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A hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp) in the fifth domain of apolipoprotein H (β2-glycoprotein I) is crucial for cardiolipin binding: Binding of a hydrophobic sequence of apoH to cardiolipin
Journal article   Open access   Peer reviewed

A hydrophobic sequence at position 313-316 (Leu-Ala-Phe-Trp) in the fifth domain of apolipoprotein H (β2-glycoprotein I) is crucial for cardiolipin binding: Binding of a hydrophobic sequence of apoH to cardiolipin

Haider Mehdi, Asma Naqvi and M. Ilyas Kamboh
European journal of biochemistry, Vol.267(6), pp.1770-1776
03/2000
DOI: 10.1046/j.1432-1327.2000.01174.x
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https://doi.org/10.1046/j.1432-1327.2000.01174.xView
Published (Version of record) Open Access

Abstract

Apolipoprotein H (apoH, protein; APOH, gene) binds to negatively charged phospholipids, which triggers the production of a subset of autoantibodies against phospholipid in patients with autoimmune diseases. We have demonstrated that two naturally occurring missense mutations in the fifth domain of apoH, Trp316Ser and Cys306Gly, disrupt the binding of native apoH to phosphatidylserine [Sanghera, D. K., Wagenknecht, D. R., McIntyre, J. A. and Kamboh, M. I. (1997) Hum. Mol. Genet. 6, 311-316]. To confirm whether these are functional mutations, we mutagenized APOH cDNAs and transiently expressed them in COS-1 cells. The cardiolipin ELISA of wild-type and mutant recombinant apoH confirmed that the Gly306 and Ser316 mutations are responsible for abolishing the binding of recombinant apoH to cardiolipin. These mutations, however, had no effect on the levels of expression or secretion of recombinant 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 position 313-316 (Leu-Ala-Phe-Trp), which is hypothesized to interact with anionic phospholipid. To test this hypothesis, we exchanged the remaining three hydrophobic amino acids with neutral amino acids by site- directed mutagenesis (Leu313Gly, Ala314Ser and Phe315Ser). Binding of the Leu313Gly and Phe315Ser mutants to cardiolipin was significantly reduced to 25% and 13%, respectively, of that of the wild-type. On the other hand, the Ala314Ser mutation showed normal cardiolipin binding. Taken together with our previous findings, these results strongly suggest that the configuration of the fifth domain of apoH, as well as the integrity of the highly conserved hydrophobic amino acids at positions 313-316, is essential for the binding of apoH to anionic phospholipid.

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