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Asparagine-linked oligosaccharides are localized to single extracytosolic segments in multi-span membrane glycoproteins.

ABSTRACT: A comprehensive survey of mammalian multi-span (polytopic) membrane proteins showed that asparagine(N)-linked oligosaccharides are localized to single extracytosolic segments. In most membrane proteins this is because potential consensus sites for N-glycosylation (Asn-Xaa-Ser/Thr, X not equal to Pro) are not found in multiple extracytosolic segments. In functional proteins where consensus N-glycosylation sites are contained within more than one extracytosolic segment, only the first segment contains N-linked carbohydrate. An exception is the alpha-subunit of the Na+ channel, which consists of a duplicated structure containing two glycosylated segments. The average size of established N-glycosylated loops connecting two transmembrane segments is 62 residues, with the smallest glycosylated loop being 33 residues in size. N-glycosylated sites are more highly conserved than non-glycosylated (primarily cytosolic) sites and are more common toward the N-terminus of the membrane domain of multi-span membrane proteins. The optimal conditions for glycosylation of consensus sites within an extracytosolic domain of a multi-span membrane protein are (i) the acceptor site is well-spaced (greater than 10 residues) from the transmembrane domain, (ii) the loop is greater than 30 residues in size and (iii) the segment is the first in the protein to contain a suitable extracytosolic consensus site. The localization of N-linked oligosaccharide chains to a single protein segment suggests either glycosylation of multiple loops may compromise protein folding or function, or only a single polypeptide domain can be optimally glycosylated during biosynthesis in vivo.

SUBMITTER: Landolt-Marticorena C

PROVIDER: S-EPMC1137217 | biostudies-other | 1994 Aug

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 are localized to single extracytosolic segments in multi-span membrane glycoproteins.

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