Gilbert Ashwell

The hepatic asialoglycoprotein receptor

Asialo- (i.e., galactose-terminal) glycoproteins are specifically and avidly recognized by a mammalian hepatic parenchymal cell receptor. This receptor, itself a glycoprotein, binds ligand molecules and directs their delivery to lysosomes for catabolism. The receptor is reutilized during this process of receptor-mediated endocytosis. Ligand specificity is conferred by galactose or N-acetyl-galactosamine at the nonreducing termini of the oligosaccharide chains. The receptor appears to be a transmembrane protein and is localized both to the cell surface as well as to several membranous intracellular compartments.

Carbohydrate Recognition Systems for Receptor-Mediated Pinocytosis

Pinocytosis is a mechanism for the transport of molecules from the exterior to the interior of the cell without transfer through the plasma membrane. Transport of molecules occurs when the membrane wraps around the external material to form vesicles which are pinched off and internalized. The vesicles fuse with lysosomes, the contents of which become enriched with the material brought in from the outside. If the area of the plasma membrane which is internalized has receptors to which a particular substance is tightly bound, that substance is delivered to lysosomes far more efficiently and selectively than if it were simply trapped within the pinocytotic vesicle. In recent years, receptor-mediated pinocytosis systems have been described for substances as diverse as cholesterol esters, vitamin B12, hormones and growth factors, circulating glycoproteins, and hydrolytic enzymes. It is the last two groups, which involve the recognition of specific carbohydrate residues, that are the subject of this chapter.

Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 (O-GlcNAcase) knockout impacts O-GlcNAc cycling, metabolism, and dauer

A dynamic cycle of O-linked N-acetylglucosamine (O-GlcNAc) addition and removal acts on nuclear pore proteins, transcription factors, and kinases to modulate cellular signaling cascades. Two highly conserved enzymes (O-GlcNAc transferase and O-GlcNAcase) catalyze the final steps in this nutrient-driven “hexosamine-signaling pathway.” A single nucleotide polymorphism in the human O-GlcNAcase gene is linked to type 2 diabetes. Here, we show that Caenorhabditis elegans oga-1 encodes an active O-GlcNAcase. We also describe a knockout allele, oga-1(ok1207), that is viable and fertile yet accumulates O-GlcNAc on nuclear pores and other cellular proteins. Interfering with O-GlcNAc cycling with either oga-1(ok1207) or the O-GlcNAc transferase-null ogt-1(ok430) altered Ser- and Thr-phosphoprotein profiles and increased glycogen synthase kinase 3β (GSK-3β) levels. Both the oga-1(ok1207) and ogt-1(ok430) strains showed elevated stores of glycogen and trehalose, and decreased lipid storage. These striking metabolic changes prompted us to examine the insulin-like signaling pathway controlling nutrient storage, longevity, and dauer formation in the C. elegans O-GlcNAc cycling mutants. Indeed, we found that the oga-1(ok1207) knockout augmented dauer formation induced by a temperature sensitive insulin-like receptor (daf-2) mutant under conditions in which the ogt-1(ok430)-null diminished dauer formation. Our findings suggest that the enzymes of O-GlcNAc cycling “fine-tune” insulin-like signaling in response to nutrient flux. The knockout of O-GlcNAcase (oga-1) in C. elegans mimics many of the metabolic and signaling changes associated with human insulin resistance and provides a genetically amenable model of non-insulin-dependent diabetes.

Membrane glycoproteins and recognition phenomena

Abstract Information encoded in specific sugar residues o f membrane glycoproteins exerts a profound effect on cellular reactivity.

Quantitation of specific serum glycoproteins in malignancy

Abstract Quantitative studies have been made on the serum carbohydrate and glycoprotein levels of cancer patients and compared with the values obtained on normal volunteers and chronically ill controls. Of the 15 glycoproteins studied, 7 (transferrin, α 2 -macroglobulin, GC-globulin, IgA, IgD, IgG, IgM) remained invariant in all these categories. Three ( α 1 -acid glycoprotein, ceruloplasmin, α 1 -antitrypsin) were elevated in both the cancer and the chronically ill group. Two (haptoglobin, hemopexin) were elevated in cancer patients but not the non-malignant controls. Three ( α 2 Hs-glyco-protein, β 2 -glycoprotein I, prealbumin) were decreased in malignancy and unchanged in the pathological control group.

[87] A membrane receptor protein for asialoglycoproteins

Publisher Summary Specific receptor sites have been identified on hepatic plasma membranes that appear to be involved in the catabolism of plasma glycoproteins by virtue of their ability to bind selectively to asialoglycoprotein, i.e., glycoproteins from which the terminal sialic acid residues have been removed. These receptors have been solubilized from particulate liver preparations by extraction with the nonionic detergent Triton X-100 3 and further purified by affinity chromatography on a column of Sepharose to which asialo-orosomucoid has been covalently attached. Binding of the receptor protein to the immobilized ligand occurs at a neutral or slightly alkaline pH and has an absolute requirement for calcium; elution is accomplished either by reducing the pH to between 5.6 and 6.4 or by addition of the calcium chelator EDTA. Excess Triton X-100 can be readily removed, prior to elution of the receptor by washing the column with a neutral, calcium-containing, aqueous buffer.

Effect of x-irradiation upon the enzyme systems of the mouse spleen.

Conclusions Total whole body irradiation of white mice with 640 r (L90 dose) revealed the following changes in the metabolism of spleen homogenates: 1) no effect upon the activity of the succinoxidase system, 2) marked increase in activity of alkaline phosphatase which was especially marked with ATP as substrate and 3) decreased ability to synthesize (or maintain) the high energy phosphorus compounds coupled with the oxidation of succinate.

METABOLISM OF ASCORBIC ACID AND RELATED URONIC ACIDS, ALDONIC ACIDS, AND PENTOSES

The outlines of the biosynthetic pathway involved in the formation of Lascorbic acid in mammalian tissues are by now abundantly clear: as is evident from the overlapping reports describing it in such detail in this monograph. However, there are a t least two closely related areas on which little or no information is currently available: (1) the controversial role of 3-keto-~-gulonic acid, and (2) the subsequent metabolic fate of L-ascorbic acid in animal tissues. Consequently this report will confine itself largely to a description of recent work in these areas. In addition, there has emerged from these studies a newer appreciation of the unusually broad but nevertheless uniquely restricted specificity requirements for each of the purified enzymes participating in these reactions. On the basis of these observations, simple rules can now be adduced whereby the reactivity of a given compound may be successfully predicted on the basis of purely stereochemical considerations. A diagrammatic construction of our current conception of the metabolic relationship existing between L-ascorbic acid and the corresponding sugar acids is illustrated in FIGURE 1.

Enzymatic synthesis of galactogen in the snall, Helix, pomatia

Abstract The galactose polymer, galactogen, has been reported in numerous pulmonate snails ( Ghose, 1963 ; Horstmann, 1956 ; May, 1931 ; McMahon et , al. , 1957 ; Meenakshi, 1954 ; Rigby, 1963 ). Whereas this polysaccharide is found exclusively in the reproductive system (albumen gland) and freshly laid eggs, glycogen is present in the rest of the body. As isolated from the albumen gland of the land snail, Helix , pomatia , the polymer has been reported to contain both D- and L-galactose in the approximate ratio of 6:1 ( Bell and Baldwin, 1941 ). The methylation studies of Bell and Baldwin (1938) , yielding essentially equimolar proportions of 2, 3, 4, 6-tetramethyl- and 2, 4-dimethyl-D-galactose, are the basis for a proposed structure of galactogen consisting of either a β-1→3 or a β-1→6 linear chain wherein each galactopyranose unit bears a terminal galactose substituent on C-6 or C-3, respectively. L-Galactose is thought to be part of the side chain ( Bell and Baldwin, 1941 ; May and Weinland, 1956 ). Heretofore, however, no information relating to the mode of biosynthesis of this polysaccharide has been available. The present report describes the preparation of an extract from the albumen gland of H. , pomatia which utilizes UDP-D-galactose-C14 for the formation of a radioactive galactose polymer, tentatively identified as galactogen.

Optical activity and conformation of carbohydrates: Part III. Preparation and optical activity of methyl 2-acetamido-2-deoxy-α-and β-D-mannopyranosides and the corresponding furanosides

Abstract The methyl 2-acetamido-2-deoxy-α- and β- D -mannopyranosides and their corresponding furanosides have been prepared by treatment of 2-acetamido-2-deoxy- D -mannose with methanol in the presence of Amberlite IR-120 (H+) and separation on Dowex-1 ion-exchange resin and by paper chromatography. The optical rotatory dispersion and circular dichroism spectra of the four glycosides have been measured and compared with previous findings on the α and β anomers of methyl 2-acetamido-2-deoxy- D -glucopyranoside and -galactopyranoside. Differences were interpreted in terms of symmetry rules of optical activity and the axial orientation of the 2-acetamido-2-deoxy substituent in the D -mannose derivatives.