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Abstract
Recruitment and activation of platelets at an injured site involves crosslinking of fibrinogen between integrin molecules on adjacent platelets. Activation of integrin αIIbβ3 is essential in this process of plug formation. Calcium- and integrin-binding protein (CIB1) has been shown to facilitate both activation of αIIbβ 3 and platelet spreading on immobilized fibrinogen. In this work, 3 new members, CIB2, CIB3 and CIB4, of the CIB protein family are identified and characterized. CIB4 has the highest identity to CIB1, with 39% amino acid identity and 57% similarity. CIB2 and CIB3 are most closely related to each other, having 56% identity and 73% similarity. The CIB family is conserved, with greater than 90% identity, in the mouse genome. These proteins have 2 EF-hands in the C-terminus as well as a putative N-terminal myristoylation site. The expression pattern of the CIB family was studied in human cell lines and in mouse tissues. CIB1 and CIB2 are ubiquitously expressed, while CIB3 expression was not detected and CIB4 was detected only in lung and testis of mouse. This work outlines the generation of multiple reagents that will be essential tools for the study of these proteins, such as expression vectors that can be used to express CIB proteins in mammalian, bacterial, or insect cell systems. In order to begin to functionally characterize this novel protein family, recombinant proteins were expressed in E. coli and Sf9 cells. CIB2 soluble protein was successfully purified from the Sf9 insect cell system. In addition, two monoclonal antibodies that recognize an epitope in the CIB1 C-terminal domain were generated and characterized. These antibodies are useful reagents for Western blot, Immunoprecipitation and ELISA assays. It will be interesting to further characterize this novel calcium binding protein family and reveal the specific functions of each protein. Some suggestions are offered for future projects which could advance the characterization of this novel protein family.
