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

Three sets of polyadenylated clones were isolated extending 3' beyond the open reading frame by as N AND O-GLYCAN structures are increasingly being found to contribute to biological recognition events during development, oncogenic transformation, and cell adhesion (3, 10, 11, 37, 38, 40) . The enzymes involved in the maturation of cell surface and intracellular N-glycans are found in the endoplasmic reticulum and Golgi complex where they act upon newly synthesized glycoproteins to generate an array of different structures from a common oligosaccharide precursor (18) . The N-glycan processing pathway consists of three stages : (a) the initial synthesis of a dolichol-linked precursor oligosaccharide and the en bloc transfer of the oligosaccharide to newly synthesized polypeptide Asn-X-Ser/Thr sequons on the lumenal face of the ER; (b) the trimming of the high mannose structures by a-glucosidases and a-mannosidases in the ER and Golgi complex; and (c) the elaboration of the branched oligosaccharide chains by Golgi glycosyltransferases . The trimming phase of the pathway is accomplished by a-glucosidases I and II as well as a collection of processing a1,2-mannosidases (29) in the ER and Golgi complex. The resulting Man5GlcNAc2 structure is then modified by the addition of © The Rockefeller University Press, 0021-9525/91/12/1521/14 $2 .00 The Journal of Cell Biology, Volume 115, Number 6, December 19911521-1534 1521 much as 2,543 bp . Northern blots suggest that these polyadenylated clones totaling 6.1 kb in length correspond to minor message species smaller than the full length message. The largest and predominant message on Northern blots (7.5 kb) presumably extends another N1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the a-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of crossreactive material in a perinuclear membrane array consistent with a Golgi localization . A region within the catalytic domain of the a-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum a-mannosidase and the vacuolar a-mannosidase of Saccharomyces cerevisiae. Partial human a-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5.