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

一种能够在鳟鱼卵受精时解聚糖多蛋白的新型蛋白酶的研究

• 批准号: 63470135
• 负责人: INOUE Yasuo
• 金额: $ 4.35万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63470135/
• 关键词: Novel protease in trout eggs (PSGPase); Polysialoglycoproteins (PSGP); Proline-directed aspartyl cleaving protease; Fertilization-associated protease; 能卵の受精と卵活性化; 魚卵表層胞在プロテア-ゼ; PSGPase; PSGPaseの基質特異性; ポリシアログリコプロテイン; ポリシアログリコプロテナ-ゼ; 魚卵の受精; 卵表層胞局

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

一种新型的蛋白酶(PSGPase)，能够解聚高度糖基化的多蛋白[参见K Kitajima等人。(1986)J Biol.Chem 261,5262-5269；MR 200x10^3 PSGP]到9 kDa PSGP[见S.Inoue&Y.Inoue(1986)J Biol.[化学261,5256-5261]虹鲑卵受精后，从未受精卵中分离纯化2100倍。该酶在10~20℃温度范围内，仅在离子强度低于40 mM时，才能明显催化“Pro-导向的前天冬氨酸裂解”。这种特异性可以与N末端对一对碱性残基的裂解相比较，或者与以作用于调节肽前体的加工酶而闻名的脯氨酸单基精氨酸裂解相比较。PSGPase与其生理底物200 kDa PSGP共定位于未受精卵的亚细胞室(皮质肺泡)内，处于非活性状态。受精后，皮质肺泡发生胞吐，其内容物被转移到…200-kDa PSGP的多蛋白结构被PSGPase转化为重复单位(9-kDa PSGP)的卵周空间较大，其活性可能受盐浓度的调节。在1 mM处，PCMB几乎完全抑制PSGPase的活性，而半胱氨酸蛋白酶抑制剂E-46对PSGPase的活性几乎没有影响，而且所有反应介质中都不需要二硫苏糖醇等还原剂来维持酶的活性。这些结果共同表明PSGPase不是一种硫醇-蛋白酶。尽管1 mM的苯甲烷磺酰氟(PMSF)对PSGPase的活性几乎没有影响，但高浓度的二异丙基氟磷酸盐(DFP)对PSGPase的活性有干扰作用，表明PSGPase可能是一种丝氨酸蛋白酶。任何一种金属蛋白酶抑制剂都不能阻止200 kDa PSGP的降解。1978年，井上和岩崎在虹鱼未受精卵中发现了一种新的酸性糖蛋白。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寡聚唾液酸链，我们将其命名为聚唾液酸糖蛋白(PSGP)。我们已经证明PSGP是所有所研究的鲑科鱼类成熟和未成熟卵母细胞中普遍存在的成分。由于B组脑膜炎球菌(脑膜炎奈瑟氏菌是致病的)中存在α-2，8-连接的N-乙酰神经氨酸(NeuAc)结构均聚体，本研究首先进行了抗多唾液酸抗体的研究。众所周知，α-2，8-连接的聚(NeuAc)在包括人类在内的各种动物中都是极弱的免疫原。虹鲑卵PSGP只含有N-羟基神经氨酸(NeuGc)，因此我们用它作为免疫原在兔和BALB/c小鼠中诱导了α-2，8-连接的聚(NeuGc)抗体。我们继续注射抗原很长一段时间(长达16个月)，但用双向免疫扩散程序和酶联免疫吸附试验检测时，所有使用的动物的血清中都没有检测到抗体。观察到的免疫原性较差，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，因此免疫可能会受到免疫耐受的抑制。最近，我们成功地在雏鸡中诱导出虹鲑卵PSGP抗血清。我们还检测了α-2，8-连接聚(NeuGc)链对J.B.Robbins(N.I.H.)获得的独特抗血清的免疫反应性。在服用多年后，用福尔马林处理的B组脑膜炎双球菌作为免疫原免疫一匹马。含有多克隆IgM抗体的抗血清(H.46)可与α-2，8-连接的聚(NeuAc)结合，已被证明能形成针对α-2，8-连接的聚(NeuGc)的免疫球蛋白条带。可以认为，H.46与α-2，8-连接的聚(NeuAc)特异结合，只能作为聚(NeuAc)的探针。在这方面，我们在鸡身上的结果虽然是初步的，但可能提供针对聚(NeuGc)的特异性抗体，该抗体可能被用作在各种动物组织和物种中检测聚(NeuGc)的诊断工具。较少

项目成果

H. Sorimachi, Y. Emori, H. Kawasaki, K. Suzuki, and Y. Inoue,: "Molecular Cloning and Characterization of cDNAs Coding for Apopolysialoglycoproteins in Cherry Salmon (Oncorhyncus masou) Eggs" J. Biochem. (Tokyo),. 107-1,. 61-67, (1990)

H. Sorimachi、Y. Emori、H. Kawasaki、K. Suzuki 和 Y. Inoue，：“樱桃鲑鱼 (Oncorhyncus masou) 蛋中脱脂多唾液酸糖蛋白编码 cDNA 的分子克隆和表征” J. Biochem。

Ken Kitajima: "Proline-directed Aspartyl Cleaving Protease Acting on Salmonidae Egg Polyproteins at Fertilization" The Journal of Biological Chemistry.

Ken Kitajima：“脯氨酸导向的天冬氨酰裂解蛋白酶在受精时作用于鲑鱼卵多蛋白”《生物化学杂志》。

反町 洋之: "Organization and Primary Sequence of Multiple Genes Coding for Apopolysialoglycoproteins of Rainbow Trout" Jounal of Molecular Biology. 211. 35-48 (1990)

Hiroyuki Sorimachi：“虹鳟脱脂多唾液酸糖蛋白的多基因编码的组织和初级序列”分子生物学杂志 211. 35-48 (1990)。

Akiko Kanamori: "Isolation and Characterization of Deaminated Neuraminic Acid-rich Glycoprotein(KDN-gp-OF)in the Ovarian Fluid of Rainbow Trout" Biochemical and Biophysical Research Communications. 164. 744-749 (1989)

Akiko Kanamori：“虹鳟鱼卵巢液中富含脱氨基神经氨酸的糖蛋白 (KDN-gp-OF) 的分离和表征”生物化学和生物物理研究通讯。

Ken,Kitajima: Developmental Biology. 129. 270-274 (1988)

肯·北岛：发育生物学。