Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or
beta-chain of spectrin through in vitro dimer reconstitution
B Pothier, N Alloisio, J Marechal, L Morle, MT Ducluzeau, C Caldani, N Philippe and J Delaunay
Faculte de Medecine Grange-Blanche, Centre National de la Recherche
Scientifique URA, Lyon, France.
Partial digestion of spectrin dimers in vitro has allowed the definition of
domains. For example, the portions of the dimers that are involved in
spectrin self-association are represented by the alpha I and the beta I
domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd
fragment and, more substantially, into a 74 Kd fragment. The intensity of
the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the
74:alpha I ratio, is variable depending on the experimental conditions, eg,
in fine, on the conformation of the alpha I domain. A number of cases of
hereditary elliptocytosis (HE) are associated with an increase of the
74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality.
Several lines of evidence have suggested that the causal mutations may lie
in the alpha- or the beta- chain, a point of importance before one
undertakes studies at the gene level. In order to address this question, we
reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of
various origins, and then carried out partial digestion and assayed the Sp
alpha I/74 abnormality. The patterns obtained with reconstituted dimers
were nearly identical to those of native dimers. We applied the assay to
three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we
previously designated spectrin Nice and whose beta-chain lacks a 4 Kd
fragment in its C-terminal region; and two distinct variants that we found
in two unrelated white families and that we provisionally designated
spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in
all kinds of dimers that harbored the beta- chain of spectrin Nice, or the
alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we
confirmed that spectrin Nice is a (alpha I/74) beta-variant, and
established that both spectrin Lyon and spectrin Culoz are (alpha I/74)
alpha-variants. The present assay may be extended to any spectrin variant
displaying the Sp alpha I/74 abnormality.
Volume 75,
Issue 10,
pp. 2061-2069,
05/15/1990
Copyright © 1990 by The American Society of Hematology
