Expression, glycosylation, and intracellular distribution of human beta-galactosidase in recombinant baculovirus-infected Spodoptera frugiperda cells

Biochemical and Biophysical Research Communications
K ItohY Suzuki

Abstract

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.

References

Sep 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·H TowbinJ Gordon
Jan 2, 1987·European Journal of Biochemistry·F W VerheijenH Galjaard
Apr 14, 1987·Biochemical and Biophysical Research Communications·E NanbaY Suzuki
Apr 29, 1988·Biochemical and Biophysical Research Communications·E NanbaY Suzuki
Nov 30, 1988·Biochemical and Biophysical Research Communications·A OshimaY Suzuki
May 31, 1968·Science·S Okada, J S O'Brien

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Citations

Nov 3, 1995·The Journal of Biological Chemistry·E J BontenA d'Azzo
Sep 13, 1991·Journal of Chromatography·Y Suzuki

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