Sadler J E, Shelton-Inloes B B, Sorace J M, Harlan J M, Titani K, Davie E W
Proc Natl Acad Sci U S A. 1985 Oct;82(19):6394-8. doi: 10.1073/pnas.82.19.6394.
A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.
用人脐静脉内皮细胞原代培养物中分离得到的聚腺苷酸加尾（poly(A)+）RNA构建了λgt11噬菌体cDNA文库。对约250万个独立重组体进行了筛选，发现其中2个能合成与β-半乳糖苷酶的融合蛋白，该融合蛋白可与抗人血管性血友病因子的兔抗体发生反应。将两个克隆的cDNA插入片段所翻译的氨基酸序列与通过蛋白质的埃德曼降解法确定的氨基酸序列进行比较，确定这两个噬菌体分离株均编码血管性血友病因子。第一个克隆（λHvWF1）包含一个404个核苷酸的插入片段，除了部分前原导序列（24个氨基酸）外，该片段对应于血液中循环的成熟蛋白的1 - 110个氨基酸残基。第二个cDNA克隆（λHvWF3）包含一个4.9千碱基的插入片段，编码血管性血友病因子的羧基末端1525个氨基酸、一个TGA终止密码子、134个核苷酸的3'非编码序列以及一个150个核苷酸的聚腺苷酸尾巴。这两个克隆共同编码血液中循环的该分子的80%以上。在成熟蛋白以及cDNA中鉴定出相同的羧基末端赖氨酸残基，这表明血管性血友病因子生物合成和组装过程中发生的所有蛋白水解加工都与前体蛋白的氨基末端部分相关。血管性血友病因子的氨基酸序列表明存在两种不同的内部基因重复和一种三倍重复。这些重复的氨基酸序列约占成熟蛋白中氨基酸的一半。在血管性血友病因子的羧基末端部分还鉴定出了介导纤连蛋白细胞附着和血小板结合活性的精氨酸 - 甘氨酸 - 天冬氨酸 - 丝氨酸四肽序列。
