Abstract
During erythropoiesis, the molecular chaperone alpha-hemoglobin-stabilizing protein (AHSP) sequesters free alpha-hemoglobin (αHb) and prevents precipitation of excess αHb. While AHSP is linked to hereditary anemia, the pattern of expression during specific erythroblast stages is poorly understood. We investigated gene and protein expressions of AHSP throughout progressive maturation stages of erythroblasts in biphasic cultures of blood and bone marrow samples from healthy donors. Differentiating erythroblasts were periodically subjected to flow cytometry, Amnis imaging and RT-qPCR analyses. We made parallel in vivo validations from naive murine bone marrow cells. Percentages of AHSP+ erythroblasts, protein expressions and AHSP gene expressions are negligible on culture day 6 (CFU-Es) and progressively increases from culture days 8–12 (peaks on day 12) and declines on day 14. Notably, sub-cellular location of AHSP is both in the cytoplasm and nucleus in the early erythroblasts while in the late stages of maturation AHSP is found predominantly in the nucleus, being expelled with it during enucleation. As both human bone marrow and peripheral blood mononuclear cells (PBMC) derived erythroblasts demonstrated similar expression patterns, sampling of erythroblasts from day 11 cultures could portray erythroblast chronology and provide optimum representative stage specific expression patterns. PBMCs may be suitable for comparison studies of AHSP expression in pathologic erythropoiesi
Highlights
Alpha-hemoglobin-stabilizing protein (AHSP) is a molecular chaperone protein that sequesters free alpha-hemoglobin
When bone marrow cells from a healthy human donor were placed in ex vivo erythropoiesis culture, there was no detectable AHSP measured prior to day 8
To our knowledge this is the first publication depicting AHSP protein expression from two different sources namely naïve bone marrow-derived and peripheral blood mononuclear cells (PBMC)-derived erythroblasts. We concurrently validated these ex vivo contrived observations in in vivo murine total bone marrow cells and confirmed that among nucleated erythroblasts, late erythroblasts expressed higher protein and gene expression for AHSP compared to early erythroblasts
Summary
Alpha-hemoglobin-stabilizing protein (AHSP) is a molecular chaperone protein that sequesters free alpha-hemoglobin (αHb). The pattern of AHSP protein expression during erythropoiesis is not fully understood, but gene expression during erythroid maturation is reported by Dos Santos et al [8]. Little is known regarding AHSP as it is still a relatively new protein in the field of erythropoiesis. In this methodological study we have portrayed a stage specific expression pattern of AHSP in erythroblast chronology. Results underscore AHSP gene expression, protein expression and localization over its developmental trajectory in ex vivo culturing from human bone marrow cells and human PBMCs. For validation, we have compared our ex vivo observations with in vivo early and late stage erythroblasts obtained from murine bone marrow
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