Identificação bioquímica e molecular das variantes de eliptocitose hereditária: importância da digestão tríptica da espectrina

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Abstract Hereditay elliptocytosis (HE) is a relatively common,clinically and genetically heterogenous disorder, characterized bythe presennce of a large number of ellypticlly-shaped red bloodcells in the peripheral blood.From the biochemical point of view,several alterations of the major membrane skeleton proteins suchas spectrin (Sp) and protein 4.1 have been observed, influencingthe structure or quantity of these proteins in affected erythrocytes.Defects within the Sp molecule include shortening of the α or β-chain and, in most cases, alterations in the Sp-dimer self-associationprocess.The N terminus of α -spectrin and the C terminus of βspectrin are known to play important roles in the self-associationof spectrin dimers into tetramers and larger oligomers, aphenomenon that confers stability to the membrane skeleton. SpαI domain mutants have been detected after limited tryptic digestionof Sp, which shows the presence of peptides derived from thenormal αI domain of 80 kDa. Each variant has been classifiedaccording to the molecular weight and isoelectric point of theabnormal peptide: Sp αI/78, Sp αI/74, Sp αI/65, Sp αI/46, Sp αI/50b and Sp αI/43. In this study, six propositi and eleven familymembers with elliptocytic red cells were evaluated. Limited trypticdigestion of spectrin, followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) revealed both adecrease in the αI domain (80 kDa) and the presence of abnormalpeptides with molecular weights of 65 kDa and 46 kDa. In onestructural variant, there was an increase in a 74 kDa peptide, withoutalterations in the αI domain. The v¤ 41 polymorphism, with a low-expression, was also encountered in one propositus and her mother.All of these spectrin variants inhibit self-association to formtetramers and higher oligomers, a process that is known to bedependent upon an intact αI domain, and one which is thought tobe important in the formation of the spectrin-actin-4.1 network onthe inner surface of the red blood cell membrane.