Characterization of a mannosidase acting on alpha 1----3- and alpha 1----6-linked mannose residues of oligomannosidic intermediates of glycoprotein processing.
Baby hamster kidney (BHK) cell extracts catalyze the conversion of [3H]mannose-labelled (Man)5GlcNAc and (Man)6GlcNAc oligosaccharides to a (Man)3GlcNAc species that retains affinity for concanavalin-A-Sepharose and appears to be Man alpha 1----3[Man alpha 1----6]Man beta 1----4GlcNAc. The properties of the (Man)5GlcNAc-hydrolase activity differ from lysosomal alpha-mannosidases as well as previously described processing mannosidases acting on oligosaccharide intermediates of N-glycan assembly. Mosquito cell extracts catalyze hydrolysis of (Man)6GlcNAc but lack the (Man)5GlcNAc hydrolase activity detected in BHK cell extracts. Glycopeptide analysis has been carried out on a ricin-resistant BHK mutant RicR14 that lacks N-acetylglucosaminyl transferase I and fails to convert oligomannosidic N-glycans to complex-type chains, and mosquito cells that constitutively lack N-acetylglucosaminyl transferase I. In both cell lines, the cellular glycoproteins contain (Man)5GlcNAc oligosaccharide as the major stable component equivalent to a 15-20-fold increase compared with normal BHK cells. Although containing very high amounts of asparagine-linked (Man)5(GlcNAc)2, RicR14 cells exhibit (Man)5GlcNAc hydrolase activity at levels similar to wild-type BHK cells. This result, together with previous work [Foddy, L., Feeney, J. & Hughes, R.C. (1986) Biochem. J. 233, 697-706] showing the complete inhibition of conversion of oligomannosidic intermediates to complex-type N-glycans in BHK cells treated with swainsonine, an inhibitor of mannosidase II but not the (Man)5GlcNAc hydrolase activity, argues against a major role for the (Man)5GlcNAc hydrolase activity in N-glycan assembly and suggesting other functions for the mannosidase activity in BHK cells.
Monis
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E
National Institute for Medical Research, London, England.
Bonay
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P
Hughes
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RC
D000330:Aedes; D000818:Animals; D001216:Asparagine / Q000096:biosynthesis; D002478:Cells, Cultured; D002854:Chromatography, Paper; D006224:Cricetinae; D006023:Glycoproteins / Q000096:biosynthesis*; D006868:Hydrolysis; D007700:Kinetics; D008358:Mannose / Q000378:metabolism*; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*
D006023:Glycoproteins; D009844:Oligosaccharides; D001216:Asparagine; D008361:Mannosidases; D008358:Mannose