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Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing.

ABSTRACT: Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.

SUBMITTER: Hughes RC

PROVIDER: S-EPMC1148447 | biostudies-other | 1987 Nov

REPOSITORIES: biostudies-other

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Project description:Previous studies have established that metastatic tumour cells express high levels of beta 1-6-branched Asn-linked oligosaccharides which can be detected with the lectin leucoagglutinin (L-PHA) [Dennis, Laferté, Waghorne, Breitman & Kerbel (1987) Science 236, 582-585]. In order to identify L-PHA-binding glycoproteins which may play a role specifically in colon cancer, we have prepared monoclonal antibodies (MAbs) to the moderately well-differentiated human colon carcinoma cell line HT29. In this paper we present the initial characterization of a family of structurally related L-PHA-binding glycoproteins detected by MAb 1H9 which are differentially expressed and processed by HT29 cells and by two other human colon carcinoma cell lines, SW480 and SW620. In contrast to HT29, the SW480 and SW620 cell lines were established from a poorly differentiated grade III/IV primary tumour and one of its lymph node metastases respectively. MAb 1H9 detects in HT29 cells a conformational determinant present on three L-PHA-binding glycoproteins of 100, 70 and 25kDa, as well as a 74 kDa glycoprotein with high-mannose-type Asn-linked oligosaccharides. Pulse-chase experiments and peptide mapping analyses revealed that the 74 kDa and 100 kDa species are related by carbohydrate processing and are probably derived from a common 76 kDa precursor. On the other hand, the 70 kDa glycoprotein is synthesized from an endoglycosidase H-sensitive precursor of 56 kDa which is structurally related to, but distinct from, the aforementioned 76 kDa precursor. In addition, the 100 kDa species is secreted into the culture medium, whereas the 70 kDa glycoprotein is retained intracellularly. SW480 and SW620 cells showed qualitative and quantitative differences from HT29 cells, including increased secretion of a smaller L-PHA-binding glycoprotein of 92 kDa into the culture medium, as well as apparent differences in glycosylation of the intracellular 66 kDa glycoprotein. These results suggested that the expression, glycosylation and subcellular localization of this family of L-PHA-binding glycoproteins may correlate with the differentiation status of colon cancer cells and/or reflect biochemical changes. characteristic of more progressive metastatic tumours.

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Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing.

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