Isolation of a mouse Golgi mannosidase cDNA, a member of a gene family conserved from yeast to mammals.
The amino acid sequence of the specific alpha-mannosidase involved in N-oligosaccharide processing in Saccharomyces cerevisiae was found to have a high degree of similarity to the deduced amino acid sequence of a rabbit liver alpha-mannosidase partial cDNA, demonstrating that processing mannosidases have been conserved through eukaryotic evolution. Regions of sequence identity were chosen to design degenerate oligonucleotide primers that can be used to prepare probes using the polymerase chain reaction (PCR) for cloning processing mannosidases from other eukaryotes. Using these primers for PCR with mouse liver cDNA as template, two related but distinct PCR products were obtained. The amino acid sequences of PCR1 and PCR2 were 88 and 65% identical with the corresponding sequence of the rabbit enzyme, respectively. Southern blot analysis of mouse genomic DNA using PCR1 and PCR2 as probes revealed that they are derived from two different genes, indicating the existence of a mammalian mannosidase gene family with at least two members. Using PCR2 as a probe, a novel mouse cDNA was isolated from a 3T3 cDNA library. It contains an open reading frame which encodes a type II membrane protein of 73 kDa with a cytoplasmic region of about 35 amino acids, a Ca2+ binding consensus sequence, and a single N-glycosylation site. Northern blot analysis of mouse tissues and L cells revealed tissue-specific expression of multiple transcripts, ranging in size from 4.2 to 8.5 kilobases, that suggests a complex pattern of gene regulation. Transient expression of the influenza hemagglutinin epitope-tagged cDNA in COS cells followed by indirect immunofluorescence with monoclonal antibody 12CA5 showed that the cloned mannosidase is primarily localized in a juxtanuclear position corresponding to the Golgi. The C-terminal domain lacking the putative transmembrane region was shown to have alpha-mannosidase activity when expressed in COS cells as a secreted Protein A fusion product.
Herscovics
A
A
McGill Cancer Centre, McGill University, Montréal, Québec, Canada.
Schneikert
J
J
Athanassiadis
A
A
Moremen
K W
KW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D017124:Conserved Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification; D006056:Golgi Apparatus / Q000201:enzymology*; D008322:Mammals / Q000235:genetics*; D008361:Mannosidases / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction; D011817:Rabbits / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D043323:alpha-Mannosidase
D017931:DNA Primers; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D004247:DNA; D008361:Mannosidases; D043323:alpha-Mannosidase