Journal of Lipid Research, Vol 35, 1511-1523, Copyright © 1994 by Lipid Research, Inc.

ARTICLES

Lipoprotein lipase and hepatic lipase: the role of asparagine-linked glycosylation in the expression of a functional enzyme

O Ben-Zeev, G Stahnke, G Liu, RC Davis and MH Doolittle
Veterans Administration Wadsworth Medical Center, Los Angeles, CA 90073.

Lipoprotein lipase (LPL) and hepatic lipase (HL) share two conserved asparagine-linked glycosylation sites, located at the amino- and carboxy-terminal domains of the protein. Human HL contains two additional sites, preceding each conserved site by 36 and 35 amino acids, respectively. The utilization of these sites for glycan-binding and the role of each glycan chain for the catalytic function of human LPL, rat HL, and human HL was investigated. To accomplish this aim, potential Asn glycosylation sites were changed to Gln by site-directed mutagenesis and the resulting constructs were expressed in a mammalian (COS) cell system. We demonstrate the following. 1) All potential glycosylation sites in human LPL, rat HL, and human HL are utilized. 2) Lack of glycosylation at the two non-conserved sites in human HL has no effect on enzyme expression. 3) Glycosylation at the conserved Asn sites in the N-terminal domain of LPL and HL is required for the synthesis of a fully active and secreted lipase. While this is an absolute requirement for LPL, a portion (approximately 25%) of HL molecules lacking glycosylation at this essential site still becomes active and secreted. However, the simultaneous elimination of both glycosylation sites at the N-terminal domain of human HL results in the virtual abolishment of enzymatic activity and secretion. 4) Glycosylation at the conserved sites in the C-terminal domain is not essential for the expression of active lipases. 5) Eliminating all glycosylation sites in LPL and HL results in the synthesis of inactive enzymes that are retained intracellularly; however, a small portion (2%) of unglycosylated rat HL was active and secreted. We conclude that glycosylation overall plays an important role in the formation of functional LPL and HL.
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