Expression, glycosylation, and intracellular distribution of human beta-galactosidase in recombinant baculovirus-infected Spodoptera frugiperda cells.
A full-length cDNA encoding human acid beta-galactosidase was inserted into the baculovirus genome under transcriptional regulation of the viral polyhedrin gene promoter. The Spodoptera frugiperda cells infected with the recombinant virus expressed the beta-galactosidase activity 300-fold higher than human fibroblasts. Immunoblot analysis revealed an 82-kDa protein band, which was modified in molecular size by deglycosylating enzymes; an 80-kDa band appeared after N-glycanase digestion, and two bands (80-kDa and 81-kDa) appeared after endoglycosidase H digestion. This result suggested that the enzyme molecule was glycosylated, partly with high-mannose type oligosaccharides. The intracellular distribution of the enzyme observed by indirect immunofluorescence staining was perinuclear or diffusely cytoplasmic, and not characteristic of lysosomes; the enzyme was secreted to the culture medium in large quantities, and not translocated to lysosomes. Possible application of this expression system to the studies of the structure and function of normal and mutant human beta-galactosidases was discussed.
Itoh
K
K
Department of Clinical Genetics, Tokyo Metropolitan Institute of Medical Science, Japan.
Oshima
A
A
Sakuraba
H
H
Suzuki
Y
Y
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005696:Galactosidases / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D007304:Insect Viruses / Q000235:genetics*; D007700:Kinetics; D008970:Molecular Weight; D009036:Moths; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D005696:Galactosidases; D001616:beta-Galactosidase