Isolation and expression of murine and rabbit cDNAs encoding an alpha 1,2-mannosidase involved in the processing of asparagine-linked oligosaccharides.
We have isolated a full-length cDNA clone encoding a murine alpha 1,2-mannosidase involved in the processing of mammalian Asn-linked oligosaccharides. Oligonucleotide primers were designed based on peptide sequences derived from the purified rabbit liver enzyme and were used to generate a 1011-base pair probe using the polymerase chain reaction. This probe was used to isolate clones from rabbit and mouse cDNA libraries. The full-length murine cDNA clone encodes a 655-amino acid type II transmembrane protein with a 43-amino acid cytoplasmic tail, a single transmembrane domain, and a large COOH-terminal catalytic domain containing two potential N-glycosylation sites. Stable transfection of the murine alpha 1,2-mannosidase cDNA into mouse L cells resulted in a approximately 22-fold overexpression of alpha 1,2-mannosidase activity. Three transcripts were detected in rabbit tissues, whereas two were found in rat and mouse tissues. The sequences of the rabbit and mouse cDNA clones indicate that the multiple transcripts differ in the length of their 3' sequences as a result of the use of multiple polyadenylation signals. Immunolocalization detected cross-reactive material in a juxtanuclear pattern consistent with the Golgi complex. The catalytic portion of the murine alpha 1,2-mannosidase was found to bear a strong similarity to the processing alpha 1,2-mannosidase from Saccharomyces cerevisiae.
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Complex Carbohydrate Research Center, University of Georgia, Athens 30602.
Schutzbach
J S
JS
Forsee
W T
WT
Neame
P J
PJ
Moremen
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000302:isolation & purification* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D007739:L Cells; D008099:Liver / Q000201:enzymology*; D008361:Mannosidases / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009844:Oligosaccharides / Q000096:biosynthesis*; D016366:Open Reading Frames; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D009844:Oligosaccharides; D001216:Asparagine; D004247:DNA; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase