Cloning, expression and characterization of a cDNA (6A8) encoding a novel human alpha-mannosidase.
A 3300-bp cDNA (6A8) has been isolated from a human tonsil cell lambdagt11 cDNA library (GenBank accession number: AF044414). The 6A8 gene is localized on human chromosome 13q31-32. Its cDNA has an open reading frame from position 57 bp to 3243 bp, encoding a 1062 amino-acid polypeptide. The sequence of the polypeptide has 89% identity to rat liver ER alpha-mannosidase. Homogenates of COS-7 cells transfected with 6A8 cDNA showed an enhanced enzymatic activity with p-nitro-phenyl-alpha-D-mannopyranoside, which was not inhibited by swainsonine. These data suggest that 6A8 alpha-mannosidase belongs to the class II alpha-mannosidase. Western blot analysis showed a band for 6A8 cDNA encoded protein of approximately 120 kDa. Northern blot analysis revealed two 6A8 mRNA transcripts with different tissue distribution. Enhanced concanavalin A (ConA) binding to CNE-2L2 cells transfected with a reverse 6A8 DNA was observed, indicating that the 6A8 protein is an important cellular alpha-mannosidase.
Li
B
B
Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Wang
Z Z
ZZ
Ma
F R
FR
Shi
G X
GX
Zhang
L X
LX
Zeng
X
X
Liu
Y
Y
Zhao
F T
FT
Zhu
L P
LP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002874:Chromosome Mapping; D002882:Chromosomes, Human, Pair 13; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D043323:alpha-Mannosidase
D018076:DNA, Complementary; D008361:Mannosidases; D043323:alpha-Mannosidase