Pothier B, Alloisio N, Maréchal J, Morlé L, Ducluzeau M T, Caldani C, Philippe N, Delaunay J
Faculté de Médecine Grange-Blanche, Centre National de la Recherche Scientifique URA, Lyon, France.
Blood. 1990 May 15;75(10):2061-9.
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.
血影蛋白二聚体在体外的部分消化使得结构域得以明确。例如，参与血影蛋白自我缔合的二聚体部分由αI和βI结构域代表。αI结构域（80千道尔顿）进一步裂解为一个较小的78千道尔顿片段，更主要的是裂解为一个74千道尔顿片段。后者的强度，我们表示为74:(80 + 78 + 74)比率，或74:αI比率，根据实验条件而变化，例如，最终取决于αI结构域的构象。许多遗传性椭圆形红细胞增多症（HE）病例与74:αI比率增加有关，也称为SpαI/74异常。几条证据线索表明，致病突变可能位于α链或β链，这在进行基因水平研究之前是一个重要问题。为了解决这个问题，我们在体外重组血影蛋白二聚体，将各种来源的α链和β链组合在一起，然后进行部分消化并检测SpαI/74异常。重组二聚体得到的模式与天然二聚体的模式几乎相同。我们将该检测方法应用于三种导致SpαI/74 HE的血影蛋白变体：（1）我们之前命名为血影蛋白尼斯的变体，其β链在C末端区域缺少一个4千道尔顿片段；以及我们在两个不相关的白人家庭中发现的两个不同变体，我们临时命名为血影蛋白里昂和血影蛋白库洛。SpαI/74异常出现在分别含有血影蛋白尼斯的β链、血影蛋白里昂或血影蛋白库洛的α链的所有类型二聚体中。因此，我们证实血影蛋白尼斯是一种（αI/74）β变体，并确定血影蛋白里昂和血影蛋白库洛都是（αI/74）α变体。目前的检测方法可扩展到任何显示SpαI/74异常的血影蛋白变体。
