MOLECULAR CHARACTERIZATION OF HUMAN HEPATIC LIPASE DEFICIENCY - IN-VITRO EXPRESSION OF 2 NATURALLY-OCCURRING MUTATIONS

Individuals with hepatic lipase (HL) deficiency are often characterize d by elevated levels of triglycerides and cholesterol and may be subje ct to premature atherosclerosis. Missense mutations in the HL gene hav e been identified in two affected families: substitutions of serine fo r phenylalanine at amino acid 267 and threonine for methionine at amin o acid 383 (S267F and T383M, respectively). To confirm the role of S26 7F and T383M in HL deficiency, we introduced these mutations separatel y into human HL cDNA by site-directed mutagenesis, and the resulting c onstructs were independently expressed in COS cells. HL activity and m ass were measured and compared with wild-type HL transfectants to dete rmine the effect of these mutations on lipase activity and secretion. Although similar amounts of HL protein were detected intracellularly a fter transfection with the wild-type and mutant constructs, S267F and T383M HL;activity levels were markedly decreased: in S267F, no HL acti vity was detected, and activity levels in T383M were 38% of wild-type HL. Heparin-induced secretion of the two HL mutants was also severely affected: no detectable activity could be measured in the media of S26 7F, although some inactive mass (12% of wild-type HL) was secreted; mu tant T383M secreted 4% and 20% of wild-type activity and mass, respect ively. These results indicate that the single amino acid substitution present in HL S267F is sufficient to render the enzyme completely nonf unctional; in contrast, the T383M mutant retains partial activity but is poorly secreted. Thus, these defects appear capable of accounting f or the HL-deficient phenotypes exhibited by individuals carrying the T 383M and S267F mutations.