Molecular and biological study on sialidase

在对纯化的人胎盘唾液酸酶部分相关蛋白的cDNA进行分子克隆的过程中，我们发现其中一个表观分子量为46 kDa的蛋白实际上是α-N-乙酰半乳糖苷酶。全长基因PCD-HS1204编码358个氨基酸，前17个残基代表一个可能的信号肽。预测的氨基酸序列与人α-半乳糖苷酶A和酵母α-半乳糖苷酶有显著的同源性。根据底物特异性和46 kDa蛋白在羟基磷灰石层析上的行为，证实46 kDa蛋白实际上是α-N-乙酰半乳糖胺酶。从人成纤维细胞cDNA文库中分离到两种人α-N-乙酰半乳糖胺酶基因。这两个物种在编码区插入了70个碱基，这两个物种彼此不同。在COS细胞中的瞬时表达研究表明，只有没有70bp插入的c DNA表达α-N-乙酰半乳糖…更多的转氨酶活性。通过聚合酶链式反应分析发现，人胎盘唾液酸酶的mRNA大部分不含70bp的插入片段，在人脑中仅有少量存在。探讨了人胎盘唾液酸酶的激活机制。我们观察到，无论是粗酶制剂，还是部分纯化的唾液酸酶制剂，都能在37ﾟC、pH 4.8的条件下被激活。阿司他丁是氨基肽酶A和亮氨酸氨基肽酶的抑制剂，可抑制这种激活。用于激活的部分纯化唾液酸酶制剂从氨基酸的甲基香豆胺(MCA)衍生物中释放亮氨酸和谷氨酸，而不释放精氨酸、赖氨酸和丙氨酸。锌离子显著抑制唾液酸酶活性，也抑制亮氨酸-MCA的水解酶活性。唾液酸酶的激活似乎是由一种内源性的蛋白酶引起的，可能是氨基肽酶。从人胎盘中分离出来的唾液酸酶与几种蛋白质组分有关，这些蛋白质组分在分离唾液酸酶时被认为是一个聚集的复合体。其中一种60 kDa的蛋白质是最近由Potier等人鉴定的。作为唾液酸酶蛋白，该蛋白还与抗丙皂素抗体发生交叉反应。我们已经分离了这个蛋白，并从以下证据中鉴定它是免疫球蛋白G的重链成分，而不是唾液酸酶或丙皂苷的衍生物。凝胶过滤高效液相色谱分析表明，唾液酸酶活性与60 kDa蛋白有明显的分离。该60 kDa蛋白不仅与人皂苷A、C、D抗体发生交叉反应，还与第二抗体(山羊抗兔免疫球蛋白G抗体)发生交叉反应。该60 kDa蛋白与抗人免疫球蛋白G抗体发生强烈交叉反应。60 kDa蛋白N-末端的前15个氨基酸序列与免疫球蛋白G重链蛋白Tie(Gamma1)的序列相同。较少

In the process of molecular cloning of cDNA for proteins associated with a purified human placental sialidase fraction, we discovered one of the proteins with apparent molecular weight of 46 kDa is in reality alpha-N-acetylgalactosaminidase. The full length cDNA, pcD-HS1204, codes for 358 amino acids with the first 17 residues representing a putative signal peptide. The predicted amino acid sequence shows striking homology with human alpha-galactosidase A and yeast alpha-galactosidase. The substrate specificities as well as the behavior of the 46 kDa protein on hydroxylapatite chromatography confirmed that the 46 kDa protein is in reality alpha-N-acetylgalactosaminidase.Two species of cDNAs for human alpha-N-acetylgalactosaminidase were isolated from a human fibroblast cDNA library. The two species differ each other by a 70 bp insertion in the coding region. Transient expression study in COS cells demonstrated that only the cDNA without the 70 bp insertion expressed alpha-N-acetylgalac … More tosaminidase activity. Analysis of mRNA species utilizing polymerase chain reaction revealed that the majority of the mRNA does not contain the 70 bp insertion, and the mRNA containing the 70 bp insertion is present only in a minor amount in human brain.The activation mechanism of human placental sialidase was studied. We observed that not only the crude enzyme preparation but also the partially purified preparation of sialidase was activated by the incubation at 37ﾟC in pH 4.8 medium. The activation inhibited by amastatin which is an inhibitor of aminopeptidase A and leucine aminopeptidase. The partially purified sialidase preparation used for the activation released leucine and glutamic acid but not arginine, lysine and alanine from methylcoumarlamide(MCA) derivatives of amino acid. Zinc ion significantly depressed the activation of sialidase and also inhibited the hydrolysis of leucine-MCA. The activation of sialidase seems to be caused by an endogeneous protease, probably aminopeptidase.Sialidase isolated from human placenta is associated with several protein components, which are thought to form an aggregated complex during isolation of sialidase. One of the proteins of 60 kDa was recently identified by Potier et al. as a sialidase protein; this protein also cross-reacted with anti-prosaposin antibodies. We have isolated this protein and from the following evidence identified it as a heavy chain component of immunoglobulin G and not sialidase or a derivative of prosaposin. On gel filtration HPLC, sialidase activity and the 60 kDa protein were clearly separated from one another. The 60 kDa protein cross-reacted not only with antibodies raised against human saposine A,C, and D, but also with second antibody (goat anti-rabbit immunoglobulin G antibody) alone. This 60 kDa protein strongly cross-reacted with anti-human immunoglobulin G antibodies. The sequence of the initial 15 amino acids from the N-terminus of the 60 kDa protein was identical to the sequence of an immunoglobulin G heavy chain protein Tie(gamma1). Less