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

Several lines of evidence indicate that the isolated cDNA clone MII-8 encodes the authentic Man II polypeptide. The NH2-terminal sequence of the intact purified enzyme aligns to amino acid position 6 of the translated cDNA at the junction of the cytoplasmic tail and the transmembrane domain. Whether the cleavage of the five amino acids segment represents an in vivo or in vitro event is difficult to determine since it is not possible to distinguish the cleaved from uncleaved forms based on size. The remaining six rat polypeptide sequences can be identified in the translation of the murine cDNA with several conservative amino acid substitutions between the two species . The Man II cDNA clone transcribed and translated in vitro was found to yield a polypeptide identical in size, glycosylation pattern, and topology in microsomal membranes to the rat liver (28) or biosynthetically labeled 3T3 enzyme (27) . Expression of the Man II cDNA in COS cells resulted in a 8-12-fold overexpression of enzyme activity with the same pH optimum, sensitivity to inhibition by swainsonine, and crossreactivity with the anti-rat Man II antibody as the endogenous 3T3 enzyme (27). Finally, the enzyme expressed in COS cells was localized by immunofluorescence to a perinuclear membrane array characteristic of the Golgi complex suggesting an appropriate localization of the transfected cDNA translation product . The open reading frame predicts a type II transmembrane topology with a 5 amino acid cytoplasmic tail, a single transmembrane domain, a "stem" region of at least 80 amino acids based on proteolysis studies, and a 1,044 amino acid catalytic domain. Although the combined "stem" and catalytic domains are two to three times larger than most Chromosome Concordant Discordant Percent discordant 1 6 19 76 2 4 21 84 3 5 20 80 4 5 20 80 5 25 0 0 6 7 18 72 7 5 20 80 8 6 19 76 9 6 19 76 10 6 19 76 11 6 19 76 12 7 18 72 13 9 16 64 14 10 15 60 15 7 18 72 16 6 19 76 17 3 22 88 18 3 22 88 19 10 15 60 20 6 19 76 21 10 15 60 22 7 18 72 X 6 19 76 Y 7 18 72 of the glycosyltransferases, the general features of membrane topology are identical between the two classes of enzymes (37) . Transmembrane topologies of plasma membrane and ER proteins are quite varied (55) suggesting thatthere is no obvious selective advantage of a single topology for membrane protein function. Many of the Golgi glycosyltransferases (37) , as well as Man II (28) , retain full catalytic activity when released from their membrane anchoring domains by selective proteolysis either in vivo or in vitro suggesting that the cleaved regions exert little, if any, influence on the catalytic activities of these enzymes . The conservation of topological features between the collection of Golgi glycosyltransferases and a processing hydrolase, Man II, might therefore suggest a functional role for the common domain structure beyond a simple attachment ofthe catalytic domain to the membrane surface. One function that has been proposed (37) for the "stem" region is to confer flexibility to the catalytic domains ofthe Golgi enzymes in order to allow accessibility to lumenal and membrane associated substrates. Although this hypothesis provides a logical resolution to the problem of substrate accessibility, evidence for this "hinge" or "stem" has yet to be demonstrated either in vitro or in vivo. A Golgi GDPase from S. cereWsia