Fourth International Symposium on Neuroacanthocytosis

Abstracts from the Fourth International Symposium on Neuroacanthocytosis

July 1-2, 2008
London and Oxford

Chairs: Prof. Kailash Bhatia, MD, FRCP, Institute of Neurology, University College London; Prof. Anthony P. Monaco, MD, PhD, Wellcome Trust Centre for Human Genetics, University of Oxford

Organizers: Antonio Velayos-Baeza, PhD; Susanne Schneider, MD; Glenn Irvine

5-2 Analysis of functional and post-translational modifications in red cells from neuroacanthocytosis
L. de Franceschi
Dept. of Clinical and Experimental Medicine, Section of Internal Medicine, University of Verona, Verona, Italy

Abnormally spurn-thorn shaped red cells, known as acanthocytes, characterise neuroacanthocytosis syndromes (NA). The enormous gap between genotype and phenotype clinical manifestations in NA suggests a possible important role of post-translation protein modifications in abnormal cell function. Here, we studied red cells from patients with chorea-acanthocytosis (ChAc) or McLeod syndrome (MLS). We evaluated hematological parameters, red cell index by ADVIA -Technicon (Bayer) and red cell cation content. In both ChAc (n=4) and MLS (n=5) patients we observed the presence of dense red cell fraction with marked significant reduction in red cell K+ content and a slight but significant increase in red cell Na+ content. We then fractioned red cells according to their density and we further analysed red cells membrane proteome and post-translational modifications focusing on red cell membrane phosphotyrosine (Tyr-) profile. We considered two fractions: F1 corresponding to red cells density < 1.074 and F2: corresponding to red cells density > 1.092, which contains denser red cells and acanthocytes. We generated bidimensional gels of membrane proteins from fractioned red cells of normal and ChAc patients. We analysed 3378 spots and a total of 91 spots significantly differently expressed were identified by MALDI-TOF MS/MS analysis. The identified proteins were divided into 6 major clusters according to their functions: (i) membrane-cytoskeleton proteins; (ii) metabolic enzymes; (iii) ubiquitin-proteasome system; (iv) membrane channel and transports; (v) phosphatase-kinases and (vi) chaperones. The differences in the spots of membrane-cytoskeleton proteins in red cell fractions (F1, F2) from ChAc subjects were mainly related to 2D mobility shift most likely due to post-translation modifications such as for ankyrin, band 4.1, protein p55 in F1 and F2 or for dematin in F2. We then moved to analysis of Tyr-phosphoproteome of fractioned red cells from both normal and ChAc patients. In both F1 and F2 fractions from ChAc red cells, we observed increase red cell Tyr-phosphorylation state of red cell membrane protein compared to normal controls. The perturbation of Tyr phosphorylation profile in dense ChAc red cells involved integral membrane protein, cytoskeleton proteins but also anchoring proteins. In addition, the increased membrane association of chaperone proteins such as heat shock proteins 70 and 27, support a perturbation of red cell membrane organisation participating in the generation of acanthocytic red cells in ChAc. Further studies need to be carried out to progress on the functional analysis of mechanisms involved in red cell volume regulation processes and in post-translational events in red cells from NA patients.