Crystal Structure and Functional Study of Thalassemic mouse/human Transgenic Hemoglobins.

Department of Chemistry, Center for Portein Structure and Function, Mahidol University* Chulabhorn Research Institute, Vibhavadee-Rangsit Highway, Bangkok 10210, THAILAND** Department of Biochemistry, Center for Protein Structure and Function, Faculty of Science, Mahidol University and Chulabhorn Research Institute, Vibhavadee-Rangsit Highway, Bangkok 10210, THAILAND*** Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Nakorn Pathom 73170, THAILAND.****
â—‹Chariwat Samanchat* Lukana Ngiwsara** Jisnuson Svasti*** Duangporn Jamsai**** Suthat Fucharoen**** Palangpon Kongsaeree*

Hemoglobin E (HbE), the most common β-thalassemic disease in Southeast Asia, is caused by a substitution of Glu by Lys at codon 26 of the β-globin gene. Clinically they are very heterogeneous in severity. Individuals homozygous HbE exhibit mild thalassemia, while the combination of HbE with a β-thalassemia mutation exhibit highly variable severity. A novel C57BL/6 Transgenic murine model of HbE were generated by S. Fucharoen's group with knock out mice that had deletions of the β-globin genes in order to create mice expressing chimeric hemoglobin (muα2:huβ2E). Evidently, human βE-transgene can be expressed and functioned in vivo. The transgenic HbE and HbA can be purified to homogeneity with DEAE anion-exchange chromatography. The transgenic hemoglobins were successfully crystallized, the crystals belong to the monoclinic P21 space group with unit-cell parameters of a=53.81, b=151.25, c=76.81 Å and β =97.38o and the same space group with unit cell parameters of a=53.60, b=148.65, c=77.70 Å and β = 97.67o for αm2E2 and αm2A2, respectively. The structures were solved by using MR-method and refined to the 2.3 Å resolution. In the transgenic αm2E2 structure, Lys26 in human βE-globin chain lost two H-bonds with Arg30 and His117 with respect to the Glu26 in the normal β-globin. Structural analysis of the difference between both hybrid molecules will be discussed. The structural investigation of transgenic hemoglobin would eventually reveal the involved mechanism in atomic level leading to better understanding of thalassemic mouse model before the development for testing antisense therapy and some antioxidant drugs instead of testing in human.