Qi X, Behrens B X, West P R, Mort A J
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078, USA.
Plant Physiol. 1995 Aug;108(4):1691-701. doi: 10.1104/pp.108.4.1691.
Extensin, a major hydroxyproline (Hyp)-rich glycoprotein in walls of cultured cells of dicotyledonous plants, is very difficult to solubilize. To learn about the nature of the insolubilization, we have tested the ability of a variety of selective hydrolytic methods, and combinations of them, to liberate extensin or fragments of extensin from suspension-culture cell walls. After the complete deglycosylation of cotton (Gossypium hirsutum L.) walls, trypsinization solubilized 80% of the Hyp. The sequences of three abundant peptides were: (a) serine-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-serine-Hyp-Hyp-lysine, (b) serine-Hyp-Hyp-Hyp-Hyp-valine-lysine, and (c) serine-Hyp-Hyp-serine-alanine-Hyp-lysine. After a sequential treatment of walls with endopolygalacturonase, cellulase, -73 degrees C anhydrous hydrogen fluoride solvolysis, and ammonium bicarbonate extraction, only sugars indicative of rhamnogalacturonan I and protein remained insoluble. Trypsin treatment of this residue liberated 50% of the Hyp. A significant proportion of rhamnogalacturonan-associated sugars co-solubilized and co-purified along with the extensin fragments following the trypsinization. By sodium dodecyl sulfate gel electrophoresis and gel filtration, the glycopeptides fell into two classes. One class contained distinctly sized molecules with relative molecular weights in the range of 4,000 to 24,000. The other class did not enter the resolving gel and was hetero-disperse. After complete deglycosylation by a 0 degrees C anhydrous hydrogen fluoride treatment, the first class was little affected in its electrophoretic mobility, whereas the larger heterogeneous material mostly entered the separating gel. After further trypsinization of the deglycosylated peptides and analysis by capillary zone electrophoresis, the peptides in both size classes were shown to contain the sequences described above. From our observations we suggest that cotton extensin becomes insolubilized into cell walls in part by pectin-protein cross-links in addition to the protein-protein (or protein-phenolic-protein) cross-links that have been repeatedly suggested.
伸展蛋白是双子叶植物培养细胞细胞壁中一种主要的富含羟脯氨酸（Hyp）的糖蛋白，极难溶解。为了解其不溶性的本质，我们测试了多种选择性水解方法及其组合从悬浮培养细胞壁中释放伸展蛋白或伸展蛋白片段的能力。棉（陆地棉）细胞壁完全去糖基化后，用胰蛋白酶处理可溶解80%的Hyp。三种丰富肽段的序列为：（a）丝氨酸-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-丝氨酸-Hyp-Hyp-赖氨酸，（b）丝氨酸-Hyp-Hyp-Hyp-Hyp-缬氨酸-赖氨酸，以及（c）丝氨酸-Hyp-Hyp-丝氨酸-丙氨酸-Hyp-赖氨酸。在用内切多聚半乳糖醛酸酶、纤维素酶、-73℃无水氟化氢溶剂解和碳酸氢铵提取依次处理细胞壁后，仅鼠李半乳糖醛酸聚糖I的指示性糖类和蛋白质仍不溶解。用胰蛋白酶处理该残渣可释放50%的Hyp。胰蛋白酶处理后，相当一部分与鼠李半乳糖醛酸聚糖相关的糖类与伸展蛋白片段共溶解并共纯化。通过十二烷基硫酸钠凝胶电泳和凝胶过滤，糖肽分为两类。一类包含相对分子质量在4000至24000范围内大小明显的分子。另一类未进入分离胶且是异质分散的。经0℃无水氟化氢处理完全去糖基化后，第一类分子的电泳迁移率几乎不受影响，而较大的异质物质大多进入分离胶。对去糖基化肽段进一步用胰蛋白酶处理并通过毛细管区带电泳分析后，两类大小的肽段均显示含有上述序列。根据我们的观察，我们认为棉伸展蛋白除了已被反复提及的蛋白质-蛋白质（或蛋白质-酚类-蛋白质）交联外，部分通过果胶-蛋白质交联而固定在细胞壁中。
