Occurrence of sulfate in an asparagine-linked complex oligosaccharide of chicken adipose lipoprotein lipase.
After adipocytes were labeled with Na2[35SO4], immunoadsorbed with immobilized antilipoprotein lipase, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, a labeled band was identified at 59,700 daltons, the molecular mass of chicken lipoprotein lipase (LPL). Excess unlabeled LPL prevented the immunoadsorption of this labeled species, hence the labeled species was determined to be LPL. Digestion of LPL with endo-beta-N-acetylglucosaminidase H (Endo H) caused a shift in mobility of LPL in SDS-PAGE with no loss of radioactivity, whereas digestion with glycopeptidase F resulted in removal of 99% of the radioactivity. Adipocytes cultured with Trans35S-label and tunicamycin produced an LPL species of 52,000 daltons, but tunicamycin abolished the incorporation of 35SO4 into LPL. This established that 35SO4 was incorporated into an N-linked oligosaccharide of LPL. Endo H digestion of pulse-chase labeled LPL revealed the presence of two complex and one high mannose-type N-linked oligosaccharides. A single 35SO4-labeled tryptic peptide was isolated by reverse phase chromatography. The amino acid sequence of the peptide established that the 35SO4 oligosaccharide is conjugated at Asn-45. Behavior of the 35SO4-labeled oligosaccharide on concanavalin A-agarose, sequential exoglycosidase digestion, and chemical analysis of the 35SO4 oligosaccharide confirms that this moiety is of the complex type. Sequential exoglycosidase digestion, thin layer chromatography of the released monosaccharides, and the use of glycosylation inhibitors established that the sulfated sugar is a core N-acetylglucosamine (GlcNAc). The data show that chicken LPL contains two complex and one high mannose N-linked oligosaccharides and that 35SO4 is incorporated into LPL on a GlcNAc residue of a complex oligosaccharide located at Asn-45.
Hoogewerf
A J
AJ
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.
Bensadoun
A
A
D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002645:Chickens; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013431:Sulfates / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
D009844:Oligosaccharides; D013431:Sulfates; D014415:Tunicamycin; D001216:Asparagine; D008071:Lipoprotein Lipase