Y. T. Pan

Trehalose synthase converts glycogen to trehalose

Trehalose (α,α‐1,1‐glucosyl‐glucose) is essential for the growth of mycobacteria, and these organisms have three different pathways that can produce trehalose. One pathway involves the enzyme described in the present study, trehalose synthase (TreS), which interconverts trehalose and maltose. We show that TreS from Mycobacterium smegmatis, as well as recombinant TreS produced in Escherichia coli, has amylase activity in addition to the maltose ↔ trehalose interconverting activity (referred to as MTase). Both activities were present in the enzyme purified to apparent homogeneity from extracts of Mycobacterium smegmatis, and also in the recombinant enzyme produced in E. coli from either the M. smegmatis or the Mycobacterium tuberculosis gene. Furthermore, when either purified or recombinant TreS was chromatographed on a Sephacryl S‐200 column, both MTase and amylase activities were present in the same fractions across the peak, and the ratio of these two activities remained constant in these fractions. In addition, crystals of TreS also contained both amylase and MTase activities. TreS produced both radioactive maltose and radioactive trehalose when incubated with [3H]glycogen, and also converted maltooligosaccharides, such as maltoheptaose, to both maltose and trehalose. The amylase activity was stimulated by addition of Ca2+, but this cation inhibited the MTase activity. In addition, MTase activity, but not amylase activity, was strongly inhibited, and in a competitive manner, by validoxylamine. On the other hand, amylase, but not MTase activity, was inhibited by the known transition‐state amylase inhibitor, acarbose, suggesting the possibility of two different active sites. Our data suggest that TreS represents another pathway for the production of trehalose from glycogen, involving maltose as an intermediate. In addition, the wild‐type organism or mutants blocked in other trehalose biosynthetic pathways, but still having active TreS, accumulate 10‐ to 20‐fold more glycogen when grown in high concentrations (≥ 2% or more) of trehalose, but not in glucose or other sugars. Furthermore, trehalose mutants that are missing TreS do not accumulate glycogen in high concentrations of trehalose or other sugars. These data indicate that trehalose and TreS are both involved in the production of glycogen, and that the metabolism of trehalose and glycogen is interconnected.

Inhibition of processing of plant N-linked oligosaccharides by castanospermine

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that inhibits lysosomal alpha- and beta-glucosidase. It also inhibits processing of influenza viral glycoproteins by inhibiting glucosidase I and leads to altered glycoproteins with Glc3Man7GlcNAc2 structures. Castanospermine was tested as an inhibitor of glycoprotein processing in suspension-cultured soybean cells. Soybean cells were pulse-labeled with [2-3H]mannose and chased for varying periods in unlabeled medium. In normal cells, the initial glycopeptides contained oligosaccharides having Glc3Man9GlcNAc2 to Glc1Man9GlcNAc2 structures and these were trimmed during the chase to Man9GlcNac2 to Man7GlcNAc2 structures. In the presence of castanospermine, no trimming of glucose residues occurred although some mannose residues were apparently still removed. Thus, the major oligosaccharide in the glycopeptides of castanospermine-incubated cells after a 90-min chase was a Glc3Man7GlcNAc2 structure. Smaller amounts of Glc3Man6GlcNAc2 and Glc3Man5GlcNAc2 were also identified. Thus, in plant cells, castanospermine also prevents the removal of the outermost glucose residue.

Chapter 5 Biosynthesis 7. How Can N-Linked Glycosylation and Processing Inhibitors be Used to Study Carbohydrate Synthesis and Function

Publisher Summary This chapter discusses how N-linked glycosylation and processing inhibitors can be used to study carbohydrate synthesis and its function. Glycoproteins are widely distributed in nature, not only in animal cells but also in plants, microorganisms and viruses. The oligosaccharide chains of the N-linked glycoproteins are believed to be involved in a wide variety of biological functions. Using various inhibitors to prevent synthesis of or to modify the carbohydrate portion of the glycoprotein has provided a useful way to determine the function of the oligosaccharide portion of a given glycoprotein. Such inhibitors are also useful for studies on the mechanism of biosynthesis of the oligosaccharide chains, since they may give rise to various intermediates that can provide important structural information. The assembly of the various types of N-linked oligosaccharides involves two distinct series of reactions. In the first stage of synthesis, a precursor oligosaccharide is synthesized on a lipid carrier by the stepwise addition of sugars from their nucleoside diphosphate derivatives or from a lipid-linked monosaccharide intermediate to the lipid oligosaccharide, and then this oligosaccharide is transferred “en bloc” to the polypeptide chain.

Control of N-linked oligosaccharide synthesis: cellular levels of dolichyl phosphate are not the only regulatory factor [Erratum to document cited in CA113(13):112839p]

When MDCK cells were incubated in the presence of the protein synthesis inhibitor puromycin or cycloheximide, there was a rapid and concentration-dependent inhibition in the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and into protein. However, mannose incorporation into dolichyl-P-mannose was not affected. Interestingly, these inhibitors did block [6-3H]glucosamine incorporation into dolichyl-PP-GlcNAc as well as into lipid-linked oligosaccharides. Similar results were obtained when other cell lines were used and also when inhibitors of protein glycosylation such as beta-hydroxynorvaline and beta-fluoroasparagine were used. Cells incubated in puromycin did not show any changes in the levels of sugar nucleotides, GDP-mannose or UDP-GlcNAc, or in the in vitro activities of the glycosyltransferases that add mannose to the lipid-linked oligosaccharides. The inhibition of mannose incorporation into lipid-linked oligosaccharides could not be overcome by addition of dolichyl-P to the inhibited cells, even though the addition of dolichyl-P to control cells stimulated mannose incorporation into dolichyl-P-mannose, lipid-linked oligosaccharides, and protein from 3- to 5-fold. Thus, limitations in the levels of dolichyl-P do not appear to be a major factor in this inhibition. On the other hand, addition of the tripeptide acceptor N-acyl-Asn-Try-Thr did overcome the puromycin inhibition to some extent, suggesting that accumulation of some intermediate such as lipid-linked oligosaccharides might be involved in the inhibition.