Isolation, Characterization, and Expression of cDNAs Encoding Murine α-Mannosidase II, a Golgi Enzyme That Controls Conversion of High Mannose to Complex N-Glycans
Golgi α-mannosidase II (GlcNAc transferase I-dependent α1,3[α1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures. We have isolated overlapping clones from a mu...
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Veröffentlicht in: | The Journal of cell biology 1991-12, Vol.115 (6), p.1521-1534 |
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Sprache: | eng |
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Zusammenfassung: | Golgi α-mannosidase II (GlcNAc transferase I-dependent α1,3[α1,6] mannosidase, EC 3.2.1.114) catalyzes the final hydrolytic step in the N-glycan maturation pathway acting as the committed step in the conversion of high mannose to complex type structures. We have isolated overlapping clones from a murine cDNA library encoding the full length α-mannosidase II open reading frame and most of the 5′ and 3′ untranslated region. The coding sequence predicts a type II transmembrane protein with a short cytoplasmic tail (five amino acids), a single transmembrane domain (21 amino acids), and a large COOH-terminal catalytic domain (1,124 amino acids). This domain organization which is shared with the Golgi glycosyl-transferases suggests that the common structural motifs may have a functional role in Golgi enzyme function or localization. Three sets of polyadenylated clones were isolated extending 3′ beyond the open reading frame by as 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 ∼1.4-kb downstream beyond the longest of the isolated clones. Transient expression of the α-mannosidase II cDNA in COS cells resulted in 8-12-fold overexpression of enzyme activity, and the appearance of cross-reactive material in a perinuclear membrane array consistent with a Golgi localization. A region within the catalytic domain of the α-mannosidase II open reading frame bears a strong similarity to a corresponding sequence in the rat liver endoplasmic reticulum α-mannosidase and the vacuolar α-mannosidase of Saccharomyces cerevisiae. Partial human α-mannosidase II cDNA clones were also isolated and the gene was localized to human chromosome 5. |
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ISSN: | 0021-9525 1540-8140 |
DOI: | 10.1083/jcb.115.6.1521 |