A recombinant human hemoglobin with anti-sickling properties greater than fetal hemoglobin.
A new recombinant, human anti-sickling beta-globin polypeptide designated beta(AS3) (betaGly(16) --> Asp/betaGlu(22) --> Ala/betaThr(87) --> Gln) was designed to increase affinity for alpha-globin. The amino acid substitutions at beta22 and beta87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The beta16 substitution confers the recombinant beta-globin subunit (beta(AS3)) with a competitive advantage over beta(S) for interaction with the alpha-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (alpha(2)beta(AS3)(2)) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that beta(AS3) has a distinct advantage over beta(S) for dimerization with alpha-globin. When equal amounts of beta(S)- and beta(AS3)-globin monomers compete for limiting alpha-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the beta(AS3) polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of beta(AS3)-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 30% percent of these individuals who coinherit alpha-thalassemia. Therefore, constructs expressing the beta(AS3)-globin gene may be suitable for future clinical trials for sickle cell disease.
Levasseur
Dana N
DN
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Ryan
Thomas M
TM
Reilly
Michael P
MP
McCune
Steven L
SL
Asakura
Toshio
T
Townes
Tim M
TM
D000328:Adult; D000755:Anemia, Sickle Cell / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D000818:Animals; D000986:Antisickling Agents / Q000737:chemistry / Q000494:pharmacology*; D004195:Disease Models, Animal; D004913:Erythrocytes, Abnormal / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005319:Fetal Hemoglobin / Q000494:pharmacology*; D005914:Globins / Q000235:genetics*; D006403:Hematologic Tests; D006454:Hemoglobins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D008099:Liver / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013154:Spleen / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure
D000986:Antisickling Agents; D006454:Hemoglobins; D011994:Recombinant Proteins; D005914:Globins; D005319:Fetal Hemoglobin