Luo Shi-Zhong, Mo Xi, Afshar-Kharghan Vahid, Srinivasan Sankaranarayanan, López José A, Li Renhao
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, MSB 6.130, 6431 Fannin St, Houston, TX 77030, USA.
Blood. 2007 Jan 15;109(2):603-9. doi: 10.1182/blood-2006-05-024091. Epub 2006 Sep 28.
It is widely accepted that glycoprotein (GP) Ib contains one Ibalpha and one Ibbeta subunit that are connected by a disulfide bond. It is unclear which Cys residue in Ibalpha, C484 or C485, forms the disulfide bond with Ibbeta. Using mutagenesis studies in transfected Chinese hamster ovary (CHO) cells, we found that both C484 and C485 formed a disulfide bond with C122 in Ibbeta. In the context of isolated peptides containing the Ibalpha or Ibbeta transmembrane domain and nearby Cys residue, C484 and C485 in the Ibalpha peptide were both capable of forming a disulfide bond with the Ibbeta peptide. Furthermore, coimmunoprecipitation of epitope-tagged subunits showed that at least 2 Ibbeta subunits but only 1 Ibalpha and 1 IX subunit were present in the GP Ib-IX complex. Finally, the size difference between GP Ib from transfected CHO cells and human platelets was attributed to a combination of sequence polymorphism and glycosylation difference in Ibalpha, not the number of Ibbeta subunits therein. Overall, these results demonstrate that Ibalpha is covalently connected to 2 Ibbeta subunits in the resting platelet, necessitating revision of the subunit stoichiometry of the GP Ib-IX-V complex. The alphabeta2 composition in GP Ib may provide the basis for possible disulfide rearrangement in the receptor complex.
人们普遍认为糖蛋白（GP）Ib含有一个通过二硫键连接的Ibalpha和一个Ibbeta亚基。目前尚不清楚Ibalpha中的哪个半胱氨酸残基，即C484还是C485，与Ibbeta形成二硫键。通过在转染的中国仓鼠卵巢（CHO）细胞中进行诱变研究，我们发现C484和C485都与Ibbeta中的C122形成了二硫键。在含有Ibalpha或Ibbeta跨膜结构域及附近半胱氨酸残基的分离肽的情况下，Ibalpha肽中的C484和C485都能够与Ibbeta肽形成二硫键。此外，表位标记亚基的共免疫沉淀显示，GP Ib-IX复合物中至少存在2个Ibbeta亚基，但只有1个Ibalpha和1个IX亚基。最后，转染的CHO细胞中的GP Ib与人血小板中的GP Ib之间的大小差异归因于Ibalpha中的序列多态性和糖基化差异的组合，而不是其中Ibbeta亚基的数量。总体而言，这些结果表明在静息血小板中Ibalpha与2个Ibbeta亚基共价连接，这需要对GP Ib-IX-V复合物的亚基化学计量进行修订。GP Ib中的alphabeta2组成可能为受体复合物中可能的二硫键重排提供基础。
