Inhibition of beta(S)-chain dependent polymerization by synergistic complementation of contact site perturbations of alpha-chain: application of semisynthetic chimeric alpha-chains

Mouse alpha(1-30)-horse alpha(31-141) chimeric alpha-chain, a semisynthetic super-inhibitory alpha-chain, inhibits beta(S)-chain dependent polymerizat ion better than both parent alpha-chains. Although contact site sequence di fferences are absent in the alpha(1-30) region of the chimeric chain, the f our sequence differences of the region alpha(17-22) could induce perturbati ons of the side chains at alpha(16), alpha(20) and alpha(23) the three cont act sites of the region. A synergistic complementation of such contact site perturbation with that of horse alpha(31-141) probably results in the supe r-inhibitory activity of the chimeric alpha-chain. The inhibitory contact s ite sequence differences, by themselves, could also exhibit similar synergi stic complementation. Accordingly, the polymerization inhibitory activity o f Hb Le-Lamentin (LM) mutation [His20(alpha)--> Gln], a contact site sequen ce difference, engineered into human-horse chimeric alpha-chain has been in vestigated to map such a synergistic complementation. Gln20(alpha) has litt le effect on the O-2 affinity of HbS, but in human-horse chimeric alpha-cha in it reduces the O-2 affinity slightly. In the chimeric alpha-chain, Gln20 (alpha) increased sensitivity of the beta beta cleft for the DPG influence, reflecting a cross-talk between the alpha(1)beta(1) interface and beta bet a cleft in this semisynthetic chimeric HbS. In the human alpha-chain frame, the polymerization inhibitory activity of Gln20(alpha) is higher compared with horse alpha(1-30), but lower than mouse alpha(1-30). Gln20(alpha) syne rgistically complements the inhibitory propensity of horse alpha(31-141). H owever, the inhibitory activity of LM-horse chimeric alpha-chain is still l on er than that of mouse-horse chimeric alpha-chain. Therefore, perturbatio n of multiple contact sites in the alpha(1-30) region of the mouse-horse ch imeric alpha-chain and its linkage with the inhibitory propensity of horse alpha(31-141) has been now invoked to explain the super-inhibitory activity of the chimeric alpha-chain. The 'linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contac t sites into alpha-chains as a strategy for generating super-inhibitory ant isickling hemoglobins for gene therapy of sickle cell disease.