McCall M R, Nichols A V, Morton R E, Blanche P J, Shore V G, Hara S, Forte T M
Molecular and Nuclear Medicine Department, Lawrence Berkeley Laboratory, Berkeley, CA 94720.
J Lipid Res. 1993 Jan;34(1):37-48.
We have previously shown that lecithin:cholesterol acyltransferase (LCAT) can transform ultracentrifugally isolated HepG2 lipoproteins (d < 1.235 g/ml) into particles that differ substantially from their nascent precursors. Transformed high density lipoprotein (HDL) subpopulations, as judged by nondenaturing gradient gel electrophoresis (GGE), resemble plasma HDL, i.e., HDL2a- and HDL3a-sized particles predominate. In HepG2 conditioned medium (CM), 60-70% of apoA-I is in the d > 1.235 g/ml fraction (lipid-poor apoA-I); hence we investigated whether inclusion of d > 1.235 g/ml fraction in LCAT incubations altered HDL subpopulations. After 18 h incubation of CM (containing lipoproteins and d > 1.235 g/ml fraction) with purified LCAT, the major transformation product on GGE was a large 9.7-nm particle (HDL2b pattern); a minor component appeared at 7.4 nm (HDL3c). Differences in particle size distribution between CM and isolated lipoprotein incubations were not the result of differences in LCAT activity; mass ratios of unesterified cholesterol:cholesteryl ester and phospholipid:cholesteryl ester were similar. Removal of apoA-I from the d > 1.235 g/ml fraction by immunoaffinity chromatography prior to incubation with the d < 1.235 g/ml fraction produced the same products (i.e., HDL2b pattern) as incubations performed with the unaltered d > 1.235 g/ml fraction; therefore, lipid-poor apoA-I does not influence nascent HDL transformation. Cholesteryl ester was transferred from HepG2 HDL to LDL in CM incubations; however, cholesteryl ester transfer protein was not immunochemically identified. Removal of HepG2 LDL from CM prior to incubation with LCAT still resulted in the HDL2b pattern. We conclude that HepG2 cells secrete a factor(s) that modifies nascent HDL transformation products into a predominantly HDL2b subpopulation.
我们之前已经表明，卵磷脂胆固醇酰基转移酶（LCAT）能够将超速离心分离的HepG2脂蛋白（d < 1.235 g/ml）转化为与其新生前体有显著差异的颗粒。通过非变性梯度凝胶电泳（GGE）判断，转化后的高密度脂蛋白（HDL）亚群类似于血浆HDL，即HDL2a和HDL3a大小的颗粒占主导。在HepG2条件培养基（CM）中，60 - 70%的载脂蛋白A-I（apoA-I）存在于d > 1.235 g/ml的组分中（低脂apoA-I）；因此，我们研究了在LCAT孵育中加入d > 1.235 g/ml的组分是否会改变HDL亚群。将CM（含有脂蛋白和d > 1.235 g/ml的组分）与纯化的LCAT孵育18小时后，GGE上的主要转化产物是一个大的9.7纳米颗粒（HDL2b模式）；一个次要成分出现在7.4纳米处（HDL3c）。CM与分离的脂蛋白孵育之间颗粒大小分布的差异不是LCAT活性差异的结果；未酯化胆固醇与胆固醇酯以及磷脂与胆固醇酯的质量比相似。在与d < 1.235 g/ml的组分孵育之前，通过免疫亲和色谱从d > 1.235 g/ml的组分中去除apoA-I，产生的产物（即HDL2b模式）与用未改变的d > 1.235 g/ml的组分进行孵育产生的产物相同；因此，低脂apoA-I不影响新生HDL的转化。在CM孵育中，胆固醇酯从HepG2 HDL转移到LDL；然而，未通过免疫化学方法鉴定出胆固醇酯转移蛋白。在与LCAT孵育之前从CM中去除HepG2 LDL仍然产生HDL2b模式。我们得出结论，HepG2细胞分泌一种因子，该因子将新生HDL转化产物修饰为主要是HDL2b亚群。
