GLYCOSYLATION STRUCTURE AND ENZYME-ACTIVITY OF LECITHIN-CHOLESTEROL ACYLTRANSFERASE FROM HUMAN PLASMA, HEPG2 CELLS, AND BACULOVIRAL AND CHINESE-HAMSTER OVARY CELL EXPRESSION SYSTEMS

The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared g lycosylation structure, enzyme kinetics, and phosphatidylcholine (PC) acyl specificity of human LCAT from four sources: human plasma (pLCAT) , media from HepG2 cells (HepG2 LCAT), media from SF21 cells infected with a recombinant baculovirus (bLCAT) and media from stably transfect ed Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycos ylated (molecular weight similar to 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (similar to 68 kDa and similar to 70-75 kD a) compared to pLCAT (similar to 65 kDa). CHO LCAT, like pLCAT, was se nsitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosi dase F. Apparent K-m values for all four enzymes were similar (1.4-9.2 mu M cholesterol) for recombinant high density lipoproteins (rHDL) co ntaining sn-1 16:0, sn-2 18:1 PC (POPC). Apparent V-max values (nmol c holesteryl ester formed/h per mu g) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl speci ficity in the presence and absence of cholesterol were characterized b y comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, s n-2 20:4 PC (PAPC) or POPC (PAPC/POPC activity ratio). The ratios for pLCAT, bLCAT, CHO LCAT, and HepG2 LCAT activity were 0.63, 0.49, 0.56, and 0.51 with cholesterol and 0.34, 0.29, 0.36, and 0.99 without chol esterol, respectively. We conclude that LCAT source influences glycosy lation structure, which affects the apparent V-max for cholesteryl est er formation with only minor changes in apparent K-m or acyl substrate specificity.