Expression of Human Lysosomal Alpha-Mannosidase Activity in Transfected Murine Cells and Human Alpha-Mannosidase Deficient Fibroblasts |
Department of Human Genetics
Department of Molecular Genetics and Biochemistry
University of Pittsburgh School of Medicine
Pittsburgh, PA 15261
| 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 a-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 a-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 a-mannosidase. Molecular characterization of mannosidosis and approaches to gene therapy are now possible using this cDNA. |