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

5-3 Analysis of functional and post-translational modifications in red cells from neuroacanthocytosis
G. Bosman
Dept. of Biochemistry, Radboud University Medical Center and Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands

The presence of abnormally shaped erythrocytes in the blood of patients with various forms of neuroacanthocytosis (NA) suggests that elucidation of the cause of the acanthocyte shape may yield clues to the mechanism underlying the neurodegeneration that is associated with mutations in chorein, XK protein, and junctophilin-3. Previous data suggested a central role in acanthocyte formation for alterations in erythrocyte band 3.This integral membrane protein occupies a central position in regulation of erythrocyte function, shape and removal. Recent data from the Verona and Nijmegen groups indicated the involvement of post-translational modifications of band 3, but also of other membrane and cytoskeleton proteins. Preliminary data showed proteomic approaches to be highly informative in these analyses. Therefore, in order to accelerate the discovery of novel clues on the pathophysiology of NA, the major aim of this project is to generate a comparative proteomic inventory of the membrane fractions of erythrocytes from NA patients with chorea-acanthocytosis (ChAc), McLeod syndrome, and Huntington disease-like 2 (HDL-2).
We performed proteomic analysis using the peptides obtained by tryptic digestion of gel slices after protein separation using one-dimensional SDS gel electrophoresis. Peptide sequences were determined using a nano-HPLC system connected to a LTQ-Fourier transform mass spectrometer (FTMS), and proteins were identified by searching an in-house data base, using Mascot 2.1. Semi-quantitative analysis was performed with a label-free, spectral counting exponentially modified protein abundance index (emPAI). The combination of these methods for identification and quantification was shown to be able to detect proteins in the picomolar range, and were validated by immunoblot analysis.
Since proteomic analyses are expensive and time-consuming, we first invested in the development of a method to generate reliable data with a minimal number of samples. In the resulting procedure, protein separation is restricted, which reduces the necessary number of FTMS runs sixfold. This yields results that are comparable to those obtained previously by much more elaborate methods, although there is some loss in the amount of - mainly qualitative - information. Using this method, we then determined the protein composition of the erythrocyte membrane preparations obtained from four healthy control donors. These were identical to those obtained before by us and others. The inter-individual concentrations varied 5-10 percent, apparently dependent on the number of molecules per cell, the degree of hydrophobicity, and/or the degree and type of glycosylation.
A first analysis of the membrane protein composition of erythrocytes from patients with ChAc and HDL-2 showed considerable differences in the concentration of a number of proteins between ChAc and HDL-2 erythrocytes and control erythrocytes, but also between ChAc and HDL-2 erythrocytes. The proteins in question are mainly components of the cytoskeleton and the cytoskeleton-membrane interface (increased concentrations of ankyrin, spectrin, band 4.2 in ChAc and HDL-2; increased p55 in ChAc but not in HDL-2). There were also considerable differences in the association of cytosolic proteins such as hemoglobin and glyceraldehyde 3-phosphate dehydrogenase, and in the amount of membrane-bound stomatin.
These analyses are presently being repeated and extended with samples from other patients. The first results of this project presented here confirm previous data from a pilot study reported in Kyoto, and complement and extend the data obtained by the Verona group. In combination with the latter, they strongly suggest that this approach is likely to inspire new theories and lead to more insight into the pathophysiology of neuroacanthocytosis.