Expression, purification, and ligand-binding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding protein found overexpressed in neoplastic skin cells
Expression, purification, and ligand-binding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding protein found overexpressed in neoplastic skin cells
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DOI：
10.1021/bi952476e
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发表时间：
1996-03-05
期刊：
影响因子：
2.9
通讯作者：
Bernlohr, DA
中科院分区：
文献类型：
--
作者：
Kane, CD;Coe, NR;Bernlohr, DA
角质形成细胞脂质结合蛋白（keratinocyte lipid-binding protein，KLBP）是细胞内脂质结合蛋白（intracellular lipid-binding protein，iLBP）多基因家族的新成员。为了表征KLBP并确定其配体结合特性，将其cDNA亚克隆到大肠杆菌中，并通过酸提取、凝胶渗透和离子交换色谱的组合过量表达和纯化KT-BP至均一。纯化的KT-BP表现出与荧光疏水探针1-苯胺基萘-8-磺酸盐（1，8-ANS）的高亲和力结合，显示表观解离常数为390 +/-90 nM（n = 0.74 +/-0.2），使用基于结合荧光团的置换的测定，发现KLBP最强烈地结合长链脂肪酸;油酸（18：1）以248 +/-12 nM的表观K-d结合，花生四烯酸（20：1）以248 +/-12 nM的表观K-d结合。4）表现出318 +/-14 nM的解离常数。随着脂肪酸长度的减少，结合亲和力降低;肉豆蔻酸（14：0）结合的Kd为1409 +/-423 nM，但中链（癸酸，10：0）和短链（辛酸，8：0）脂质完全不结合，该蛋白质不以任何可测量的亲和力结合前列腺素E（2），但确实与类二十烷酸如5-氢过氧二十碳四烯酸结合（5-HPETE; Kd为848 +/-211 nM）和15-HPETE（K-d为463 +/-243 nM）以及在较小程度上它们的羟基衍生物，5-HETE和15-HETE（Kd分别为1560 +/-115 nM和大于4 μ M），全反式视黄酸是KLBP的弱配体，结合Kd为3600 nM，全反式视黄醇不能置换1，8-ANS，几种iLBP的X-射线晶体结构的KLBP序列的分子建模表明，一个或多个半胱氨酸残基的侧链可能存在于推定的配体结合腔。与此一致，在存在和不存在油酸的情况下，在pH 8.0下用5，5'-二硫代双（2-硝基苯甲酸）对纯化的KLBP进行巯基滴定，显示至少一个残基被保护免于被脂肪酸修饰。这些结果描述了KLBP的配体结合特性的首次纯化和表征，并表明该蛋白质是具有可能类似于iLBP多基因家族的其他成员的三级结构的脂肪酸结合蛋白。
The keratinocyte lipid-binding protein (KLBP) has been identified on the basis of nucleotide sequence analysis of its cloned cDNA as a new member of the intracellular lipid-binding protein (iLBP) multigene family. To characterize KLBP and determine its ligand-binding properties, its cDNA was subcloned into Escherichia coli, and the protein was overexpressed and purified to homogeneity by a combination of acid extraction, gel permeation, and ion-exchange chromatographies, Purified KT-BP exhibited high-affinity binding of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (1,8-ANS), displaying an apparent dissociation constant of 390 +/- 90 nM (n = 0.74 +/- 0.2), Using an assay based upon displacement of the bound fluorophore, KLBP was found to bind long chain fatty acids most avidly; oleic acid (18:1) bound with an apparent K-d of 248 +/- 12 nM, and arachidonic acid (20:4) exhibited a dissociation constant of 318 +/- 14 nM. As the length of the fatty acid decreased, the binding affinity was reduced; myristic acid (14:0) bound with a K-d of 1409 +/- 423 nM, but medium-chain (decanoic acid, 10:0) and short-chain (octanoic acid, 8:0) lipids were not bound at all, The protein did not bind prostaglandin E(2) with any measurable affinity but did associate with eicosanoids such as 5-hydroperoxyeicosatetraenoic acid (5-HPETE; K-d of 848 +/- 211 nM) and 15-HPETE (K-d Of 463 +/- 243 nM) and to a lesser extent their hydroxy derivatives, 5-HETE and 15-HETE (K-d of 1560 +/- 115 nM and greater than 4 mu M, respectively), all-trans-Retinoic acid was a weak ligand for KLBP, binding with a K-d of 3600 nM, and all-trans-retinol did not displace 1,8-ANS, Molecular modeling of the KLBP sequence upon the X-ray crystal structures of several iLBP's suggested that thr side chains of one or more cysteine residues may reside within the putative ligand-binding cavity. Consistent with this, sulfhydryl titration of purified KLBP with 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 in the presence and absence of oleic acid revealed that at least one residue was protected from modification by the fatty acid. These results describe the first purification and characterization of the ligand-binding properties of KLBP and indicate that the protein is a fatty acid binding protein with a tertiary structure likely to be similar to other members of the iLBP multigene family.