Miller K R, Wang J, Sorci-Thomas M, Anderson R A, Parks J S
Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.
J Lipid Res. 1996 Mar;37(3):551-61.
The glycosylation state of lecithin:cholesterol acyltransferase (LCAT) may be important in determining its enzymatic activity. We compared glycosylation 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 transfected Chinese hamster ovary (CHO) cells (CHO LCAT). bLCAT was underglycosylated (molecular weight approximately 50 kDa) and resistant to digestion by N-glycanase F, endoglycosidase F, and neuraminidase. CHO and HepG2 LCAT were overglycosylated (approximately 68 kDa and approximately 70-75 kDa) compared to pLCAT (approximately 65 kDa). CHO LCAT, like pLCAT, was sensitive to N-glycanase F and neuraminidase but not to endoglycosidase F. HepG2 LCAT demonstrated resistance to N-glycanase F and endoglycosidase F. Apparent Km values for all four enzymes were similar (1.4-9.2 microM cholesterol) for recombinant high density lipoproteins (rHDL) containing sn-1 16:0, sn-2 18:1 PC (POPC). Apparent Vmax values (nmol cholesteryl ester formed/h per micrograms) were 52.6 for pLCAT, 48.6 for CHO LCAT, 15.3 for bLCAT, and 8.3 for HepG2 LCAT. Changes in PC acyl specificity in the presence and absence of cholesterol were characterized by comparing the ratio of LCAT activity on rHDL containing sn-1 16:0, sn-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 cholesterol, respectively. We conclude that LCAT source influences glycosylation structure, which affects the apparent Vmax for cholesteryl ester formation with only minor changes in apparent Km or acyl substrate specificity.
卵磷脂胆固醇酰基转移酶（LCAT）的糖基化状态可能对其酶活性的决定具有重要意义。我们比较了来自四种来源的人LCAT的糖基化结构、酶动力学和磷脂酰胆碱（PC）酰基特异性：人血浆（pLCAT）、HepG2细胞培养基（HepG2 LCAT）、感染重组杆状病毒的SF21细胞培养基（bLCAT）以及稳定转染的中国仓鼠卵巢（CHO）细胞培养基（CHO LCAT）。bLCAT糖基化不足（分子量约50 kDa），对N - 聚糖酶F、内切糖苷酶F和神经氨酸酶的消化具有抗性。与pLCAT（约65 kDa）相比，CHO和HepG2 LCAT糖基化过度（约68 kDa和约70 - 75 kDa）。CHO LCAT与pLCAT一样，对N - 聚糖酶F和神经氨酸酶敏感，但对内切糖苷酶F不敏感。HepG2 LCAT对N - 聚糖酶F和内切糖苷酶F具有抗性。对于含有sn - 1 16:0、sn - 2 18:1 PC（POPC）的重组高密度脂蛋白（rHDL），所有四种酶的表观Km值相似（1.4 - 9.2 microM胆固醇）。表观Vmax值（每微克每小时形成的胆固醇酯的纳摩尔数）对于pLCAT为52.6，对于CHO LCAT为48.6，对于bLCAT为15.3，对于HepG2 LCAT为8.3。通过比较含有sn - 1 16:0、sn - 2 20:4 PC（PAPC）或POPC（PAPC/POPC活性比）的rHDL上的LCAT活性比，来表征在有和没有胆固醇的情况下PC酰基特异性的变化。pLCAT、bLCAT、CHO LCAT和HepG2 LCAT活性的比值在有胆固醇时分别为0.63、0.49、0.56和0.51，在没有胆固醇时分别为0.34、0.29、0.36和0.99。我们得出结论，LCAT来源影响糖基化结构，这会影响胆固醇酯形成的表观Vmax，而表观Km或酰基底物特异性仅有微小变化。
