Expression of human lysosomal alpha-mannosidase activity in transfected murine cells and human alpha-mannosidase deficient fibroblasts.
We studied the human lysosomal alpha-mannosidase (MANB) by expressing the putative cDNA in mammalian cells, using the eucaryotic expression vector pCDE. The construct pCDE-MANB and pSV2-Neo were cotransfected into human alpha-mannosidase deficient fibroblasts and into a murine cell line and selected by culture in the presence of G418. Six G418 resistant 3T3 clones had increased alpha-mannosidase activity 2 to 3 times above the controls. Two clones from transfected human fibroblasts showed a 2 fold increase in enzyme activity. The human MANB cDNA gene was demonstrated in the target cells by Southern blot analysis and the expression of the gene was shown by RT-PCR analysis. This study is the first to successfully express the MANB gene in a human and a murine cell line. The results confirm that the putative MANB cDNA encodes the full length of lysosomal alpha-mannosidase. Molecular characterization of mannosidosis and approaches to gene therapy are now possible using this cDNA.
Wang
W
W
Department of Human Genetics, University of Pittsburgh, Pennsylvania 15261, USA.
Nebes
V L
VL
Schmidt
M C
MC
Barranger
J A
JA
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002999:Clone Cells; D018076:DNA, Complementary; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014162:Transfection; D043323:alpha-Mannosidase
D018076:DNA, Complementary; D008361:Mannosidases; D043323:alpha-Mannosidase