α-Mannosidases in Asparagine-Linked Oligosaccharide Processing and Catabolism

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse–chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin–cruzipain interaction. This result is consistent with the known pathway of proteinN-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-β-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin–cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

Download Full-text

Inhibition of myoblast fusion by the glucosidase inhibitor N-methyl-1-deoxynojirimycin, but not by the mannosidase inhibitor 1-deoxymannojirimycin

Biochemical Journal ◽

10.1042/bj2380335 ◽

1986 ◽

Vol 238 (2) ◽

pp. 335-340 ◽

Cited By ~ 11

Author(s):

P C Holland ◽

A Herscovics

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Myoblast Fusion ◽

Fusion Process ◽

Cell Surface Expression ◽

Recognition Process ◽

Glucosidase Inhibitor ◽

Surface Recognition ◽

Oligosaccharide Processing ◽

High Mannose

The effects of N-linked-oligosaccharide-processing inhibitors on the fusion of rat L6 myoblasts to form myotubes were examined. The glucosidase inhibitor N-methyl-1-deoxynojirimycin (MDJN) greatly inhibited fusion, whereas the mannosidase inhibitor 1-deoxymannojirimycin (ManDJN) had relatively little effect, although both compounds prevented the formation of N-linked complex oligosaccharides. These results indicate that complex oligosaccharides on glycoproteins do not play a role in myoblast fusion. With MDJN, high-mannose oligosaccharides containing three glucose residues and seven to eight mannose residues were found at the cell surface, whereas with ManDJN, non-glucosylated high-mannose oligosaccharides with seven to nine mannose residues were obtained. These results indicate that the persistence of glucose residues on high-mannose oligosaccharides may be responsible for the inhibition of fusion. It is suggested that glucose either masks the cell-surface recognition process leading to fusion or prevents the cell-surface expression of specific glycoprotein(s) essential to the fusion process.

Download Full-text

Possible role for peptide-oligosaccharide interactions in differential oligosaccharide processing at asparagine-107 of the light chain and asparagine-297 of the heavy chain in a monoclonal IgG1.kappa.

Biochemistry ◽

10.1021/bi00311a026 ◽

1984 ◽

Vol 23 (16) ◽

pp. 3736-3740 ◽

Cited By ~ 38

Author(s):

Georgina Savvidou ◽

Michel Klein ◽

Arthur A. Grey ◽

Keith J. Dorrington ◽

Jeremy P. Carver

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Oligosaccharide Processing

Download Full-text

Effects of glycosylation inhibitors on human growth hormone receptor in cultured human lymphocytes

Acta Endocrinologica ◽

10.1530/acta.0.1190517 ◽

1988 ◽

Vol 119 (4) ◽

pp. 517-524 ◽

Cited By ~ 9

Author(s):

Kumiko Asakawa ◽

Jose A. Hedo ◽

Phillip Gorden ◽

Kazuo Shizume

Keyword(s):

Molecular Weight ◽

Growth Hormone ◽

Human Growth Hormone ◽

Growth Hormone Receptor ◽

Human Lymphocytes ◽

Human Growth ◽

Control Value ◽

Receptor Complexes ◽

Oligosaccharide Processing ◽

Receptor Number

Abstract. IM-9 cultured human lymphocytes were treated with N-linked glycosylation inhibitors, N-linked oligosaccharide processing inhibitors, or neuraminidase to study the effect of glycosylation modification on human growth hormone binding and molecular weight of surface hGH receptor. One mg/l tunicamycin and 20 mmol/l glucosamine decreased 125I-hGH binding to the cells to 46.3 ± 2.4% (mean ± sem) and 21.9 ± 0.2% of the controls, respectively. The hGH binding was 33.0 ± 18.4% of the control value in the cells treated with monensin. The inhibition of binding was due to a decrease in the hGH receptor number without any affinity changes in these cells. Neither 1 mg/l swainsonine nor 100 mg/l castanospermine had any effect on the hGH binding. On the other hand, 125I-hGH binding to neuraminidase-treated cells was significantly enhanced with accompanying affinity changes. When 125I-hGH was cross-linked to IM-9 cells, there were no differences in the molecular weight of hGH receptor complexes (140K) between untreated cells and cells treated with tunicamycin, glucosamine, monensin, or castanospermine. However, the 128K hGH-receptor complex appeared in swainsonine-treated cells; this complex was sensitive to endoglycosidase H. These data show that the altered carbohydrate moiety changed hGH binding and the size of surface hGH receptor and suggest that glycosylation of receptor is important for the binding of hGH and for its physiological action.

Download Full-text