Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor
Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor
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DOI：
10.1074/jbc.m002654200
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发表时间：
2000-08-18
影响因子：
4.8
通讯作者：
Sadler, JE
中科院分区：
文献类型：
--
作者：
Katsumi, A;Tuley, EA;Sadler, JE
血管性血友病因子（VWF）是正常止血所需的多聚体糖蛋白。在易位进入内质网后，proVWF亚基通过其C-末端胱氨酸结样（CK）结构域之间的二硫键二聚化。CK结构域的特征在于六个保守的半胱氨酸，半胱氨酸2和5之间以及半胱氨酸3和6之间的二硫键限定了被半胱氨酸1和4之间的二硫键穿透的环。二聚化通常由CK结构域亚家族中不同的另外的半胱氨酸介导。当在杆状病毒系统中表达时，重组VWF CK结构域（残基1957-2050）以二聚体形式分泌，通过三个不保守的半胱氨酸残基（Cys（2008）、CyS 2010和Cys（2048））的选择性还原和烷基化将其转化为单体。通过部分还原和烷基化、化学和蛋白水解消化、质谱和氨基酸测序，对剩余的链内二硫键进行了表征：Cys（1961）-Cys（2011）（1-4）、Cys（1987）-Cys（2041）（2-5）、Cys（1991）-Cys（2045）（3-6）和Cys（1976）- Cys（2025）。突变C2008 A或C2010 A阻止了二聚化，而突变C2048 A则没有。基于转化生长因子-β结构的对称性考虑和分子建模表明，每个亚基中的残基Cys（2008）、Cys（2010）和Cys（2048）中的一个或三个介导proVWF的共价二聚化。
von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines, Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), CyS2010 and Cys(2048). BY partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (1-4), Cys(1987)-Cys(2041) (2-5), Cys(1991)-Cys(2045) (3-6), and Cys(1976)- Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF.