Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase.
We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library. Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe. The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization. We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs. A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences. The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain. The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide. The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142.
J
J
Bischoff
K
K
Moremen
H F
HF
Lodish
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008361:Mannosidases / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D043323:alpha-Mannosidase
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D004247:DNA; D008361:Mannosidases; D043323:alpha-Mannosidase