Inspection of the conserved regions of plant chitinases participating in yieldin activity : Site-directed mutagenesis and computer-aided prediction of a three-dimensional structure

Yieldin是从豇豆伸长下胚轴中纯化的一种细胞壁蛋白，在酸性条件下可降低细胞壁的屈服阈值张力（y）。我们构建了重组yieldin（rYLD）在大肠杆菌中的高效表达系统。rYLD恢复酸诱导的热变性甘油空心圆柱体（GHC）的豇豆下胚轴的y的变化。Yieldin与植物几丁质酶糖基水解酶家族18的氨基酸序列有40%以上的同源性，并且有两个保守的共有区域，涉及一个推定的活性位点。而yieldin没有几丁质酶活性。计算机辅助预测的三维结构表明，yieldin与concanavalin B和hevamine一样，具有一个（β/α）_8桶形折叠的单结构域。我们通过定点突变研究了在yieldin的保守共有区中取代谷氨酸残基的影响。重组YLD（E128 Q）在胁迫-应变实验中丧失了屈服蛋白活性。位于该家族中保守的两个共有区的其他氨基酸残基的取代也导致yieldin活性的松动。然而，在非共有区域中具有定点突变的rYLD难以表达。因此，该保守区在屈服阈值张力调节活性中的作用仍有待阐明。其中一种抑制yieldin的功能。我们正在研究它的抗原决定簇。

Yieldin, a cell wall protein purified from elongating hypocotyls of cowpea, decreases the yield threshold tension (y) of the wall in an acidic condition. We constructed a high-level expression system of recombinant yieldin (rYLD) in Eseherichia coli. rYLD restored the acid-induced changes of y of heat-denatured glycerinated hollow cylinders (GHCs) of cowpea hypocotyls. Yieldin shares over 40% identity in the amino acid sequence with plant chitinases belonging to the glycosyl hydrolase family 18, and it has two conserved consensus regions involving a putative active site. However, no chitinase activity could be detected for yieldin. A computer-aided prediction of a 3D structure suggested that yieldin has a single domain with a (β/α)_8 barrel fold as concanavalin B and hevamine. We investigated effects of substitution of a glutamic acid residue in the conserved consensus regions of yieldin by site-directed mutagenesis. The resulted rYLD (E128Q) lost the yieldin activity in the stress-strain experiments. Substitutions of other amino acid residues located in the two consensus regions conserved in the family also resulted in loosening the yieldin activity. However, rYLD having site-directed mutation in other than the consensus regions was difficult to be expressed. Thus the role of the conserved region in the regulatory activity of yield threshold tension remained to be clarified Monoclonal antibodies against rYLD were successfully produced. One of them was found to inhibit the function of yieldin. We are now investigating its antigenic determinant.