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

主要胰酶已发现新的碳水化合物结合活性。哺乳动物的a-淀粉酶、胰蛋白酶、胰蛋白酶原、凝乳胰蛋白酶、弹性酶、脂肪酶和核糖核酸酶对糖蛋白的n -低聚糖具有相同的结合活性，这与底物识别不同。通过表面等离子体共振（SPR）相互作用分析，胰蛋白酶通常与含有N-聚糖的糖蛋白结合，而不是与0连接的粘蛋白型聚糖结合，而胰蛋白酶原被发现与含有0-聚糖和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，通过对胎儿蛋白的den -糖基化，胰蛋白酶对胎儿蛋白的结合常数（KA）从101 M-1降低到104M"。与生物素-聚合物（BP-）糖探针的结合表明，每种胰酶都具有特定的糖结合活性，其特异性略有差异，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。我们发现这种结合活性是胰腺酶所特有的，而人类唾液中的a-淀粉酶、植物和真菌中的a-淀粉酶和真菌中的脂肪酶都没有观察到这种结合活性，这表明这种活性具有胰腺特异性功能。糖蛋白或碳水化合物的结合非竞争性或非竞争性地增强了酶的活性，表明n -聚糖的识别位点与其催化位点不同。与BBM部分的结合研究表明，每种酶都存在糖受体，作为支架来实现有组织和系统的消化。利用预测蛋白质碳水化合物结合位点的软件和x射线晶体学的实验分析，对胰酶的碳水化合物结合位点和模式进行了研究。

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.