Inder K. Vijay

Purification and characterization of glucosidase II involved in N-linked glycoprotein processing in bovine mammary gland.

Glucosidase II is an endoplasmic-reticulum-localized enzyme that cleaves the two internally alpha-1,3-linked glucosyl residues of the oligosaccharide Glc alpha 1----2Glc alpha 1----3Glc alpha 1----3Man5-9GlcNAc2 during the biosynthesis of asparagine-linked glycoproteins. We have purified this enzyme to homogeneity from the lactating bovine mammary gland. The enzyme is a high-mannose-type asparagine-linked glycoprotein with a molecular mass of approx. 290 kDa. Upon SDS/polyacrylamide-gel electrophoresis under reducing conditions, the purified enzyme shows two subunits of 62 and 64 kDa, both of which are glycosylated. The pH optimum is between 6.6 and 7.0. Specific polyclonal antibodies raised against the bovine mammary enzyme also recognize a similar antigen in heart, liver and the mammary gland of bovine, guinea pig, rat and mouse. These antibodies were used to develop a sensitive enzyme-linked immunosorbent assay for glucosidase II.

A new fluorescent tag for labeling of saccharides.

Potential aldehyde groups of N-acetylglucosamine and its di- and tri-saccharides were coupled with 7-amino-4-methylcoumarin (AMC) by reductive amination in the presence of sodium cyanoborohydride. The products were purified by silica gel chromatography. Acid hydrolysis and digestion with glycosidases gave the expected products. One picomole of the sugar-AMC complexes could be detected after thin-layer chromatography by fluorescence under uv light. The sensitivity of the detection of the fluorescently labeled carbohydrates is comparable to that of NaB3H4 labeling.

[48] Synthesis of capsular polymers contaiing polysialic acid in Escherichia coli 07-K1☆☆☆★★★

Publisher Summary This chapter presents a procedure for synthesis of capsular polymers containing polysialic acid in Escherichia coli 07-K11. For enzyme preparation a heavily encapsulated colony of E. coli K-235 is used. Two similar procedures are used for preparation of the membranous STC. Both consist of membrane hybrids composed of inner and outer membranes. In procedure 1, an operationally defined soluble and particulate STC are resolved whereas in procedure 2, both are included in the same fraction. In preparation of undecaprenyl phosphate-depleted sialyltransferase complex delipidation of both the soluble and paniculate STC is carried out by the acetone-butanol procedure at –20 °. Preparation of exogenous acceptors involves 3 H- or U- 14 C-labeled oligo- and polysialic acid. Further, purification of oligosialic acid is carried out on polyacrylamide gel electrophoresis.

Processing alpha-glucosidase I is an inverting glycosidase.

Abstractα-Glucosidase I is a key enzyme in the biosynthesis of asparagine-linked oligosaccharides catalyzing the first processing event after the en bloc transfer of Glc3Man9GlcNAc2 to proteins. This enzyme is an inhibitor target for anti-viral agents that interfere with the formation of essential glycoproteins required in viral assembly, secretion and infectivity. Of fundamental mechanistic interest for all oligosaccharide hydrolyzing enzymes is the stereochemical course of the reaction which can occur with either retention or inversion of anomeric configuration. The stereochemistry is used to categorize enzymes and is important in designing mechanism-based inhibitors. To determine the stereochemical course of the α-glucosidase I reaction, the release of glucose from a synthetic trisaccharide substrate, Glc(α1-2)Glc(α1-3)GlcαO(CH2)4COOCH3 was directly monitored by 1H NMR spectroscopy. Both the yeast and bovine mammary gland enzymes released β-glucose concomitant with the formation of the Glc(α1-3)GlcαO(CH2)8COOCH3 disaccharide product demonstrating that both enzymes operate with inversion of anomeric configuration.

Characterization of a new isomer of lipid-linked heptasaccharide formed during in vitro biosynthesis of mammary glycoproteins

Incubation of microsomes from the lactating bovine mammary tissue with UDP-GlcNAc and GDP-Man results in the biosynthesis of lipid-linked oligosaccharides Man,r(GlcNA~)~, n = l-9 [I]. Pulse and chase kinetics indicated these to be interrelated as precursor-products for the biosynthesis of asparaginelinked glycoproteins in this tissue. Structural analyses showed that among these, Man(GlcNAch through ManS(GlcNAc)z and Man,(GlcNAc)* species were monoisomeric [ 11; however, 2 isomers of Maq(GlcNAc), and 3 isomers of Mans(GlcNA~)~ could be iden tidied [2]. The resolution of isomers among hexaand heptasaccharides was facilitated by the specificity of endo D and endo H towards oligomannosylchitobiose substrates [3,4]. The heptasaccharide cleaved by endo D was characterized as Manal+2Manal+3Maml+6(Mancul+3)Mar@l+4(3)GlcNAcf31+4(3)GlcNAc. Structural data on the octathrough decasaccharide indicated that an additional isomer might also be present in the endo-Dcleaved heptasaccharide. Using controlled acetolysis, in which only incipient cleavage of the a-1,6 linkages occurs and aided by the availability of an cr-1,2-specific mannosidase, we now report the characterization of another isomer within the heptasaccharide, Mans(GlcNAc),.

STAT5a regulates the GlcNAc-1-phosphate transferase gene transcription and expression.

The dolichyl-phosphate alpha-N-acetylglucosaminephosphotransferase 2 (Dpagt2) gene in the mouse has a housekeeping promoter, and its expression is regulated during the development and hormonally modulated lactogenesis of the mammary gland. Previous studies showed that the transcription of the mouse mammary Dpagt2 gene is stimulated by the lactogenic hormones, insulin, glucocorticoid receptor (GR), and prolactin. Transcription factors which bind to the Dpagt2 gene promoter region can influence the expression level of the Dpagt2 gene. It is supposed that the Dpagt2 gene promoter region (bases pairs -1462 to -5) maybe contain 10 putative STAT (signal transducer and activator of transcription) binding sites: TTN (5/6) AA. In order to identify the STAT factors involved in the transcription of the GPT gene, 32P labeling probes and lactating mouse nuclear extracts were prepared. Electrophoretic Mobility Shift Assays (EMSA) show that the region (bases pairs -386 to -322, where there is a STAT binding site, TTTCAAAAA) binds to STAT5a, not to glucocorticoid receptor (GR) or other STAT factors. The involvement of STAT5a in regulating the expression of the mouse Dpagt2 gene was further investigated by transient transfections of various Dpagt2 promoter/luciferase (Luc) constructs into COS 7 cells. The results showed that co-transfection of STAT5a or prolactin receptor can enhance Dpagt2 promoter activities in the promoter construct pGL-MX6 (from base pairs -386 to -5), but not in the promoter construct pGL-MX7 (from base pairs -322 to -5). This paper first reports that STAT5a is involved in the binding between -386 and -322 base pairs of the Dpagt2 gene promoter and stimulates the expression of the Dpagt2 gene transcription in the mouse lactating mammary gland.

Accumulation of pentamannose oligosaccharides in human mononuclear leukocytes by action of swainsonine, an inhibitor of glycoprotein processing

Swainsonine, a known inhibitor of the alpha-mannosidase II involved in processing of asparagine-linked glycoproteins, causes accumulation of hybrid-type oligosaccharide-containing glycoproteins in mammalian cells. Swainsonine augments lymphokine-activated, killer-cell induction at suboptimal doses of interleukin-2; the amount needed to increase LAK activity is 100-1000 fold higher than required to completely inhibit mannosidase II. Human mononuclear lymphocytes, when treated with these relatively high (58 microM) concentrations of swainsonine showed a 3-4 fold increase in D-[3H]mannose incorporation into the glycan as compared to glycans of untreated cells. Analysis indicated accumulation of high-mannose type, free oligosaccharides in the soluble fractions of the cell. Chromatographic analysis of glycan obtained by D-[2-3H]mannose labeling of human mononuclear lymphocytes showed synthesis of a new oligosaccharide, at 58 microM of swainsonine, that contained 36% of the total radioactivity incorporated into the glycan (oligosaccharide pool). This oligosaccharide fraction was resistant to hydrolysis by endoglycosidase H, endoglycosidase F, O and N-glycanase, but was susceptible to cleavage by Jack bean alpha-mannosidase and was bound > 90% to concanavalin A-Sepharose. A similar chromatographic elution profile was obtained from glycans labeled with D-[2-3H]mannose from mouse B16F10 melanoma and baby hamster kidney cells subsequent to swainsonine treatment. Methylation analysis of free oligosaccharides obtained from MNL revealed the presence of a pentamannose. These results indicate the accumulation of a free high-mannose oligosaccharide rather than expected hybrid-type structure on treatment of cells with relatively high concentrations of swainsonine.

ATTACHMENT OF SUGARS ON LIPID-LINKED CHITOBIOSYL UNIT FOR THE INITIAL ASSEMBLY OF OLIGOSACCHARIDE MOIETIES OF ASPARAGINE-LINKED GLYCOPROTEINS IN DIFFERENT EUKARYOTIC SYSTEMS

Numerous studies in the last decade have provided evidence that chitobiosylpyrophosphodolichol serves as the initial acceptor glycolipid for the assembly of the oligosaccharide, Glc 3 Man 9 (GlcNAc) 2 prior to its transfer to nascent polypeptides for the biosynthesis of asparagine-linked glycoproteins in eukaryotic systems. The tetradecasaccharide synthesized by Chinese hamster ovary cells was shown to be a multibranched precursor; its biosynthesis in vivo was proposed to occur by an ordered sequence of reactions. In vitro preparations from the lactating bovine mammary tissue and Saccharomyces cerevisiae yielded multiple isomers of several of the intermediates leading up to Glc 3 Man 9 (GlcNAc) 2 and raised the possibility of multiple pathways of biosynthesis in different cell types. In vivo studies were therefore undertaken with slices from the lactating bovine mammary tissue, baby hamster kidney cells and Crithidia fasciculata. Our results show that the ordered sequence of assembly for the biosynthesis of the precursor oligosaccharide is the same for different cell types. Minor, multiple isomers, observed during biosynthesis in microsomal preparations, may result from lack of absolute specificities of the enzymes and/or disorganization of the glycosyltransferases in an in vitro set up. Thus, the pathway of assembly of the carbohydrate unit prior to its transfer to polypeptides of N-linked glycoproteins appears to have been conserved during evolution.