O K, Hill J S, Pritchard P H
Department of Pathology, University of British Columbia, Vancouver, Canada.
Biochim Biophys Acta. 1995 Jan 20;1254(2):193-7. doi: 10.1016/0005-2760(94)00183-y.
Lecithin:cholesterol acyltransferase (LCAT) is responsible for the formation of cholesteryl ester in plasma. LCAT is a glycoprotein which has a carbohydrate content estimated to be approx. 25% of its total mass. Previous studies of recombinant LCAT have characterized the function of the four N-linked glycosylation sites of LCAT with respect to reconstituted HDL analogue substrates. In order to investigate the relationship between N-linked glycosylation and the ability of LCAT to esterify cholesterol in native plasma lipoproteins, we have expressed a series of mutant LCAT cDNAs in which each of the four glycosylation consensus sequences was eliminated individually. All mutant LCAT proteins were secreted by stably transfected baby hamster kidney cells. The ability of mutant LCATs to esterify cholesterol in purified native lipoproteins indicated that the elimination of the carbohydrate chain at position 20 of recombinant LCAT was associated with a lower activity than the wild type enzyme when HDL was used as a substrate, but no inhibitory effect was observed when LDL was used as a substrate. A mutant enzyme with a substitution of Asn-84-->Gln or Asn-272-->Gln displayed a decreased ability to esterify cholesterol in either HDL or LDL. In contrast, the loss of a carbohydrate chain at position 384 was associated with an increase in enzyme activity for both HDL (1.5-fold) and LDL (2.5-fold) substrates. Kinetic analysis of these recombinant enzymes indicated that the apparent Km values for cholesterol in either HDL or LDL were not affected, but that the differences in activities were due to changes in the apparent Vmax. Heat inactivation studies were performed to assess the role of specific carbohydrate groups in enzyme stability. Loss of a carbohydrate chain at position 20, 272 or 384 decreased thermostability of LCAT whereas a mutation at position 84 did not affect thermostability. These results suggest that individual carbohydrate chains confer specific structural and functional properties to LCAT.
卵磷脂胆固醇酰基转移酶（LCAT）负责血浆中胆固醇酯的形成。LCAT是一种糖蛋白，其碳水化合物含量估计约占其总质量的25%。先前对重组LCAT的研究已经描述了LCAT的四个N-连接糖基化位点相对于重组高密度脂蛋白类似物底物的功能。为了研究N-连接糖基化与LCAT在天然血浆脂蛋白中酯化胆固醇能力之间的关系，我们表达了一系列突变LCAT cDNA，其中四个糖基化共有序列中的每一个都被单独去除。所有突变LCAT蛋白均由稳定转染的幼仓鼠肾细胞分泌。突变LCAT在纯化的天然脂蛋白中酯化胆固醇的能力表明，当以高密度脂蛋白作为底物时，重组LCAT第20位的碳水化合物链去除与比野生型酶更低的活性相关，但当以低密度脂蛋白作为底物时未观察到抑制作用。将天冬酰胺-84替换为谷氨酰胺或天冬酰胺-272替换为谷氨酰胺的突变酶在高密度脂蛋白或低密度脂蛋白中酯化胆固醇的能力均降低。相比之下，第384位碳水化合物链的缺失与高密度脂蛋白（1.5倍）和低密度脂蛋白（2.5倍）底物的酶活性增加相关。对这些重组酶的动力学分析表明，高密度脂蛋白或低密度脂蛋白中胆固醇的表观Km值不受影响，但活性差异是由于表观Vmax的变化。进行热失活研究以评估特定碳水化合物基团在酶稳定性中的作用。第20、272或384位碳水化合物链的缺失会降低LCAT的热稳定性，而第84位的突变不影响热稳定性。这些结果表明，单个碳水化合物链赋予LCAT特定的结构和功能特性。
