Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2)

Abstract

The online version of this article has a Supplementary Appendix. BackgroundProtein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. Design and MethodsWe used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. ResultsWe report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship. ConclusionsWe have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.

Characteristic phenotypes associated with congenital dyserythropoietic anemia (type II) manifest at different stages of erythropoiesis

“…During the early stages of terminal differentiation, there is a significant secretory pathway burden whereby expression of erythroid specific proteins increases dramatically and must be delivered to the plasma membrane. 29,34 Thus this work suggests that there is an important role for SEC23B in general COPII mediated trafficking or in the transport of cargo with function specific to the erythroid cellular context that cannot be completely compensated by the decreasing levels of SEC23A.…”

“…Rabbit polyclonal antibodies to RhAG, Glut1, GPA, CD47, 29,30 SEC24C, 31 SEC24D 32 and SEC31A 31 were available in house. Anti-mouse band 3 was from Prof. Carsten Wagner (University of Zurich), anti GM130 (BD Transduction), anti-GAPDH (Santa Cruz) and anti-GRP78 (Sigma).…”

“…Erythroblasts derived from peripheral blood mononuclear cells were isolated, expanded and differentiated as previously described 29,33,34 for initial experiments and then subsequently modified according to Bell et al…”

The majority of the in vitro erythroid expansion potential resides in CD34- cells, outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples

“…Importantly, terminal differentiation of PBMC derived erythroblasts to enucleated reticulocytes is of a similar nature within all the parameters studied here (hemoglobinization, morphology, proliferation and erythroid marker expression). The culture of PBMC described here will prove invaluable for investigating the alterations that occur during blood diseases such as hereditary spherocytosis 15 or congenital dyserythropoietic anemia type II (CDAII) where only small amounts of blood may be available for study.…”

Changes in Erythrocyte Surface Marker Cd44 During Hypothermic and Low Temperature Storage

“…Зв'язок СD44 з цито-скелетом в еритроцитах, вочевидь, може відігравати певну роль у модуляції механо-еластичних властивостей мембрани. Це по-бічно підтверджується тим, що за наявності генетичної модифікації, яка призводить до повної втрати цитоскелетного білка смуги 4.2 і супроводжується підвищенням асоціації СD44 з цитоскелетом, спостерігається від-носно м'який фенотип анемії [10].…”