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是从豇豆的细长下胚轴中纯化出来的细胞壁蛋白，在酸性条件下降低细胞壁的产率阈值张力(y)。构建了重组产率蛋白（rYLD）在大肠杆菌中的高水平表达体系。rYLD恢复了豇豆下胚轴热变性甘油空心柱体（GHCs）酸致y的变化。Yieldin在氨基酸序列上与属糖基水解酶家族18的植物几丁质酶有40%以上的一致性，并且它有两个保守的共识区域，涉及一个假定的活性位点。但产率酶未检测到几丁质酶活性。三维结构的计算机辅助预测表明，yieldin具有（β/α）_8桶折叠的单一结构域，与concanavalin B和hevamine相同。我们研究了谷氨酸残基在产率保守一致区域被定点诱变取代的影响。所得的rYLD （E128Q）在应力应变实验中丧失了屈服活性。在家族中保守的两个共识区的其他氨基酸残基的取代也导致了产率活性的降低。然而，在共识区以外的位点定向突变的rYLD很难表达。因此，保守区在产率阈值张力调控活性中的作用尚不清楚，抗rYLD单克隆抗体已成功制备。其中一种被发现能抑制屈服蛋白的功能。我们现在正在研究它的抗原决定因素。

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.