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The biological significance of the carbohydrate-binding activities found for pancreatic enzymes in secretion and digestion

胰酶在分泌和消化中发现的碳水化合物结合活性的生物学意义

基本信息

批准号：
17570109
负责人：
OGAWA Haruko
金额：
$ 2.24万
依托单位：
Ochanomizu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570109/
关键词：
biomolecule sugar chain enzyme carbohydrate-binding pancreatic enzyme 膵臓分泌消化

项目摘要

Novel carbohydrate-binding activities have been discovered for principal pancreatic enzymes. Mammalian a-amylases, trypsins, trypsinogen, chymotrypsin, elastase, lipase, and ribonuclease were found to share the binding activity toward N-linked oligosaccharides of glycoproteins, which is different from the substrate recognition. Trypsins were commonly shown by interaction analyses using surface plasmon resonance (SPR) to bind with glycoproteins possessing N-glycans, but not 0-linked mucin-type glycans, while trypsinogen was found to bind to the gycoproteins possessing 0-glycans as well as N -glycans. The binding constants (KA) between trypsins and glycoproteins were 106-1010 M-1 and Ka of trypsin toward fetuin was decreased from101 M-1 to 104M" by deN-glycosylation of fetuin. Binding with biotin-polymer (BP-) sugar probes revealed that each pancreatic enzyme exhibits specific carbohydrate-binding activity with slight differences among their specificities and the glycoprotein-binding activities were due to the affinity of the enzymes toward the component sugars of Nglycans or 0-glycans. The binding activity was found to be unique to pancreatic enzymes and not observed for a-amylase from human saliva, plants, and fungus and lipase from fungi suggesting a pancreas- specific function of the activity. The binding of glycoproteins or carbohydrates enhanced the enzyme activity noncompetitively or uncompetitively, indicating that the recognition site for N-glycans is different from its catalytic site. Binding studies with the BBM fraction indicated the presence of glycoreceptors for each enzyme that serve as scaffolds to achieve an organized and systematic digestion. The sites and the modes of the carbohydrate-binding of pancreatic enzymes are under investigation by using the software developed for the prediction of the carbohydrate-binding site of proteins and the experimental analyses by x-ray crystallography.

新的碳水化合物结合活性已被发现的主要胰腺酶。哺乳动物的α-淀粉酶、胰蛋白酶、胰蛋白酶原、胰凝乳蛋白酶、弹性蛋白酶、脂肪酶和核糖核酸酶被发现对糖蛋白的N-连接寡糖具有与底物识别不同的结合活性。胰蛋白酶通常通过使用表面等离子体共振（SPR）的相互作用分析显示与具有N-聚糖的糖蛋白结合，但不与0-连接的粘蛋白型聚糖结合，而胰蛋白酶原被发现与具有0-聚糖以及N -聚糖的糖蛋白结合。胰蛋白酶与糖蛋白的结合常数（KA）为106-1010 M ~（-1），胎球蛋白去N-糖基化后，胰蛋白酶与胎球蛋白的结合常数（KA）由101 M ~（-1）降至104 M ~（-1）。与生物素-聚合物（BP-）糖探针的结合显示，每种胰酶都表现出特异性碳水化合物结合活性，其特异性之间略有差异，并且糖蛋白结合活性是由于酶对N-聚糖或0-聚糖的组分糖的亲和力。发现结合活性对于胰腺酶是独特的，并且对于来自人唾液、植物和真菌的α-淀粉酶和来自真菌的脂肪酶没有观察到，表明活性的胰腺特异性功能。糖蛋白或碳水化合物的结合非竞争性或非竞争性地增强酶活性，表明N-聚糖的识别位点不同于其催化位点。与BBM级分的结合研究表明，作为支架，以实现有组织的和系统的消化每种酶的糖受体的存在。本文利用为预测蛋白质糖结合位点而开发的软件和X射线晶体学实验分析，研究了胰蛋白酶糖结合的位点和模式。