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

Abstract

AbstractHuman erythroid α-spectrin alleles responsible for hereditary elliptocytosis (αHE alleles) undergo increased incorporation into red blood cell membranes when the polymorphism αLELY (LELY: Low Expression LYon) occurs in trans. The αLELY polymorphism is characterized by a mutation in exon 40 at codon 1857 (CTA → GTA, Leu → Val) and the partial (50%) skipping of exon 46, which encodes residues 2177-2182 (Wilmotte et al, J Clin Invest 91:2091, 1993). Both of these peptide sequence alterations are located within the region of the α-chain involved in initiating heterodimer assembly, and either or both mutations could potentially contribute to decreased incorporation of α-chains from the αLELY allele in heterozygotes into red blood cell membranes. These possibilities were evaluated by testing the protease resistance and in vitro binding properties of normal and mutant recombinant 4-motif α subunit peptides containing the dimer initiation region. The two forms of α spectrin produced by alternative mRNA splicing of the αLELY allele were represented by α18-211857, a peptide with the codon 1857 mutation and retaining the exon 46 encoded sequence, and α18-211857-Δ46, a peptide carrying both the 1857 codon mutation and the exon 46 deletion. The properties of these two recombinant peptides were compared with α18-21, a peptide with the normal sequence at codon 1857 and retaining the exon 46 encoded sequence. The codon 1857 mutation does not adversely affect dimer formation, but it is responsible for the increased trypsin cleavage between the αIV and αV domains that was the characteristic feature initially used to identify the αLELY (SpαV/41) polymorphism (Alloisio et al, J Clin Invest 87:2169, 1991). Deletion of the six amino acids encoded by exon 46 perturbs folding of the α21 motif, because this region of the α18-211857-Δ46 peptide is rapidly degraded and this recombinant peptide is unusually prone to self-aggregation. Exon 46 deletion reduces, but does not eliminate, dimerization. Comparison of mild trypsin proteolytic products from an αLELY homozygote and the two αLELY recombinant peptides strongly suggests that little, if any, of the 50% of the α chains from the αLELY allele that contain the exon 46 deletion are incorporated into the mature erythroid membrane. Based on the in vitro analysis of recombinant αLELY peptides, the inability of detectable amounts of exon 46− α chains to assemble into the mature membrane skeleton in vivo is probably due to a combination of decreased dimer binding affinity and increased proteolytic degradation of these mutant chains.