Throwing out the baby

In this issue of Blood, Salomao and colleagues reveal the novel mechanism of aberrant protein sorting during enucleation, resulting in additional membrane protein deficiencies in hereditary elliptocytosis and spherocytosis.¹ 

Whereas red cells of all birds, fish, reptiles, and amphibians retain their nucleus, the unique signature of definitive mammalian red cells is that they lose their nucleus before entering the blood stream. Investigators in the 1960s argued over the mechanism by which this occurred, some favoring karyolysis. Microcinematography of bone marrow cell suspensions by Bessis and Bricka in 1952 provided evidence in favor of expulsion,²  later confirmed by electron microscopy studies. But questions remained: Was this just a curious elimination of the unwieldy nuclear structure to prepare the flexible but strong red cell for its travels, or did this process have any clinical relevance? Careful study of this process has revealed that enucleation involves regulated segregation of cytoskeletal and cell-surface proteins between the plasma membrane of the extruded nucleus and the reticulocyte. Major cytoskeletal proteins, including band 3, spectrin, ankyrin, and 4.1, segregate to the reticulocyte, leaving the extruded nucleus devoid of these proteins.³  In contrast, the adhesion molecules Emp^4,5  and Beta-1-integrin⁴  partition to the exiting nucleus, facilitating its elimination by macrophages. Remarkably, the current study by Salomao et al directly links the failure of this coordinated membrane protein distribution to the pathogenesis of an important hematologic disease.

A curious feature of hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), true also in the murine models of these disorders, is the deficiency of red cell membrane proteins beyond those encoded for by the mutant gene. For example, HE reticulocytes resulting from 4.1R gene mutations predictably lack protein 4.1R. What is not so predictable is that they also lack glycophorin C (GPC), a membrane spanning protein and binding partner that cooperates in linking the cytoskeleton to the lipid bilayer.⁶  Similarly in HS resulting from ankyrin-1 gene mutations, there are also deficiencies of band 3, Rh-associated antigen (RhAG), and glycophorin A (GPA).⁷  Because these proteins are related to each other in macromolecular complexes, several mechanisms, singly or in combination, could account for their absence. Possibilities include improper assembly, aberrant sorting during enucleation, excessive degradation, or release in exosomes. Using immunofluorescence microscopy, the authors provide a snapshot of the dynamic enucleation process in erythroblasts from the bone marrow of normal and mutant HS and HE mice. They reveal various normal proteins being misdirected to the extruded nucleus, effectively “throwing out” these valuable integral membrane proteins. The end result of these events is a reticulocyte with a reduced “deformability endowment,” certainly a critical factor impacting its survival.

The study very nicely delineates a mechanism for greater disease severity in HS/HE over what would be predicted by the genotype. It is exciting that the study highlights the notion of membrane loss during reticulocyte formation, challenging the conventional wisdom of membrane loss solely during circulation. Temporal differences in membrane loss between HS and immune hemolytic anemia have been described before.⁸  The decreased surface area of the reticulocyte in HS, but not immune hemolytic anemia, distinguishes the 2 illnesses that share the common phenotype of spherocytic red cells.

This study opens up the door to various further scientific queries. What governs the quantity of integral membrane protein loss, and could those factors contribute to phenotypic variability? Quantitation of integral protein deficiencies at the level of the erythroblast, reticulocyte, and mature red cell could identify the most vulnerable erythroid population.⁴  Might this type of analysis identify the relative contributions to the anemia from ineffective erythropoiesis, ineffective “reticulocytopoiesis,” and extravascular hemolysis resulting from sequestration of the mature red cells in the spleen? It is intriguing to speculate that these types of studies might eventually help predict response to splenectomy and inform the clinician's decision in trading the benefit of reduced anemia with the increased risk of untoward side effects.