Interspecies hybrid HbS: Complete neutralization of Val6(β)-dependent polymerization of human β-chain by pig α-chains

M. Janardhan Rao, Ashok Malavalli, Belur N. Manjula, Ramesh Kumar, Muthuchidambaran Prabhakaran, D. Philip Sun, Nancy T. Ho, Chien Ho, Ronald L. Nagel, A. Seetharama Acharya

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Abstract

Interspecies hybrid HbS (α2/(P)β2/(S)), has been assembled in vitro from pig α-globin and human β(S)-chain. The (α2/(P)β2/(S)) retains normal tetrameric structure (α2β2) of human Hb and an O2 affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O2 affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The 1H-NMR spectroscopy detected distinct differences between the heme environments and α1β1 interfaces of pig Hb and HbS, while their α1β2 interfaces appear very similar. The interspecies hybrid (α2/(P)β2/(S)) resembles pig Hb; the pig β-chain dictated the conformation of the heme environment of the human α-subunit, and to the α1β1 interfaces of the hybrid. In the (α2/(P)β2/(S)) hybrid, β(S)-chain dictated the conformation of human heme environment to the pig α-chain in the hybrid; but the conformation of α1β1 interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the α1β1 interface conformation is identical to that of HbS. More important, the (α2/(P)β2/(S)) does not polymerize when deoxygenated; pig α-chain completely neutralizes the β(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig α-chain is higher when it is present in the cis α(P)β(S) dimer relative to that in a trans α(P)β(A) dimer. The semisynthetically generated chimeric pig-human and human-pig α-chains by exchanging the α1-30 segments of human and pig α-chains have established that the sequence differences of pig α31-141 segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the α-chain surfaces of HbS and α2/(P)β2(S) suggests that the differences in electrostatic potential energy surfaces on the α-chain of α2/(P)β2(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig α-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric α-chains can serve as blueprints for the design of a new generation of variants of α-chain(s) suitable for the gene therapy of sickle cell disease. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)1389-1406
Number of pages18
JournalJournal of Molecular Biology
Volume300
Issue number5
DOIs
StatePublished - Jul 28 2000

Keywords

  • Antisickling globins
  • Gene therapy
  • Hybrid Hb
  • Molecular modeling
  • Semisynthetic chimeric α-chains

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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    Rao, M. J., Malavalli, A., Manjula, B. N., Kumar, R., Prabhakaran, M., Sun, D. P., Ho, N. T., Ho, C., Nagel, R. L., & Acharya, A. S. (2000). Interspecies hybrid HbS: Complete neutralization of Val6(β)-dependent polymerization of human β-chain by pig α-chains. Journal of Molecular Biology, 300(5), 1389-1406. https://doi.org/10.1006/jmbi.2000.3898