A hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp) in the fifth domain of apolipoprotein H (beta(2)-glycoprotein I) is crucial for cardiolipin binding

Apolipoprotein H (apoH, protein; APOH, gene) binds to negatively charged ph ospholipids, which triggers the production of a subset of autoantibodies ag ainst phospholipid in patients with autoimmune diseases. We have demonstrat ed that two naturally occurring missense mutations in the fifth domain of a poH, Trp316Ser and Cys306Gly, disrupt the binding of native apoH to phospha tidylserine [Sanghera, D. K., Wagenknecht, D. R., McIntyre, J. A. & Kamboh, M. I. (1997) Hum. Mol. Genet. 6, 311-316]. To confirm whether these are fu nctional mutations, we mutagenized APOH cDNAs and transiently expressed the m in COS-1 cells. The cardiolipin ELISA of wild-type and mutant recombinant apoH confirmed that the Gly306 and Ser316 mutations are responsible for ab olishing the binding of recombinant apoH to cardiolipin. These mutations, h owever, had no effect on the levels of expression or secretion of recombina nt apoH in transfected COS-1 cells. While the Cys306Gly mutation disrupts a disulfide bond between Cys306 and Cys281, which appears to be critical for clustering positively charged amino acids, the Trp316Ser mutation affects the integrity of an evolutionarily conserved hydrophobic sequence at positi on 313-316 (Leu-Ala-Phe-Trp), which is hypothesized to interact with anioni c phospholipid. To test this hypothesis, we exchanged the remaining three h ydrophobic amino acids with neutral amino acids by site-directed mutagenesi s (Leu313Gly, Ala314Ser and Phe315Ser). Binding of the Leu313Gly and Phe315 Ser mutants to cardiolipin was significantly reduced to 25% and 13%, respec tively, of that of the wild-type. On the other hand, the Ala314Ser mutation showed normal cardiolipin binding. Taken together with our previous findin gs, these results strongly suggest that the configuration of the fifth doma in of apoH, as well as the integrity of the highly conserved hydrophobic am ino acids at positions 313-316, is essential for the binding of apoH to ani onic phospholipid.