ISOLATION AND CHARACTERIZATION OF A CDNA CODING FOR HUMAN FACTOR-IX
ISOLATION AND CHARACTERIZATION OF A CDNA CODING FOR HUMAN FACTOR-IX
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DOI：
10.1073/pnas.79.21.6461
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发表时间：
1982-01-01
期刊：
影响因子：
--
通讯作者：
DAVIE, EW
中科院分区：
文献类型：
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作者：
KURACHI, K;DAVIE, EW
从人肝脏制备的c[互补]DNA文库中筛选因子IX（圣诞因子），这是一种参与血液凝固中期的凝血因子。用从狒狒因子IX的富集mRNA和合成寡核苷酸混合物制备的单链DNA筛选文库。鉴定了含有编码人因子IX的1466个碱基对的cDNA插入物的质粒。插入物的两侧分别是在5“和3”末端的11和18个碱基对的G-C尾。它还包括138个碱基对，编码氨基端的前导序列，1248个碱基对，编码成熟蛋白，一个终止密码子和48个碱基对的非编码序列在3“端。前导序列含有46个氨基酸残基;该序列显然包括信号序列和在血浆中循环的成熟蛋白质的原序列。1248个碱基对编码由416个氨基酸组成的多肽链。该蛋白的氨基末端区域在成熟蛋白中含有12个谷氨酸。该蛋白质的氨基末端区域含有12个谷氨酸残基，其被转化为γ-谷氨酸。成熟蛋白中的羧基谷氨酸。这些谷氨酸残基由GAA和GAG编码。通过因子XIa将人因子IX转化为因子IXa时裂解的N-酰基肽键被鉴定为Arg 145-Ala 146和Arg 180-Val 181。这2个内部肽键的裂解导致形成活化肽（35个氨基酸）和因子IXa，因子IXa是一种由轻链（145个氨基酸）和H链（236个氨基酸）组成的丝氨酸蛋白酶;这2条链通过二硫键连接在一起。包括组氨酸、天冬氨酸和丝氨酸的活性位点残基分别位于H链的221、270和366位。这些氨基酸与胰凝乳蛋白酶活性部位的His 57、Asp 102和Ser 195同源。在激活肽中鉴定了两个潜在的碳水化合物结合位点（Asn-X-Thr）;它们位于Asn 157和Asn 167。人和牛因子IX之间的氨基酸序列同源性为83%。
A c[complementary]DNA library prepared from human liver was screened for factor IX (Christmas factor), a clotting factor that participates in the middle phase of blood coagulation. The library was screened with a single-stranded DNA prepared from enriched mRNA for baboon factor IX and synthetic oligonucleotide mixture. A plasmid was identified that contained a cDNA insert of 1466 base pairs coding for human factor IX. The insert is flanked by G-C tails of 11 and 18 base pairs at the 5'' and 3'' ends, respectively. It also included 138 base pairs that code for an amino-terminal leader sequence, 1248 base pairs that code for the mature protein, a stop codon and 48 base pairs of noncoding sequence at the 3'' end. The leader sequence contains 46 amino acid residues; this sequence apparently includes both a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 1248 base pairs code for a polypeptide chain composed of 416 amino acids. The amino-terminal region for this protein contains 12 glutamic acid in the mature protein. The amino-terminal region for this protein contains 12 glutamic acids residues that are converted to .gamma.-carboxyglutamic acid in the mature protein. These glutamic acid residues are coded for by both GAA and GAG. The arginyl peptide bonds cleaved in the conversion of human factor IX to factor IXa by factor XIa were identified as Arg145-Ala146 and Arg180-Val181. The cleavage of these 2 internal peptide bonds results in the formation of an activation peptide (35 amino acids) and factor IXa, a serine protease composed of a light chain (145 amino acids) and a H chain (236 amino acids); these 2 chains are held together by a disulfide bond(s). The active site residues including histidine, aspartate and serine are located in the H chain at positions 221, 270 and 366, respectively. These amino acids are homologous with His57, Asp102 and Ser195 in the active site of chymotrypsin. Two potential carbohydrate binding sites (Asn-X-Thr) were identified in the activation peptide; these were located at Asn157 and Asn167. The homology in the amino acid sequence between human and bovine factor IX is 83%.