STRUCTURE AND FUNCTION OF THE RECOMBINANT 5TH DOMAIN OF HUMAN BETA(2)-GLYCOPROTEIN-I - EFFECTS OF SPECIFIC CLEAVAGE BETWEEN LYS77 AND THR78

In order to elucidate the mechanism of binding of beta(2)-glycoprotein I (beta(2)-GPI) to cardiolipin (CL), we constructed a high-level expr ession system for the C-terminal domain (Domain V) of beta(2)-GPI usin g Pichia pastoris and studied its conformation and liposome-binding ac tivity. Purified Domain V was found to have the native disulfide bonds . It had a compactly folded conformation, judging from the circular di chroism spectrum, and exhibited a cooperative unfolding transition ind uced by pH or urea. Also, it bound liposomes containing CL. Commercial ly available human beta(2)-GPI is known to be selectively cleaved betw een Lys 317 and Thr 318. We found that bovine factor Xa weakly but spe cifically cleaves the corresponding site of recombinant Domain V, i.e. , the peptide bond between Lys 77 and Thr 78. The conformation of the ''nicked'' Domain V, which was cleaved at this site, was examined by c ircular dichroism and fluorescence measurements, and concluded to be s imilar to that of the intact protein. The stability of the nicked Doma in V to urea was slightly lower than that of the intact protein. Altho ugh both Domains V bound to liposomes containing CL, the affinity of t he nicked Domain V was greatly reduced in comparison with the intact p rotein, indicating that the cleavage of the peptide bond between Lys 7 7 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indic ated that Trp 76 is involved in the binding site. These results sugges t that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.