environment. Asparagine-linked (N-glycosyl) oligosaccharides can be subdivided into at least 12 classes according to their branching patterns. It is presently believed that these classes all stem from a common precursor oligosaccharide containing three D-glucose, nine D-mannose, and two N-acetyl-D-glucosamine residues. This precursor is incorporated into the protein backbone in the rough endoplasmic reticulum and is then processed within the endoplasmic reticulum and Golgi apparatus by a series of highly specific glycosidases and glycosyltransferases to yield the various classes of N-glycosyl oligosaccharides. The branches that occur in N-glycosyl oligosaccharides are usually initiated by the incorporation of a N-acetylglucosamine (GlcNAc) residue. Our laboratory has studied four of the N-acetylglucosaminyltransferases (GlcNAc-transferases) involved in this initiation process. We have defined various factors which determine the synthetic pathway. There are at least three types of control that are commonly found. (i) Tissues differ in the relative activities of the different glycosyltransferases and glycosidases and, therefore, competition between two or more enzymes for a common intermediate often determines the synthetic route. (ii) The incorporation of a key glycosyl residue into an oligosaccharide may convert a nonsubstrate to a substrate for either a glycosyltransferase or a glycosidase. (iii) Conversely, the incorporation of a key residue may convert a substrate into a nonsubstrate. Other controls are undoubtedly operative during glycoprotein synthesis: e.g., the effect of the polypeptide sequence on transferase specificity, the distribution of transferases along the endomembrane system, and compartmentation and the availability of substrates and cofactors. These factors have not been studied in our laboratory. However, the oligosaccharides made by the hen oviduct correlate quite well with the control factors elucidated by our approach; other tissues are presently under investigation. Recent studies on the three-dimensional structures of N-glycosyl oligosaccharides have enabled us to explain certain features of glycosyltransferase substrate specificity on the basis of steric factors.
Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanavalin A (Con A) – Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains:[Formula: see text]Such glycopeptides have only a single mannose residue capable of interacting with Con A – Sepharose; an interacting mannose residue is either an α-linked nonreducing terminal residue or an α-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl α-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures:[Formula: see text]where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A – Sepharose and elution as a sharp peak with 0.1 M methyl α-D-glucopyranoside; glycopeptides giving this pattern had the structures:[Formula: see text]where R2 is either H, GlcNAc, Gal-β1,4-GlcNAc, or sialyl-Gal-β1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the minimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl α-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures:[Formula: see text]where R3 is either GlcNAc, Gal-β1,4-GlcNAc, or siaryl-Gal-β1,4-GlcNAc. Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A – Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.