Assembly of Recently Translated Human γ-Globin Chains with a Pool of α-Globin Chains To Form Hb F in a Cell-Free System.

A pool of free α chains as well as hemin appears compatible with cellular homeostasis and important for ensuring efficient assembly with non-α globin chains to form functional hemoglobin tetramers in erythroid cells. Furthermore, globin-chain assembly with partner chains and efficient assembly of subunits to form functional hemoglobin is critical to prevent accumulation of unstable single globin chains which could lead to cell destruction. Efficient assembly also is critical for optimizing gene therapy for the treatment of hemoglobinopathies, since one therapeutic scenario involves expression of excess β-chain variants or γ-globin chains. We studied determinants for efficient assembly of αγ hetero-dimers followed by tetrameric Hb F formation. Indeed, γ-globin chains in Hb F are negatively charged; and, therefore, they would be expected to assemble efficiently with positively charged α chains. However, Hb F formation in vitro using purified γ- and α-globin chains is very slow compared to Hb A formation from purified β- and α-globin chains. This slow Hb F formation in vitro is to some extent due to stable γ2-dimer formation. Differences in amino acids at α1β1 and α1γ1 interaction sites and differences in surface charge also lead to different in vitro assembly rates for Hb A and Hb F. In contrast, expressed radiolabeled γ- and β-globin chains made in a wheat germ, coupled cell-free transcription/translation system using γ- and β-globin cDNA expression vectors with excess unlabeled α chains added prior to translation initiation showed similar formation rates for radiolabeled Hb A and Hb F. These results suggested in the absence of free α chains the newly translated monomeric γ chains form stable homo-dimers in erythroid cells, which leads to lower levels of αγ dimers. Therefore, we assessed formation of Hb A and Hb F after expression of radiolabeled β- or γ-globin chains in a wheat germ, coupled cell-free transcription/translation system. Results indicate that assembly of Hb F, but not Hb A, is enhanced by early addition of excess free α chains that act by inhibiting the formation of stable γ2 homo-dimers. Mutational analysis of truncated γ-globin chains showed that removal of more than 11 but not 2 amino acids from the carboxy terminus of γ chains inhibits assembly with α chains. Polysome profile studies showed that all carboxy-terminal deleted γ-chain mutants retained on polysomes due to removal of the translation termination codon in the cDNA expression vector do not assemble with unlabeled holo α chains. However, the two amino acid-deleted γ chains coded from vectors lacking a stop codon only assembled with α chains after puromycin-induced polysome release. In addition, small amounts of radiolabeled α γ dimers were detected on polysomes in reactions using wild type γ-chain templates only when excess unlabeled α chains were present. Together, our results suggest that Hb F formation is enhanced by a stable pool of free α chains that interact with newly synthesized γ chains, either before or soon after their release from the ribosome and prior to formation of stable γ2 homo-dimers.