Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity
Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity
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DOI：
10.1111/j.1742-4658.2006.05553.x
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发表时间：
2006-12-01
期刊：
影响因子：
5.4
通讯作者：
Edqvist, Johan
中科院分区：
文献类型：
--
作者：
Viitanen, Lenita;Nylund, Matts;Edqvist, Johan
甾醇载体蛋白-2 （SCP-2）是一个小的细胞内碱性蛋白结构域，与过氧化物酶体β -氧化有关。我们通过表征Euphorbia lagascae中的SCP-2来扩展我们对植物SCP-2的认识。该蛋白由122个氨基酸组成，包括一个PTS1过氧化物酶体靶向信号。它的分子量为13.6 kDa， pI为9.5。它与拟南芥中的SCP-2具有67%的相似性和84%的相似性。蛋白质组学分析表明，lagascae SCP-2在种子萌发过程中在胚乳中积累。结果表明，bodipy -磷脂酰胆碱（BODIPY-PC）具有体外转移活性。添加磷脂酰肌醇、棕榈酸、硬脂酰辅酶a和油酸后，BODIPY-PC的转移几乎被完全抑制，而添加甾醇后，BODIPY-PC的转移仅具有非常微弱的抑制作用。我们使用蛋白质模型和定点诱变技术来研究为什么E. lagascae SCP-2介导的BODIPY-PC转移对甾醇不敏感，而A. thaliana SCP-2介导的BODIPY-PC转移对甾醇敏感。蛋白质模型表明，拟南芥SCP-2的配体结合腔有4个蛋氨酸（Met12、14、15和100），而拟南芥SCP-2的配体结合腔则被亮氨酸（Leu11、13、14和99）取代。将Leu99转化为Met99足以使E. lagascae SCP-2转化为对甾醇敏感的bodipy - pc -转移蛋白，相应地，将Met100转化为Leu100可以消除a . thaliana SCP-2对甾醇的敏感性。
Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.