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

已发现主要胰腺酶具有新的碳水化合物结合活性。哺乳动物的α-淀粉酶、胰酶、胰酶原、糜蛋白酶、弹力酶、脂肪酶和核糖核酸酶与糖蛋白N-连接的寡糖具有不同于底物识别的结合活性。利用表面等离子体共振(SPR)的相互作用分析表明，胰蛋白酶通常与含有N-糖链的糖蛋白结合，但不与0-连接的粘蛋白类型的糖链结合，而胰酶原则与含有0-糖链和N-糖链的糖蛋白结合。胰酶与糖蛋白的结合常数(Ka)为106-1010M-1，经胎牛蛋白去糖基化后，胰酶对胎蛋白的结合常数由101M-1降至104M“。与生物素-聚合物(BP-)糖探针的结合表明，每种胰酶都表现出特异性的糖结合活性，但它们的特异性略有不同，糖蛋白结合活性是由于酶对N-聚糖或0-聚糖的组成糖的亲和力所致。这种结合活性被发现是胰腺酶所特有的，而对来自人唾液、植物和真菌的α-淀粉酶和来自真菌的脂肪酶则没有观察到，这表明该活性具有胰腺特有的功能。糖蛋白或碳水化合物的结合非竞争性或非竞争性地增强了酶的活性，表明N-糖链的识别部位与其催化部位不同。与BBM组分的结合研究表明，每种酶都存在糖受体，作为支架实现有组织和系统的消化。利用为预测蛋白质的碳水化合物结合部位而开发的软件和X射线结晶学的实验分析，对胰腺酶的碳水化合物结合部位和方式进行了研究。

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.