Figure 2.
The E91K variant α-spectrin causes marked reduction in spectrin contents and mechanical stability of RBC membranes. (A) Japanese black cattle (JB, n = 136) and HF cattle (n = 31) were genotyped for the SPTA1 alleles. All these animals were free from the premature termination mutation R664X, causative of band 3 deficiency in cattle. Alleles SpαA and SpαB, major alleles in both bovine species, are different in the nucleotide sequence, resulting in amino acid substitutions at 14 amino acid residues. The allele SpαBK91 has SpαB backbone and contains an additional nucleotide change which results in the E91K substitution. SpαBK91 was found only in JB cattle so far examined. A minor allele SpαAB, possessing a chimeric sequence of SpαA and SpαB, was also found in both breeds during the process of genotyping. (B-C) RBC membrane proteins from JB cattle (n = 136) were analyzed by SDS-PAGE, and the abundance of spectrin relative to band 3 was determined by densitometric scanning. The data are summarized for the animals free from (E/E) and heterozygous (E/K) and homozygous (K/K) for E91K substitution in panel B, or for different genotypes in panel C. In panel C, alleles SpαA, SpαB, SpαAB, and SpαBK91 are abbreviated as A, B, AB, and BK91, respectively. The data are indicated for each individual with the mean ± SD. Sample numbers are found in parentheses. Statistical significances were determined using Kruskal-Wallis ANOVA with the Dunn multiple comparison test. ∗P < .05; ∗∗P < .01. (D-E) Ektacytometry analysis for the expandability in panel D and mechanical stability in panel E of RBCs from animals with different E91K phenotypes. In panel D, the data demonstrate the change in deformability index (DI) values of RBCs in response to increasing shear stress in rpm and expressed as the mean ± SD for E/E (n = 3) and E/K (n = 3). K/K represents independent samples (n = 2). The Mann-Whitney test was used to calculate statistical significance between E/E and E/K. ∗∗∗∗P < .0001. The data in panel E indicate the change in DI values as a function of time under a high shear stress (750 dynes per cm2). Representative data for E/E (n = 2), E/K (n = 1), and K/K (n = 1) RBCs are illustrated. HF, Holstein Friesian.

The E91K variant α-spectrin causes marked reduction in spectrin contents and mechanical stability of RBC membranes. (A) Japanese black cattle (JB, n = 136) and HF cattle (n = 31) were genotyped for the SPTA1 alleles. All these animals were free from the premature termination mutation R664X, causative of band 3 deficiency in cattle. Alleles SpαA and SpαB, major alleles in both bovine species, are different in the nucleotide sequence, resulting in amino acid substitutions at 14 amino acid residues. The allele SpαBK91 has SpαB backbone and contains an additional nucleotide change which results in the E91K substitution. SpαBK91 was found only in JB cattle so far examined. A minor allele SpαAB, possessing a chimeric sequence of SpαA and SpαB, was also found in both breeds during the process of genotyping. (B-C) RBC membrane proteins from JB cattle (n = 136) were analyzed by SDS-PAGE, and the abundance of spectrin relative to band 3 was determined by densitometric scanning. The data are summarized for the animals free from (E/E) and heterozygous (E/K) and homozygous (K/K) for E91K substitution in panel B, or for different genotypes in panel C. In panel C, alleles SpαA, SpαB, SpαAB, and SpαBK91 are abbreviated as A, B, AB, and BK91, respectively. The data are indicated for each individual with the mean ± SD. Sample numbers are found in parentheses. Statistical significances were determined using Kruskal-Wallis ANOVA with the Dunn multiple comparison test. ∗P < .05; ∗∗P < .01. (D-E) Ektacytometry analysis for the expandability in panel D and mechanical stability in panel E of RBCs from animals with different E91K phenotypes. In panel D, the data demonstrate the change in deformability index (DI) values of RBCs in response to increasing shear stress in rpm and expressed as the mean ± SD for E/E (n = 3) and E/K (n = 3). K/K represents independent samples (n = 2). The Mann-Whitney test was used to calculate statistical significance between E/E and E/K. ∗∗∗∗P < .0001. The data in panel E indicate the change in DI values as a function of time under a high shear stress (750 dynes per cm2). Representative data for E/E (n = 2), E/K (n = 1), and K/K (n = 1) RBCs are illustrated. HF, Holstein Friesian.

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