The Exon 46-Encoded Sequence Is Essential for Stability of Human Erythroid a-Spectrin and Heterodimer Formation

Human erythroid a-spectrin alleles responsible for heredinormal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adtary elliptocytosis (a alleles) undergo increased incorporation into red blood cell membranes when the polymorphism versely affect dimer formation, but it is responsible for the increased trypsin cleavage between the aIV and aV domains a (LELY: Low Expression LYon) occurs in trans. The a polymorphism is characterized by a mutation in exon 40 at that was the characteristic feature initially used to identify the a (Spa) polymorphism (Alloisio et al, J Clin Invest codon 1857 (CTA r GTA, Leu r Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wil87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the a21 motif, because this remotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of gion of the a18-21 peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. the a-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to deExon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from creased incorporation of a-chains from the a allele in heterozygotes into red blood cell membranes. These possian a homozygote and the two a recombinant peptides strongly suggests that little, if any, of the 50% of the bilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recoma chains from the a allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. binant 4-motif a subunit peptides containing the dimer initiation region. The two forms of a spectrin produced by alterBased on the in vitro analysis of recombinant a peptides, the inability of detectable amounts of exon 46 a chains native mRNA splicing of the a allele were represented by a18-21, a peptide with the codon 1857 mutation and to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding retaining the exon 46 encoded sequence, and a18-21, a peptide carrying both the 1857 codon mutation and the affinity and increased proteolytic degradation of these mutant chains. exon 46 deletion. The properties of these two recombinant peptides were compared with a18-21, a peptide with the q 1997 by The American Society of Hematology.