Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the αβ spectrin self-association binding site

Gallagher, Patrick G.; Zhang, Zhushan; Morrow, Jon S.; Forget, Bernard G.

doi:10.1038/labinvest.3700029

Cited by 14 publications

(10 citation statements)

References 43 publications

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“…Genetic variants associated with hereditary elliptocytosis and hereditary pyropoikilocytosis have been identified in the α I and β I regions of the spectrin genes, which encode the self‐association binding site of spectrin. This site corresponds to exon 2 of SPTA1 and exons 30–31 of SPTB (Gallagher et al , ).…”

Section: Discussionmentioning

confidence: 99%

Deciphering molecular heterogeneity of Indian families with hereditary spherocytosis using targeted next‐generation sequencing: First South Asian study

et al. 2019

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Summary Defects in various erythrocyte membrane proteins genes (ankyrin, band‐3, β‐ and α‐spectrin and protein 4·2) can cause hereditary spherocytosis (HS). This molecular heterogeneity of HS, together with co‐inherited genetic modifiers, results in marked phenotypic variability among patients. We studied the molecular spectrum and genotype‐phenotype correlations in 73 families (with 113 patients) with HS. Deleterious variants including nonsense (42%), deletions (18%), splice site (20%), missense (10%) and duplication/insertion (10%) were found in 47 patients. The variants detected included sporadic and dominantly‐inherited defects in ANK1 (53·2%), SPTB (36·2%) and SLC4A1 (4·2%). Compound heterozygous variants in SPTA1 (6·4%) showed autosomal recessive inheritance. Alpha‐spectrin variants were associated with severe anaemia and splenectomy alleviated symptoms. Co‐inherited glucose‐6‐phosphate dehydrogenase (G6PD) deficiency was found in 15%. G6PD variants (n = 5) led to greater transfusion requirements (1‐8 times) in males with HS. Homozygosity (41%) for the promoter variant of UGT1A1 (Gilbert syndrome) led to a significantly higher mean bilirubin level (126·54 µmol/l) with a higher frequency of cholelithiasis (30%) (P < 0·001). This first‐ever south Asian study on the molecular spectrum of HS found ANK1 and SPTB genes variants to be the commonest with inheritance being sporadic/dominant. Next‐generation sequencing provided a relatively sensitive and rapid tool for molecular diagnosis with a diagnostic yield of 64·4%.

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Section: Discussionmentioning

confidence: 99%

Deciphering molecular heterogeneity of Indian families with hereditary spherocytosis using targeted next‐generation sequencing: First South Asian study

et al. 2019

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“…Available literature indicates that this self-association domain is where the heterodimers of spectrin link up to form tetramers (Shammas, Rogers, Hill, & Clarke, 2012) and mutations in this region lead to hemolytic diseases (Nicolas et al, 1998). Moreover, available data shows this region to have hydrophobic residues present on the peptide surface (Gallagher, Zhang, Morrow, & Forget, 2004;Henniker & Ralston, 1994). This presence of surface exposed hydrophobic patches has led us to include these two domains.…”

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confidence: 99%

Localizing the chaperone activity of erythroid spectrin

Bose

Chakrabarti

2019

Cytoskeleton

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Spectrin, the major protein of the erythrocyte membrane skeleton has canonically been thought to only serve a structural function. We have previously described a novel chaperone‐like property of spectrin and also hypothesized that the chaperone activity and binding of a hydrophobic ligand, Prodan are localized in the self‐association domain. Here we probe the location and molecular origin of the chaperone activity of multi‐domain spectrin using a selection of individual recombinant spectrin domains, which we have characterized using intrinsic tryptophan fluorescence and CD spectroscopy to show their identity to native spectrin. Aggregation assays using insulin, ADH, α‐ and β‐globin as well as enzyme refolding assays using alkaline phosphatase and α‐glucosidase show that the chaperone activity is not only localized in the self‐association domain but is a generalized property of spectrin domains. This is to our understanding, a unique feature in the case of modular multi‐repeat proteins, possibly implicating that the large family of “spectrin‐repeat” domain containing proteins may also have chaperone like property. Substrate selectivity of chaperone activity as evidenced by the preferential protection of α‐ over β‐globin chains is seen; which has implications in hemoglobin diseases. Moreover, enzyme‐refolding assays also indicate alternate modes of chaperone action. We propose that the molecular origin of chaperone activity resides in the surface exposed hydrophobic patches of the spectrin domains as shown by ANS (1‐anilinonaphthalene‐8‐sulfonic acid) and Prodan (6‐propionyl‐2[dimethylamino]‐naphthalene) binding. We also show that Prodan does indeed have a unique binding site on spectrin located at the self‐association domain.

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“…Erythrocyte membranes were prepared from peripheral blood as previously described (39). Membrane proteins were analyzed and spectrin content determined as described (40). Membrane proteins were separated by SDS-PAGE on 3.5%-17% gradient polyacrylamide gels and stained with Coomassie blue.…”

Section: Methodsmentioning

confidence: 99%

Aberrant splicing contributes to severe α-spectrin–linked congenital hemolytic anemia

Gallagher¹,

Maksimova²,

Lezon-Geyda³

et al. 2019

Journal of Clinical Investigation

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with the NMD inhibitor cycloheximide (100 μg/ml, MilliporeSigma, C1988) for 4 hours, or with a combination of both. The amount of total α-spectrin mRNA transcripts and α LEPRA mRNA transcripts were determined by real-time RT-PCR with fluorescent probes as described above. Analyses included 3 biologic and 3 technical replicates. RNA-seq data. RNA-seq data sets accessed from the Gene Expression Omnibus database were GSE61566 and GSE53983 for human erythroid cells and GSM958729 for K562 cells. Statistics. GraphPad Prism 8 software (Graph Pad Software) was used for statistical analyses. All data were reported as mean ± SD in tables or as error bars. Comparisons between 2 groups were performed using the 2-tailed Student's t test. Multiple test correction was performed using the Benjamini and Hochberg method (51). Significance was set at P less than 0.05. Human subjects. All human studies were approved by the Yale University Human Investigation Committee review board. Written informed consent was received from participants or their parents, as appropriate, prior to inclusion in the study.

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Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the αβ spectrin self-association binding site

Cited by 14 publications

References 43 publications

Deciphering molecular heterogeneity of Indian families with hereditary spherocytosis using targeted next‐generation sequencing: First South Asian study

Deciphering molecular heterogeneity of Indian families with hereditary spherocytosis using targeted next‐generation sequencing: First South Asian study

Localizing the chaperone activity of erythroid spectrin

Aberrant splicing contributes to severe α-spectrin–linked congenital hemolytic anemia

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