Characterization of a mannosidase acting on α1 → 3‐ and α1 → 6‐linked mannose residues of oligomannosidic intermediates of glycoprotein processing

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Abstract
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 Manα1 → 3[Manα1 → 6]Manβ1 → 4GlcNAc. The properties of the (Man)5GlcNAc‐hydrolase activity differ from lysososomal α‐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 oligoigomannosidic N‐glycans to complex‐type chains, and mosquito cells that constitutively lack N‐acetylglucosaminyl transferase I. In both cells 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 asembly and suggesting other functions for the mannosidase activity in BHK cells.
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