Grobe Kay, Pöppelmann Marco, Becker Wolf-Meinhard, Petersen Arnd
University of California San Diego, La Jolla 92093-0687, USA.
Eur J Biochem. 2002 Apr;269(8):2083-92. doi: 10.1046/j.1432-1033.2002.02856.x.
Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls. They are divided into two groups, alpha and beta, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall. Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension. Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site-directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of beta-expansins. These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties.
扩展蛋白是一类催化离体植物细胞壁pH依赖性长期伸展的蛋白质家族。它们分为α和β两组，后者由禾本科I组花粉过敏原及其营养同源物组成。扩展蛋白被认为通过干扰细胞壁中的结构蛋白或多糖网络来介导植物细胞生长。我们的研究小组报道了梯牧草（Phleum pratense）花粉β-扩展蛋白Phl p 1具有木瓜蛋白酶样特性，并提出细胞壁结构蛋白的裂解可能是扩展蛋白介导的细胞壁伸展的潜在机制。在此，我们报告了更多数据，表明β-扩展蛋白类似于古代和现代的组织蛋白酶B，它是半胱氨酸蛋白酶木瓜蛋白酶（C1）家族的成员。使用毕赤酵母表达系统，我们发现重组过敏原N-糖基化促进了其抑制性前序列的裂解，截短的、活化的过敏原具有蛋白水解活性，导致该蛋白的稳定性极低。我们还表明，去糖基化的全长过敏原不能有效活化，因此相对稳定。基序和同源性搜索工具检测到β-扩展蛋白与现代动物以及原始动物蓝氏贾第鞭毛虫的组织蛋白酶之间存在显著相似性，证实了抑制性前序列、活性位点和其他功能性氨基酸残基的存在，以及这些特征在这些分子中的保守位置。最后，我们通过定点诱变证明保守的His104残基参与了β-扩展蛋白的催化活性。这些结果表明组织蛋白酶B和β-扩展蛋白有共同的起源，特别是结合它们先前已知的生化特性来看。
