Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

The large EcoRI fragment of clone MII-8 containing the en- Moremen tire Man II open reading frame was subcloned into the COS cell expression vector pXM . This is an SV-40-based expression vector driven by the adenovirus major late promoter (58) . COS cells were transfected and assayed for Man II activity either in cell homogenates or in membrane fractions . Crude homogenates showed approximately threefold overexpression of 4-methylumbelliferyl a-mannoside activity (Table I), but Man II activity in crude homogenates is commonly masked by the lysosomal a-mannosidase or the ER a-mannosidase when using synthetic substrates . A salt washed membrane fraction was prepared which has been shown to result in the enrichment ofMan II activity (27) . Assays of this membrane fraction revealed a 8-12-fold overexpression of 4-methylumbelliferyl a-mannoside activity in COS cells transfected with MII-pXM when compared cells transfected with the vector alone or vector with an antisense insert (Table I) . This degree of overexpression is similar to the activity levels seen for the 01,4-galactosyltransferase expressed in COS cells (24) . The overexpressed Man II activity has a pH optimum of 5.5, identical to the endogenous enzyme in 3T3 cells (27), it is sensitive to inhibition by swainsonine, and is immunoprecipitable with antibody to the purified rat enzyme (Fig. 6 ) . Biosynthetic labeling of transfected COS cells resulted in a >10-fold overexpression ofimmunoprecipitable polypeptide (data not shown) reflecting the high rate of biosynthesis of the vector encoded polypeptide 48 h after transfection .