Hematopoietic protein-1 (Hem-1) is a member of the Wiskott-Aldrich syndrome verprolin-homologous protein (WAVE) complex and is found in hematopoietic cells.1 The WAVE pathway functions to regulate actin polymerization. To investigate the interactions between Hem-1 and the WAVE complex, Chan et al.2 generated knockout mice containing a point mutation in the Hem1 gene that inhibited Hem-1 expression.1
Blood was obtained from Hem-1 null mice (Hem1−/−) and mice that were phenotypically identical to wild-type mice (Hem1+/−). Wright-Giemsa-stained blood smears from Hem1−/− mice showed abnormal erythrocyte morphology that included schistocytes, dacryocytes, keratocytes, and acanthocytes. The Hem1−/− samples also included areas of poikilocytosis, polychromasia, hypochromia, and anisocytosis. Phalloidin and antispectrin confocal microscopy of Hem1−/− revealed altered erythrocyte morphology and agreed with previous studies that contained disrupted organization of the actin membrane skeleton. The Hem1−/− blood smears were also more susceptible to osmotic stress and damage when compared with Hem1+/− erythrocytes.1
For proteomic analysis, red blood cells were purified and extracted from whole blood as previously described by Beutler et al.3 Following extraction and purification of proteins, protein samples were prepared and profiled on an LTQ-FT Ultra hybrid linear ion trap FTICR mass spectrometer (Thermo Scientific). Wild-type and Hem1−/− erythrocytes were compared using a label-free comparative proteomic approach known as CRAWDAD (chromatographic retention time alignment and warping for differential analysis of LC-MS data).4
CRAWDAD uses the intensity of LC-MS/MS spectra to estimate the abundance of peptides. Fourteen peptides were identified in the insoluble fraction, and 74 proteins were present in the soluble (cytosolic) fraction. To further validate prominent membrane proteins in Hem1−/− erythrocytes, SDS-PAGE and immunoblotting was performed. Coomassie-stained gels were employed to analyze the most abundant erythrocyte proteins in Hem1−/−. Purified immunoblots of erythrocyte membrane ghosts from purified wild-type and Hem1−/− erythrocytes exhibited decreased levels of adducin, dematin, β-spectrin, ankyrin, tropomodulin1, phosphoglycerate kinase 1 (PGK-1), and p55 (MPP1).
Chan et al.2 identified Hem-1 as an important player in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton. In addition to the structural problems, Hem1−/− erythrocytes also had a reduced life span relative to wild-type erythrocytes. Further studies need to be completed to understand the changes that occur in the context of normal and abnormal actin protofilaments, F-actin, membrane skeletal proteins, and key metabolic enzymes present in erythrocyte membranes.
1. Park, H., et al. (2008) ‘A point mutation in the murine Hem1 gene reveals an essential role for Hematopoietic protein 1 in lymphopoiesis and innate immunity‘, The Journal of Experimental Medicine, 205 (12), (pp. 2899-2913)
2. Chan, M.M., et al. (2013) ‘Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes‘, Public Library of Science One, 8 (2), February 12, 2013, doi/10.1371/journal.pone.0054902.
3. Beutler, E., et al. (1976) ‘The removal of leukocytes and platelets from whole blood‘, The Journal of Laboratory and Clinical Medicine, 88 (2), (pp. 328-333)
4. Finney, G.L., et al. (2008) ‘Label-free comparative analysis of proteomics mixtures using chromatographic alignment of high-resolution muLC-MS data‘, Analytical Chemistry, 80 (4), (pp. 961-971)