Structure and function of the recombinant fifth domain of human β2-glycoprotein I: Effects of specific cleavage between Lys77 and Thr78

Yoshihisa Hagihara, Kei Ichi Enjyoji, Takeshi Ômasa, Yoshio Katakura, Ken Ichi Suga, Makoto Igarashi, Eiji Matsuura, Hisao Kato, Tetsuro Yoshimura, Yuji Goto

Research output: Contribution to journalArticlepeer-review

23 Citations (Scopus)

Abstract

In order to elucidate the mechanism of binding of β2-glycoprotein I (β2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of β2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative, unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human β2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically 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 circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.

Original languageEnglish
Pages (from-to)128-137
Number of pages10
JournalJournal of biochemistry
Volume121
Issue number1
DOIs
Publication statusPublished - Jan 1 1997
Externally publishedYes

Keywords

  • Anti-cardiolipin syndrome
  • Autoimmune diseases
  • Pichia pastoris
  • Protein-lipid interaction
  • β-glycoprotein I

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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