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Insights into Hemoglobin Assembly through in Vivo Mutagenesis of α-Hemoglobin Stabilizing Protein

journal contribution
posted on 2023-05-17, 13:24 authored by Khandros, E, Mollan, TL, Yu, X, Wang, X, Yao, Y, D'Souza, J, David GellDavid Gell, Olson, JS, Weiss, MJ
α-Hemoglobin stabilizing protein (AHSP) is believed to facilitate adult Hemoglobin A assembly and protect against toxic free α-globin subunits. Recombinant AHSP binds multiple forms of free α-globin to stabilize their structures and inhibit precipitation. However, AHSP also stimulates autooxidation of αO2 subunit and its rapid conversion to a partially unfolded bishistidyl hemichrome structure. To investigate these biochemical properties, we altered the evolutionarily conserved AHSP proline 30 in recombinantly expressed proteins and introduced identical mutations into the endogenous murine Ahsp gene. In vitro, the P30W AHSP variant bound oxygenated α chains with 30-fold increased affinity. Both P30W and P30A mutant proteins also caused decreased rates of αO2 autooxidation as compared with wild-type AHSP. Despite these abnormalities, mice harboring P30A or P30W Ahsp mutations exhibited no detectable defects in erythropoiesis at steady state or during induced stresses. Further biochemical studies revealed that the AHSP P30A and P30W substitutions had minimal effects on AHSP interactions with ferric α subunits. Together, our findings indicate that the ability of AHSP to stabilize nascent α chain folding intermediates prior to hemin reduction and incorporation into adult Hemoglobin A is physiologically more important than AHSP interactions with ferrous αO2 subunits.

Funding

National Health & Medical Research Council

History

Publication title

Journal of Biological Chemistry

Volume

287

Issue

14

Pagination

11325-11337

ISSN

0021-9258

Department/School

Menzies Institute for Medical Research

Publisher

Amer Soc Biochemistry Molecular Biology Inc

Place of publication

United States

Rights statement

Copyright 2012 American Society for Biochemistry and Molecular Biology, Inc

Repository Status

  • Restricted

Socio-economic Objectives

Diagnosis of human diseases and conditions

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