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 polymerization better than both parent alpha-chains. Although contact site sequence differences are absent in the alpha(1-30) region of the chimeric chain, the four sequence differences of the region alpha(17-22) could induce perturbations of the side chains at alpha(16), alpha(20) and alpha(23), the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse alpha(31-141) probably results in the super-inhibitory activity of the chimeric alpha-chain. The inhibitory contact site sequence differences, by themselves, could also exhibit similar synergistic complementation. Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin (LM) mutation [His20(alpha)-->Gln], a contact site sequence difference, engineered into human-horse chimeric alpha-chain has been investigated to map such a synergistic complementation. Gln20(alpha) has little effect on the O(2) affinity of HbS, but in human-horse chimeric alpha-chain it reduces the O(2) affinity slightly. In the chimeric alpha-chain, Gln20(alpha) increased sensitivity of the betabeta cleft for the DPG influence, reflecting a cross-talk between the alpha(1)beta(1) interface and betabeta 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) synergistically complements the inhibitory propensity of horse alpha(31-141). However, the inhibitory activity of LM-horse chimeric alpha-chain is still lower than that of mouse-horse chimeric alpha-chain. Therefore, perturbation of multiple contact sites in the alpha(1-30) region of the mouse-horse chimeric 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 contact sites into alpha-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.
Srinivasulu
S
S
Division of Hematology, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Malavalli
A
A
Prabhakaran
M
M
Nagel
R L
RL
Acharya
A S
AS
D000755:Anemia, Sickle Cell / Q000235:genetics; D000818:Animals; D002852:Chromatography, Ion Exchange; D006451:Hemoglobin, Sickle / Q000737:chemistry* / Q000235:genetics; D006736:Horses; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments / Q000138:chemical synthesis; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D017421:Sequence Analysis
D006451:Hemoglobin, Sickle; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D010100:Oxygen