(A) Schematic of EL protein structure. Diamonds represent potential N-linked glycosylation sites. Filled diamonds indicate validated N-linked glycosylation sites (32), and diamonds with a black circle indicate N-linked glycosylation sites conserved across lipoprotein lipase and hepatic lipase. Numbering indicates amino acid residues and differs from previous publications due to the inclusion of the 20–amino acid signal peptide (SP) in the numbering. RNKR indicates the site of proteolytic cleavage of the 68-kDa full-length EL into a 40-kDa N-terminal fragment and a 28-kDa C-terminal fragment. The “‘lid” domain and conserved catalytic triad are indicated. Nonsynonymous EL variants identified in participants with high HDL-C were created by site-directed mutagenesis and transiently expressed using 293 cells in the presence of heparin. (B) Expressed EL was visualized by immunoblotting of the conditioned media and (C) cell lysate. (D) Media from cells transiently expressing WT EL or variant EL in the presence of heparin were collected and assayed for the hydrolysis of dipalmitoylphosphatidyl choline and (E) isolated human HDL₃. Activity data for the EL variants were normalized to a percentage of WT EL activity. Assays were performed in triplicate and separate transfections were repeated at least 3 times. Presented results are from a representative experiment. 1, Fs114DelA; 2, Ala116Thr; 3, Gly176Arg; 4, Ile239Thr; 5, Met342Val; 6, Arg476Trp; 7, X501Arg. Error bars indicate ± SD. *P ≤ 0.001 compared with WT.

(A) Schematic of EL protein structure as described in Figure 1. Thr111Ile and Asn396Ser were created by site-directed mutagenesis and transiently expressed using 293 cells in the presence of heparin. (B) Expressed EL was visualized by immunoblotting of the conditioned media and cell lysate. (C) Media from cells transiently expressing WT EL or variant EL in the presence of heparin were collected and assayed for the hydrolysis of dipalmitoylphosphatidyl choline and (D) isolated human HDL₃. Activity data for the EL variants were normalized to a percentage of WT EL activity. Assays were performed in triplicate, and separate transfections were repeated at least 3 times. Presented results are from a representative experiment. 1, Thr111Ile; 2, Asn396Ser. *P ≤ 0.001 compared with WT. (E) Somatic gene transfer of WT and Asn396Ser EL into male Lipg^–/– mice was performed by administering AAV2/8, carrying the designated transgene via i.p. injection. HDL-C was measured at indicated intervals. Triangles, LacZ (n = 4); circles, Asn396Ser (n = 3); squares, WT (n = 4). Error bars indicate ± SD.