Y. Hagihara et al., STRUCTURE AND FUNCTION OF THE RECOMBINANT 5TH DOMAIN OF HUMAN BETA(2)-GLYCOPROTEIN-I - EFFECTS OF SPECIFIC CLEAVAGE BETWEEN LYS77 AND THR78, Journal of Biochemistry, 121(1), 1997, pp. 128-137
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.