Molecular and Biochemical Studies on Compensatory Mechanisms for Total Band 3 Deficiency in Japanese Black Cattle

Band 3：这一研究项目is aim of this research project is The aim of mammals. The aim of this research project isto define compensatory mechanisms for total band 3 deficiency in cattle.1) Linkage analyses showedthe genotype for R664X mutation determined by PCR-RFLP coinherited with the red cell phenotypedominantly inherited HS and band 3 deficiency，demonstrating that R664X mutation is the主要molecular cause for dominant hereditary band 3deficiency in cattle associated with HS.2) Extensive studies on the red cell membrane proteinsdemonstrated the major proximal causes for the membrane instability of homozygous andheterozygous cells appear to be the loss of band 3-ankyrin-spectrin association，and the reduction of spectrin，respectively. Quantitation of mutant mRNA coinjection of normal and mutant RNA into Xenopusoocytes,and in vitro s y / n t hesis immunoprecipitation of normal and the mutant bend 3 demonstrated adominant-negative effect of the mutant protein in vivo on the expression of normal band 3. Ahypothetical possibility for pathogenesis of the affected animals involves：(1) Band 3-independent assembly of membrane skeleton to the plasma membrane. (2) Translocation ofreduced normal band 3-ankyrin and their association with spectrin to strengthen interactions betweenthe lipid bilayer and the skeleton in heterozygous but not m homozygous red cells.3) Bovine redcells with total band 3 deficiency possessed anion transport activity mediated by AE2with substrate specificity an sensitivity to stilbene disulfonate which were extremely lower than那些在普通cells. T rapid anion exchange was not compensated at allindicating that the function of band 3 is not obligatory to 0 - D22 - D2/C0 - D22 - D2 exchange。

Band 3 has been believed to be essential to survival of mammals. The aim of this research project is to define compensatory mechanisms for total band 3 deficiency in cattle.1) Linkage analyses showed that the genotype for R664X mutation determined by PCR-RFLP coinherited with the red cell phenotype of dominantly inherited HS and band 3 deficiency, demonstrating that R664X mutation is the principal molecular cause for dominant hereditary band 3 deficiency in cattle associated with HS.2) Extensive studies on the red cell membrane proteins demonstrated that the major proximal causes for the membrane instability of homozygous and heterozygous cells appear to be the loss of band 3-ankyrin-spectrin association, and the reduction of spectrin, respectively. Quantitation of mutant mRNA co-injection of normal and mutant RNA into Xenopus oocytes, and in vitro synthesis/immunoprecipitation of normal and the mutant bend 3 demonstrated a dominant-negative effect of the mutant protein in vivo on the expression of normal band 3. A hypothetical possibility for pathogenesis of HS in the affected animals involves : (1) Band 3-independent assembly of membrane skeleton to the plasma membrane. (2) Translocation of reduced normal band 3-ankyrin and their association with spectrin to strengthen interactions between the lipid bilayer and the skeleton in heterozygous but not m homozygous red cells.3) Bovine red cells with total band 3 deficiency possessed anion transport activity mediated by AE2, with substrate specificity an sensitivity to stilbene disulfonate which were extremely lower than those in normal cells. T e rapid anion exchange was not compensated at all, indicating that the function of band 3 is not obligatory to 0ィイD22ィエD2/C0ィイD22ィエD2 exchange.