Studies on a Novel Protease, Capable of Depolymerizing Glycopolyproteins upon Fertilization of Trout Egg

一种能够解聚高糖基化多蛋白的新型蛋白酶（PSGPase） [cf., K Kitajima et al. (1986) [J]。化学261,5262-5269；Mr 200x10^3 PSGP] to 9-kDa PSGP[参见S. Inoue & Y. Inoue (1986) J biology。化学261,5256-5261]在虹鳟鱼卵受精后，从未受精卵中分离并纯化了2100倍。该酶在10 ~ 20℃的温度和低于40 mM的离子强度下明显催化“脯氨酸定向前天冬氨酸裂解”。其特异性可与n端裂解成一对碱基残基或脯氨酸定向单基精氨酸裂解相比较，后者是已知的作用于调节肽前体的加工酶。PSGPase是共定位的，在未受精卵的亚细胞区室（皮质肺泡）中处于失活状态，与其生理底物200 kda PSGP一起。受精后，皮层肺泡发生胞吐，其内容物被转移到卵泡周围空间，在那里200-kDa PSGP多蛋白结构被PSGPase转化为重复单元（9-kDa PSGP），其活性可能受盐浓度的调节。对纯化的PSGPase进行抑制剂研究。PCMB在1mM时几乎完全抑制PSGPase活性，但半胱氨酸蛋白酶抑制剂E-46对PSGPase活性的影响很小或没有影响，并且在所有反应介质中存在还原剂如二硫苏糖醇并不是酶活性的必要条件。这些结果共同表明psgase不是一种巯基蛋白酶。虽然1 mM的苯甲磺酰氟（PMSF）对PSGPase活性几乎没有影响，但高浓度的氟化二异丙基（DFP）干扰了PSGPase活性，表明PSGPase可能是一种丝氨酸蛋白酶。金属蛋白酶抑制剂均不能阻断200 kda PSGP的降解。1978年，井上和岩崎在虹鳟鱼的未受精卵中发现了一类新的酸性糖蛋白。这些糖蛋白最显著的特征是在假体碳水化合物基团中存在低聚（聚）唾液酸链，我们将它们命名为多唾液糖蛋白（PSGP）。我们已经证明，PSGP是研究的所有鲑科鱼类的成熟和未成熟卵母细胞中普遍存在的成分。目前的研究首先是为了引出针对聚唾液酸的抗体，因为在B群脑膜炎球菌（脑膜炎奈瑟菌）中发生的- 2,8 -连接的n -乙酰神经氨酸（NeuAc）结构的均聚物是致病性的。在包括人类在内的多种动物中，α - 2,8 -linked poly （NeuAc）也是一种极差的免疫原。虹鳟鱼卵PSGP中仅含有n -糖基神经氨酸（NeuGc），因此我们将其作为免疫原，在兔和BALB/c小鼠中诱导α - 2,8 -linked poly （NeuGc）抗体。我们将抗原注射持续了很长一段时间（长达16个月），但通过双重免疫扩散程序和酶联免疫吸附试验检测时，所有动物的血清中均未检测到抗体。观察到的免疫原性差再次归因于，至少部分归因于宿主动物中存在具有相似或相同结构的分子，因此免疫可能会被免疫耐受抑制。最近，我们成功地在鸡体内诱导了虹鳟鱼蛋的PSGP抗血清。我们还检测了α - 2,8链聚（NeuGc）链对一种独特的抗血清的免疫反应性，该抗血清是由j.b.罗宾斯（n.i. h）用福尔马林处理的B组脑膜炎球菌作为免疫原免疫一匹马多年后获得的。含有结合α - 2,8 -linked poly （NeuAc）的多克隆IgM抗体的抗血清（H. 46）已被证明可形成针对α - 2,8 -linked poly （NeuGc）的免疫预匹肽带。结果表明，H. 46与α - 2,8 -linked poly （NeuAc）特异性结合，只能作为poly （NeuAc）的探针。在这方面，我们在鸡身上的结果虽然是初步的，但可能提供针对poly （NeuGc）的特异性抗体，该抗体可能用作检测多种动物组织和物种中的poly （NeuGc）的诊断工具。少

A novel protease (PSGPase), capable of depolymerizing highly glycosylated polyprotein [cf., K Kitajima et al. (1986) J Biol. Chem 261, 5262-5269 ; Mr 200x10^3 PSGP] to 9-kDa PSGP[see S. Inoue & Y. Inoue (1986) J Biol. Chem 261, 5256-5261] upon fertilization of rainbow trout eggs, was isolated and purified 2100-fold from the unfertilized eggs. The enzyme apparently catalyzes the "prolinedirected pre-aspartyl cleavage" at temperature between 10 and 20^0C and only at ionic strength below 40 mM. The specificity may be compared with the cleavage of N-terminal to a pair of basic residues or , proline-directed monobasic arginyl cleavage, known for the processing enzymes acting on precursors of regulatory peptides. PSGPase is colocalized, as an inactive state within asubcellular compartment (cortical alveolus) of the unfertilized eggs together with its physiological substrates, 200-kDa PSGP. Upon fertilization exocytosis of the cortical alveoli occurred and their contents were translocated to … More perivitelline space where the polyprotein structure of 200-kDa PSGP was converted to the repeating unit (9-kDa PSGP) by PSGPase of which activity might be regulated by salt concentration.Inhibitor studies were carried out on the purified PSGPase. The PSGPase activity was inhibited almost completely by PCMB at 1mM, but the cysteine-protease inhibitor E-46 had little or no effect on PSGPase activity and the presence of reducing agents such as dithiothreitol in all reaction media is not necessary for enzyme activity. These results collectively suggest PSGPase not to be a thiol-protease. Although the presence of 1 mM of phenylmethanesulfonyl fluoride (PMSF) had almost no effect on PSGPase activity, diisopropyl phosphorofluoridate (DFP), at high concentrations interfered with the PSGPase activity, indicating PSGPase likely to be a serine-protease. None of the metalloprotease inhibitors blocked the degradation of 200-kDa PSGP.In 1978, Inoue and Iwasaki discovered a novel class of acidic glycoproteins in the unfertilized eggs of rainbow trout. The most striking feature of these glycoproteins is the presence of oligo (ploy) sialyl chains in the prosthetic carbohydrate groups, and we named them polysialoglycoproteins (PSGP). We have shown that PSGP is a ubiquitous components of both mature and immature oocytes of all species of salmonid fishes studied. The present study was first undertaken to elicit antibodies against polysialic acids simply because alpha-2, 8-linked homopolymer of N-acetylneuraminic acid (NeuAc) structure occurring in the group B meningococci (Neisseria meningitidis is pathogenic. It is also well known that alpha-2, 8-linked poly (NeuAc) is extremely poor immunogen in various animals including human. Rainbow trout egg PSGP contains exclusively N-glycolylneuraminic acid (NeuGc) , so that we used it as the immunogen to elicit alpha-2, 8-linked poly (NeuGc) antibodies in rabbits and BALB/c mouse. We continued the antigen injection for a long period (up to 16 months), but there was no detectable antibodies in the serum of all animals used when tested by a double immunodiffusion procedure and by enzyme-linked immunosorbent assays. The observed poor immunogenicity is again ascribed, at least in part, to the presence of molecules having similar or identical structures in the host animals so that immunization would likely be suppressed by immune tolerance. Recently, we succeeded in eliciting rainbow trout egg PSGP antiserum in chicken.We also examined the immunoreactivity of alpha-2, 8-linked poly (NeuGc) chains against a unique antiserum obtained by J. B. Robbins (N. I. H.) after taking for years by immunizing a horse using formalin-treated group B meningococci as immunogen. The antiserum (H. 46) containg polyclonal IgM antibodies to bind alpha-2, 8-linked poly (NeuAc) has been proven to form immunopricipitin bands against alpha-2, 8-linked poly (NeuGc). It can be concluded that H. 46 binds specifically to alpha-2, 8-linked poly (NeuAc) and can only be used as a probe for poly (NeuAc). In this respect, our results on chicken, though preliminary, may provide specific antibody against poly (NeuGc) which may possibly be used as a diagnostic tool for detecting poly (NeuGc) in a variety of animal tissues and species. Less