Purification and Chemical Characterization of β‐Trace Protein from Human Cerebrospinal Fluid: Its Identification as Prostaglandin D Synthase
Purification and Chemical Characterization of β‐Trace Protein from Human Cerebrospinal Fluid: Its Identification as Prostaglandin D Synthase
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DOI：
10.1111/j.1471-4159.1993.tb02145.x
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发表时间：
1993-08
影响因子：
4.7
通讯作者：
A. Hoffmann;H. Conradt;G. Gross;M. Nimtz;F. Lottspeich;U. Wurster
中科院分区：
文献类型：
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作者：
A. Hoffmann;H. Conradt;G. Gross;M. Nimtz;F. Lottspeich;U. Wurster
摘要：将来自合并人CSF的β-痕量蛋白纯化至均一。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上测定多肽的表观分子量为23-29 kDa。多肽的氨基末端测序产生独特的氨基酸序列APEAQVSVQPNIPQDKFLGRWFSA 24。将从胰蛋白酶肽获得的氨基酸序列与之前从其他研究人员分离的cDNA克隆推导的序列进行比对，可以将β-微量蛋白鉴定为前列腺素D合酶[前列腺素-H2 D-异构酶;（5 Z，13 E）-（15 S）-9α，11 a-表二氧-15-羟基前列腺-5，13-二烯酸D-异构酶; EC 5.3.99.2]。在相应的胰蛋白酶肽中β-trace多肽链的第154位氨基酸处检测到保守的氨基酸交换（The而不是Ser）。多肽的两个N-糖基化位点几乎被碳水化合物定量占据。碳水化合物组成和甲基化分析表明，Asn 29和Asn 56仅具有复杂型寡糖结构（用α2-3-和/或α2-6-连接的N-乙酰神经氨酸部分唾液酸化），在近端N-乙酰葡萄糖胺几乎定量α1 - 6岩藻糖基化;这些分子中约70%含有二等分N-乙酰葡萄糖胺。也存在无乳结构以及具有外周岩藻糖的结构。
Abstract: β‐Trace protein from pooled human CSF was purified to homogeneity. An apparent molecular mass of 23–29 kDa was determined for the polypeptide on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Amino‐terminal sequencing of the polypeptide yielded the unique amino acid sequence APEAQVSVQPNFQQDKFLGRWFSA24. Alignment of amino acid sequences obtained from tryptic peptides with the sequence previously deduced from a cDNA clone isolated by other investigators allowed the identification of β‐trace protein as prostaglandin D synthase [prostaglandin‐H2 D‐isomerase; (5Z, 13E)‐(15S)‐9α, 11 a‐epidioxy‐15‐hydroxyprosta‐5,13‐dienoate D‐isomerase; EC 5.3.99.2]. A conservative amino acid exchange (The instead of Ser) was detected at amino acid position 154 of the β‐trace polypeptide chain in the corresponding tryptic peptide. The two N‐glycosylation sites of the polypeptide were shown to be almost quantitatively occupied by carbohydrate. Carbohydrate compositional as well as methylation analysis indicated that Asn29and Asn56 bear exclusively complex‐type oligosaccharide structures (partially sialylated with α2–3‐ and/or α2–6‐linked N‐acetylneuraminic acid) that are almost quantitatively α1‐6 fucosylated at the proximal N‐acetylglucosamine; ∼70% of these molecules contain a bisecting N‐acetylglucosamine. Agalacto structures as well as those with a peripheral fucose are also present.