LIPOPROTEIN-LIPASE AND HEPATIC LIPASE - THE ROLE OF ASPARAGINE-LINKEDGLYCOSYLATION IN THE EXPRESSION OF A FUNCTIONAL ENZYME

resultsLipoprotein lipase (LPL) and hepatic lipase (HL) share two cons erved asparagine-linked glycosylation sites, located at the amino- and carboxy-terminal domains of the protein. Human HL contains two additi onal sites, preceding each conserved site by 36 and 35 amino acids, re spectively. 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 Gin by site-directed mutagenes is and the resulting constructs were expressed in a mammalian (COS) ce ll system. We demonstrate the following. 1) All potential glycosylatio n sites in human LPL, rat HL, and human HL are utilized. 2) Lack of gl ycosylation at the two nonconserved 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 fu lly active and secreted lipase. While this is an absolute requirement for LPL, a portion (approximately 25%) of HL molecules lacking glycosy lation at this essential site still becomes active and secreted. Howev er, the simultaneous elimination of both glycosylation sites at the N- terminal domain of human HL results in the virtual abolishment of enzy matic 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 intracellularl y; however, a small portion (2%) of unglycosylated rat HL was active a nd secreted. We conclude that glycosylation overall plays an important role in the formation of functional LPL and HL.