Proteomic approach to hereditary stomatocytosis

Hereditary stomatocytosis (HSt) describes a wide spectrum of autosomal dominantly inherited disorders in which the basal red cell membrane cation permeability is increased. The cation leak results in the regulations of cellular volume, which can lead to morphological abnormality. Clinically HSt is very heterogeneous and we can identify four principal form: Overhydrated Hereditary Stomatocytosis (OHSt), Dehydrated Hereditary Stomatocytosis (DHSt), Familial Pseudohyperkalemia (FP) and Hereditary Cryohydrocytosis (CHC). Since, the DHSt pathogenesis is linked to a defect or a decreased in a membrane protein, we studied the red blood cells membrane proteome by 2D-DIGE. In fact we selected 2 DHSt family, for a total of 5 patients to isolate the membrane proteins and compare they with membrane proteins of 10 healthy controls. Approximately, 1000 protein spots were detected, the protein spots were then filtered for the statistically relevant trend of regulation: p-value 0.075 (Student’s paired t test) and +1,35≥AV.RATIO ≤ -1.35. We compared all the spots derived from healthy controls versus all the spots derived from patients and found 65 spots of interest, of these 36 spots were upregulated in the DHSt patients and 29 downregulated. To identify the differentially expressed proteins we performed two preparative gel, but unfortunately the reproducibility of this gel was not 100% and of 65 spots of interest we identified and excised only 51 spots (33 upregulated in the DHSt patients and 18 downregulated). The analysis of peptides extracted from each spot was performed by analysis LC/MS/MS. Mass spectrometric analysis identified 24 proteins. We select 14 protein that could be involved in DHSt, and grouped them on based of their expression: Peptide C-Band3, Flotillin 1 and 2, Stomatin, Peroxiredoxin 1 and 2, Catalase, Annexin A1, Cytovillin 2, RAP2B e RAP1A that resulted up-regulated and G3PD, Aldo A, G protein beta subunit that resulted down-regulated. These data, in particular the discovery of high levels of Peroxiredoxin 1 and 2 and Catalase A1, suggest an increased oxidative stress in patients whit DHSt, also promoted by the decrease of some enzymes of glycolysis are important for the production of NADH and thus reducing potential. Another important data is the increase of Peptice c-Band3 and some proteins involved in the formation of lipid raft (Flotillin 1 and 2, Stomatin and Cytovillin 2) and in the vesciculation (RAP1A and RAP2B). Studies are currently underway to assess phosphorylation of band 3 in DHST patients and red blood cell vesciculation.