胡萝卜抗冻蛋白不具有PGIP家族的聚半乳糖醛酸酶抑制活性

ZHANG Dang-Quan , WANG Hong-Bin , LIU Bin , FENG Dong-Ru , HE Yan-Ming , WANG Jin-Fa
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引用次数: 11

摘要

胡萝卜(Daucus carota)抗冻蛋白(DcAFP)具有很强的抗冻活性,根据其序列相似性,包括富含亮氨酸的重复序列(LRR)的存在,被鉴定为属于植物多半乳糖醛酸酶抑制蛋白(PGIP)家族。本研究采用酵母双杂交技术分析胡萝卜AFP是否具有PGIP的功能。完整的DcAFP和聚半乳糖醛酸酶(PGase);利用RT-PCR方法从真菌Alternaria alternata中分离得到)的编码序列,分别克隆到诱饵载体和捕获载体上,进行酵母双杂交实验。结果显示,DcAFP与PGase之间没有相互作用的证据,这表明DcAFP可能缺乏PGIP活性。对DcAFP和其他PGIPs的静电电位分析表明,DcAFP LRR基序β-螺旋内的大量非保守残基被碱性氨基酸取代,从而使表面由负变为正。这将在静电上阻止DcAFP与PGase带正电的表面结合。这是首次报道DcAFP的LRR基序中的非保守氨基酸与其聚半乳糖醛酸酶抑制活性的丧失之间的相关性。
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Carrot Antifreeze Protein Does Not Exhibit the Polygalacturonase-inhibiting Activity of PGIP Family

The carrot (Daucus carota) antifreeze protein (DcAFP) has a strong antifreeze activity and identified as belonging to the plant polygalacturonase-inhibiting protein (PGIP) family based on its sequence similarities, including the presence of a leucine-rich repeat (LRR) motif. In this study, yeast two-hybrid technology was used to analyze whether the carrot AFP could act as a PGIP. The complete DcAFP and polygalacturonase (PGase; obtained from fungus Alternaria alternata by RT-PCR) coding sequences were cloned into the bait and capture vectors, respectively, and yeast two-hybrid assays were performed. The results revealed that there was no evidence of an interaction between DcAFP and PGase, which suggests that DcAFP probably lacks PGIP activity. An analysis of the electrostatic potential of DcAFP and other PGIPs revealed that a large number of nonconservative residues within the β-helix of the DcAFP LRR motif had been substituted to basic amino acids, thus changing the surface from negative to positive. This will electrostatically prevent DcAFP from binding with the positively charged surface of PGase. This is the first report that showed the correlation between nonconservative amino acids within the LRR motif of the DcAFP and its loss of polygalacturonase inhibiting activity.

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