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 isolation of several size classes of polyadenylated clones that differ in their degree of extension in the 3' untranslated region demonstrates the heterogeneous nature of the termination and polyadenylation of the Man 11 message. Each of Table I Figure 6 . pH profiles ofa-mannosidase activity in COS cells transfected with control (pXM, o) or Man II clone MII-8 (MII-pXM, o) constructs . Salt-washed membranes were prepared and assayed as described in Materials and Methods with the 4methylumbelliferyl a-n-mannoside substrate at the indicated pH . Membranes prepared from COS cells transfected with MII-pXM were also assayed at the indicated pH inthe presence of 10 AM swainsonine (0) . Residual a-mannosidase activity in the supernatant following immunoprecipitation of the salt-washed membrane extract with antirat Man II antibody is as indicated (m) . transcript of 7.5 kb was found in all tissues with the greatest enrichment being in adrenal and thymus . Most other tissues resulted in an autoradiographic band of -3-10-fold lesser intensity except skeletal muscle which was undetectable except with prolonged exposure. The ratio of the multiple Man II message species appeared constant across the range of rat tissues despite the >100-fold difference in the level ofexpression between adrenal and muscle tissues. Comparison with the rat ER a-mannosidase (2), reveals that while the Man II transcript is most abundant in adrenal and thymus, the ER enzyme has a highest abundance level in adrenal and testis and minor differences in other tissues except spleen, intestinal epithelia, and muscle tissues which have a slightly reduced level ofmessage. The differences with Man II message expression in the thymus, testis, and muscle suggests that there is a differential tissue expression of the two a-mannosidases at least in these tissues . A low but detectable level of Man II message was also found in rat brain tissues consistent with a report of reduced levels of Man II activity in rat brain and the presence of a novel brain a-mannosidase (51) which is not sensitive to inhibitionby swainsonine and is able to directly cleave high mannose structures to Man3GlcNAc2 without the prior addition of a G1cNAc residue by GnT I. Whether the message levels adequately reflect the levels of the Man II enzyme activity in the brain or inthe other tissues remains to be determined. Although there is a >100-fold difference in Man II autoradiographic intensity on Northern blots between thymus and muscle, it is also not clear if this merely reflects the biosynthetic level and the degree of elaboration of the Golgi complex and the secretory pathway/glycosylation machinery in the respective tissue types .