Abstract
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.
Highlights
Man,GlcNAc, can be modified byGlcNAc transferase I, the first glycosyltransferase leading to the synthesis of hybrid or complex oligosaccharides
Southern blot analysis of mouse GlcNAc,. This oligosaccharide is furthemrodified by Golgi genomic DNA using PCR, and PC& as probes revealed glycosyltransferases togeneratethevariety of N-complex that they are derived from twodifferent genes, indicat- structures found on glycoproteins
Little is known of the molecular genetics of human processing mannosidases, one form of the human heregion was shownto have a-mannosidaseactivity when reditary anemia,HEMPAS is caused by a deficiency in a-manexpressed in COS cells as a secreted Protein A fusion nosidase I1 expression (Fukuda, 1990)
Summary
Materials-Materials were obtained from the following sources: restrictionenzymes, New England Biolabs, Life Technologies Inc., or Pharmacia LKB Biotechnology Inc. (BaiDe UrfeQ, uebect)h; e GenAmpDNA amplification reagent kit, Perkin Elmer Cetus; Sequeand characterized (for reviews, see Schachter (1991), Shaper and Shaper The filters were washetwdice for 15min at room temperature, West Grove, PA).Following seven washes i0n.2% Tween 20, PBS, slides and twice for 15 min at 65"C in 2 x SSC, 0.2% SDS. The poly(A+)RNA was denatured at 65 "C in 30% formamide, 10% mented Dulbecco's modified Eagle's medium, as described above using formaldehyde in 10 m MOPS buffer, pH 7, and fractionated by elec- the DEAE-dextran plus chloroquine method with 6 pg of expression trophoresis in1% agarose/formaldehyde gels overnighatt 40 V, followed vector per plate. COS cells using pXM-139 (Yang et al, 1986) the entire coding region was incubated with7.5 pl of uniformly labeled [3HlMaqGlcNAc (8200 was isolated usingPCR with the sens5e'-oligonucleotide CAT CTCGAG cpm) prepared a s described previously (Jelinek-Kelly etal., 1985) and CCACC ATG ACT ACC CCAGCG containing an XhoI site anda Kozak 56pg of bovine serum albumin for 4.5 h at 37 "C. Duplicate samples of mouse genomic DNA digested with: 1 , EcoRI; 2, PslI; 3 , RamHI; 4, XbaI; 5, HindIII; 6, RglII were probed with random labeled PCR, or PC%, as indicated
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