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首页> 外文期刊>Biochemistry >Mass Spectrometry Analysis of Reeombinant Human ZP3 Expressed in Glycosylation-Deficient CHO Cells
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Mass Spectrometry Analysis of Reeombinant Human ZP3 Expressed in Glycosylation-Deficient CHO Cells

机译：糖基化缺陷型CHO细胞中表达的重组人ZP3的质谱分析

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The zona pellucida is an extracellular matrix that mediates taxon-specific fertilization in which human sperm will not bind to mouse eggs.The mouse zona pellucida is composed of three glycoproteins (ZP1,ZP2,ZP3).The primary structure of each has been deduced from the cDNA nucleic acid sequence,and each has been analyzed by mass spectrometry.However,determination of the secondary structure and processing of the human zona proteins have been hampered by the paucity of biological material.To investigate if taxon-specific sperm-egg recognition was ascribable to structural differences in a zona protein required for matrix formation,recombinant human ZP3 was expressed in CHO-Lec3.2.8.1 cells and compared to mouse ZP3.With nearly complete coverage,LC-QTOF mass spectrometry was used to determine the cleavage of an N-terminal signal peptide (amino acids 1-22) and the release of secreted ZP3 from a C-terminal transmembrane domain (amino acids 379-424).The resultant N-terminal glutamine was cyclized to pyroglutamate (pyrGln~(23)),and several C-terminal peptides were detected,including one ending at Asn~(350).The disulfide bond linkages of eight cysteine residues in the conserved zona domain were ascertained (Cys~(46)/Cys~(140),Cys~(78)/Cys~(99),Cys~(217)/Cys~(282),Cys~(239)/Cys~(300)),but the precise linkage of two additional disulfide bonds was indeterminate due to clustering of the remaining four cysteine residues (Cys~(319),Cys~(321),Cys~(322),Cys~(327)).Three of the four potential N-linked oligosaccharide binding sites (Asn~(125),Asn~(147),Asn~(272)) were occupied,and clusters of O-glycans were observed within two regions,amino acids 156-173 and 260-281.Taken together,these data indicate that human and mouse ZP3 proteins are quite similar,and alternative explanations of taxon-specific sperm binding warrant exploration.
机译：透明带是一种细胞外基质，可介导分类细胞特异的受精，其中人类精子不会与小鼠卵结合。小鼠透明带由三种糖蛋白（ZP1，ZP2，ZP3）组成。 cDNA核酸序列，并已通过质谱法进行了分析。然而，由于缺乏生物材料，阻碍了人类Zona蛋白的二级结构的确定和加工。由于基质形成所需的Zona蛋白结构上的差异，重组人ZP3在CHO-Lec3.2.8.1细胞中表达并与小鼠ZP3进行了比较。LC-QTOF质谱法几乎覆盖了其全部区域，确定了其裂解N端信号肽（氨基酸1-22）和分泌的ZP3从C端跨膜结构域释放（氨基酸379-424）.N端谷氨酰胺为环将其裂解为焦谷氨酸（pyrGln〜（23）），并检测到多个C端肽，其中一个终止于Asn〜（350）。确定了保守的Zona域中八个半胱氨酸残基的二硫键（Cys〜（46） ）/ Cys〜（140），Cys〜（78）/ Cys〜（99），Cys〜（217）/ Cys〜（282），Cys〜（239）/ Cys〜（300）），但是由于剩余的四个半胱氨酸残基（Cys〜（319），Cys〜（321），Cys〜（322），Cys〜（327））的聚集，两个额外的二硫键是不确定的。四个潜在的N-连接寡糖中的三个结合位点（Asn〜（125），Asn〜（147），Asn〜（272））被占据，并且在两个区域（氨基酸156-173和260-281）中观察到O-聚糖簇。数据表明，人和小鼠的ZP3蛋白非常相似，对分类群特异性精子结合的替代解释值得探索。

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