A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitors of the Biosynthesis and Processing of N-Linked Oligosaccharide Chains

Chitin is one of the most important biopolymers in nature. It is mainly produced by fungi, arthropods and nematodes. In insects, it functions as scaffold material, supporting the cuticles of the epidermis and trachea as well as the peritrophic matrices lining the gut epithelium. Insect growth and morphogenesis are strictly dependent on the capability to remodel chitin-containing structures. For this purpose, insects repeatedly produce chitin synthases and chitinolytic enzymes in different tissues. Coordination of chitin synthesis and its degradation requires strict control of the participating enzymes during development. In this review, we will summarize recent advances in understanding chitin synthesis and its degradation in insects.

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Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases

Inhibitors of the biosynthesis and processing of n-linked

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.

p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

XBP-1 Is a Cell-Nonautonomous Regulator of Stress Resistance and Longevity

Mechanism of action of tunicamycin on the UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase

Loss of Hydrogen from Dihydroxyacetone Phosphate during Glyceryl Ether Synthesis

Previous studies from this laboratory on the mechanism of O-alkyl bond formation using a microsomal system from Tetrahymena pyriformis have shown that O-alkyl lipid synthesized from dihydroxyacetone phosphate has exchanged one hydrogen stereospecifically from the 1-sn position of the glycerol moiety. Indirect evidence suggested that acyldihydroxyacetone phosphate, an intermediate in )-alkyl lipid synthesis, is probably not the locus of the exchange. In the present study in was shown that stable acyldihydroxyacetone phosphate incubated in the presence of tritiated water and Tetrahymena microsomes does not become tritiated. When hexadecanol is added to the system O-alkyl lipid is produced which has incorporated one atom of hydrogen for each mole of hexadecanol at all time periods examined. Experiments in Ehrlich ascites tumor cells have shown that the hydrogen exchange also occurs in a mammalian system. The results indicate that the mechanism of O-alkyl lipid ether bond formation involves a hydrogen exchange and that this exchange occurs after the formation of acyldihydroxyacetone phosphate.

The formation of tritiated O-alkyl lipid from acyldihydroxyacetone phosphate in the presence of tritiated water.

Cultured vascular endothelial cells incorporate 35SO4 into a class of oligosaccharides which are N-glycosidically linked to cell-surface or extracellular proteins. This type of sulfated oligosaccharide was not synthesized by smooth muscle cell cultures and may represent sulfation of vascular tissue glycoproteins which are unique to endothelial cells. These endothelial cell 35S-labeled oligosaccharides are not released from the polypeptides under alkaline conditions that cleave O-glycosidically linked chains. The incorporation of both [3H]hexosamine and 35SO4 into these oligosaccharides is inhibited by tunicamycin, an antibiotic which inhibits protein N-glycosylation. 35S-Labeled oligosaccharides, isolated as glycopeptides, were resistant to a variety of chemical and enzymatic treatments which degraded vascular cell sulfated glycosaminoglycans but appear to contain terminal sialic acid residues. Endothelial cell sulfated oligosaccharide chains could also be distinguished from sulfated glycosaminoglycans by the apparent size and charge. Endothelial cells released or secreted 35S-labeled glycoproteins into the culture medium. These soluble 35S-labeled glycoproteins were partially purified by DEAE-cellulose chromatography and separated from the 35S-labeled proteoglycans, which are also released into the medium. The major 35S-labeled glycoproteins released from the cells had apparent molecular weights of 24 000--66 000. The cell-associated 35S-labeled glycoproteins, released with ethylenediaminetetraacetate or extracted with 0.5% octyl glucoside, were partially purified on DEAE-cellulose and had apparent molecular weights of 45 000--250 000.

Aaron Heifetz

Papers

Mechanism of action of tunicamycin on the UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase

Loss of Hydrogen from Dihydroxyacetone Phosphate during Glyceryl Ether Synthesis

The formation of tritiated O-alkyl lipid from acyldihydroxyacetone phosphate in the presence of tritiated water.

Biosynthesis of cell-surface sulfated glycoproteins by cultured vascular endothelial cells

Hydrogen exchange in the synthesis of glyceryl ether and in the formation of dihydroxyacetone in Tetrahymena pyriformis.