Cloning of the human cDNA which can complement the defect of the yeast mannosyltransferase I-deficient mutant alg 1.
The assembly of the lipid-linked oligosaccharide, Glc(3)Man(9)GlcNAc(2)-P-P-Dol, occurs on the rough ER membrane in an ordered stepwise manner. The process is highly conserved among eukaryotes. In order to isolate the human mannosyltransferase I (MT-I) gene involved in the process, we used the Saccharomyces cerevisiae MT-I gene ( ALG1 ), which has already been cloned. On searching the EST database with the amino acid sequence of the ALG1 gene product, we detected seven related human EST clones. A human fetal brain cDNA library was screened by PCR using gene-specific primers based on the EST nucleotide sequences and a 430 bp cDNA fragment was amplified. The cDNA library was rescreened with this 430 bp cDNA, and two cDNA clones (HR1-3 and HR1-4) were isolated and sequenced. On a homology search of the EST database with the nucleotide sequence of HR1-3, we detected a novel human EST clone, AA675921 (GenBank accession number). Based on the nucleotide sequences of AA675921 and HR1-4, we designed gene-specific PCR primers, which allowed to amplify a 1.8 kb cDNA from human fetal brain cDNA. This cDNA was cloned and shown to contain an ORF encoding a protein of 464 amino acids. We designated this ORF as Hmat-1. The amino acid sequence deduced from the Hmat-1 gene showed several highly conserved regions shared with the yeast and nematode MT-I sequences. Furthermore, this 1.8 kb cDNA successfully complemented the S. cerevisiae alg1-1 mutation, indicating that the Hmat-1 gene encodes the human MT-I and that the function of this enzyme was conserved between yeast and human.
Takahashi
T
T
Laboratory for Glycobiology and Glycotechnology, Department of Industrial Chemistry, School of Engineering, Tokai University, 1117 Kitakaname, Hiratsuka City, Kanagawa 259-1292, Japan.
Honda
R
R
Nishikawa
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D005816:Genetic Complementation Test; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase