Retention of glucose by N-linked oligosaccharide chains impedes expression of lipoprotein lipase activity: effect of castanospermine.
The effect of castanospermine (CSTP), an inhibitor of glucosidase I, on processing, activity, and secretion of lipoprotein lipase was studied in 3T3-L1 adipocytes. Processing was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of endoglycosidase H (endo H)-digested subunits of lipoprotein lipase from cells incubated 1-2 h with [35S]methionine. Lipoprotein lipase in untreated cells consisted of two groups of subunits, M(r) = 55,000-58,000 and M(r) = 53,000-55,000. The heavier subunits were endo H-resistant, whereas the others were either totally or partially endo H-sensitive. The lipase secreted by untreated cells contained primarily endo H-resistant subunits. Immunofluorescent studies showed that lipoprotein lipase accumulated in Golgi in untreated cells. CSTP, 100 micrograms/ml for 18 h, decreased intracellular lipase activity by 80% and decreased secretion of lipase activity by 91%. Most of the lipase subunits in CSTP-treated cells were totally endo H-sensitive with M(r) = 57,000, some were partially endo H-sensitive, and a trace was endo-H resistant. Totally endo H-sensitive subunits in CSTP-treated cells had a M(r) 2,000-4,000 larger than that in untreated cells, indicating impaired trimming of sugar residues from oligosaccharide chains of the lipase in CSTP-treated cells. The small amount of lipase secreted by CSTP-treated cells consisted primarily of partially endo H-sensitive subunits, with one sensitive and one resistant chain per subunit. Immunofluorescent studies showed that lipoprotein lipase was excluded from Golgi in CSTP-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Masuno
H
H
Department of Medical Biochemistry, School of Medicine, Ehime University, Japan.
Blanchette-Mackie
E J
EJ
Schultz
C J
CJ
Spaeth
A E
AE
Scow
R O
RO
Okuda
H
H
D000273:Adipose Tissue / Q000201:enzymology*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology; D006596:Hexosaminidases / Q000378:metabolism; D007212:Indolizines / Q000494:pharmacology*; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D005947:Glucose; D006596:Hexosaminidases; D007212:Indolizines; D008071:Lipoprotein Lipase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009844:Oligosaccharides; D014415:Tunicamycin; C037806:castanospermine; D054836:Indolizidines