Journal of Lipid Research, Vol 34, 2169-2176, Copyright © 1993 by Lipid Research, Inc.

ARTICLES

Functional role of N-linked glycosylation in human hepatic lipase: asparagine-56 is important for both enzyme activity and secretion

J Wolle, H Jansen, LC Smith and L Chan
Department of Cell Biology, Baylor College of Medicine, Houston, TX.

Hepatic lipase (HL) and lipoprotein lipase (LPL) are evolutionarily related enzymes that are essential for normal lipoprotein metabolism. While much has been published on the structure-function relationship of LPL, little is known concerning the structural basis of HL action and secretion. Human HL is a glycoprotein and its predicted amino acid sequence contains four putative N-linked glycosylation sites at Asn residues 20, 56, 340, and 375. We studied the role of these residues in the secretion and catalytic activity of hHL by analysis of hHL expressed in stable CHO cell lines. Using site-specific mutagenesis, the wild-type human HL and substitution mutants of each of the four Asn residues were expressed in vitro. The relative sizes of these site- specific mutants indicate that all four putative sites are utilized for glycosylation in CHO cells. Abolition of N-linked glycosylation of three (residues 20, 340, and 375) of the four sites did not affect enzyme secretion or activity. Mutations of Asn-56 to either Gln or Ala resulted in the production of a totally inactive HL which accumulated intracellularly but was not secreted into the culture medium. Therefore, Asn-56 is required for both HL enzyme activity and secretion. The fact that the homologous N-linked glycosylation site (Asn-43) is required for both enzyme activity and secretion for human LPL (Semenkovich et al. 1990. J. Biol. Chem. 265: 5429-5433) indicates that carbohydrate chains at this site are essential for the active conformation and correct folding for secretion of these evolutionarily related lipases. Our observations provide insight into the structural basis of lipase action and secretion.
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