Marked changes in red cell membrane proteins in hereditary spherocytosis: a proteomics approach

Abstract

Hereditary spherocytosis (HS) is the most common red cell membrane defect resulting from protein abnormalities. However, changes in red cell membrane proteins in HS remain under-investigated. We therefore evaluated red cell membrane proteome in non-splenectomized, mild-degree HS patients (n = 9) compared to healthy individuals (n = 5). Proteins derived from the red cell membranes of each subject were resolved in each two-dimensional gel and visualized by Deep Purple fluorescence staining. Spot matching and quantitative intensity analysis revealed 56 differentially expressed protein spots (41 increased and 15 decreased), which were then successfully identified by quadrupole time-of-flight mass spectrometry. Among these, seven isoforms/subunits of spectrin were markedly increased (up to 10.51 folds), whereas two isoforms/subunits of band-3 protein were decreased approximately 50% as compared to normal red cells. However, two isoforms/subunits of protein 4.1 were increased, while another isoform/subunit was decreased. All these significantly altered proteins were subjected to global protein network analysis using Ingenuity Pathways Analysis tool, which revealed three important networks related to HS, including Network I: Cell death, genetic and hematological disorders; Network II: Cell cycle, carbohydrate metabolism and molecular transport; and Network III: Genetic and hematological disorders, cell-to-cell signaling and interactions. These data offer many opportunities and new roadmaps for further functional studies to better understand the biology and pathogenic mechanisms of HS.

Assessment of the red blood cell proteome in a dog with unexplained hemolytic anemia

“…The results suggested alterations in the amounts of key membrane proteins, in particular the band 3, and alpha‐ and beta‐adducin proteins, while the other protein levels, including alpha‐ and beta‐spectrin and ankyrin, were comparable with that of the control dog. Interestingly, this same protein derangement pattern was previously reported in people with primary band 3 deficiencies . The proteomic approach used here is recognized as a diagnostic approach when investigating hereditary spherocytosis in people; however, to the authors’ knowledge, this technique has not been previously applied as a diagnostic approach in dogs.…”

“…A limitation of the study was the number of samples analyzed; in similar studies of human patients with hereditary spherocytosis, small numbers of control subjects are also described with an emphasis on the quantitative alterations in proteins in these patients . In addition, lack of replication of the results prohibits statistical evaluation of the results and the fold changes reported should be interpreted in this light.…”

“…In this study, a 2‐fold change was taken as the cutoff value to identify proteins that had significantly altered amounts . This approach allowed exploration of the protein differences detected in the presence of a reticulocytosis and hemolysis as previously described . For protein amounts that increased in the affected dog compared with that of the control dog, DAVID analysis revealed an enrichment of proteins associated with the functional annotation terms “citrate cycle (TCA cycle)” and “oxidative phosphorylation,” while proteins that decreased were associated with the functional annotation terms “proteasome,” “chaperonin‐containing T‐complex,” and “intermediate filament protein” (Table ).…”

Erythrocyte and platelet proteomics in hematological disorders

“…Rabilloud established more sensitive methods of gel staining leading to visualizing greater number of protein spots in 2DE [10]. Following this, few gel-based studies on erythrocyte membrane came up, describing protein deregulation in hereditary spherocytosis [11][12][13][14][15], sickle cell anemia [16][17][18], and hemoglobin E ␤ (HbE␤) thalassemia [19]. Few novel proteins were identified in malaria parasite infected erythrocyte membrane [20].…”

Defects in Erythrocyte Membrane Skeletal Architecture

“…Recently, high through put techniques like proteomics is also used to understand the disease Peker et al 2012 ;Polprasert et al 2012 ;Saha et al 2011 ). These studies not only reveals the involvement of major MS proteins in the disease but also tells about increased association of globin chains with the membrane and up regulation of redox regulators in the erythrocyte cytosol (Saha et al 2011 ).…”