Functional characterisation of ganglioside-induced differentiation-associated pr

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Biochem Biophys Res Commun. 2006 Jul 18

Functional characterisation of ganglioside-induced differentiation-

associated protein 1 as a glutathione transferase.

Shield AJ, Murray TP, Board PG.

Molecular Genetics Group, Division of Molecular Biosciences,

Curtin School of Medical Research, Australian National University,

Canberra, ACT 2601, Australia.

Mutations in the ganglioside-induced differentiation-associated

protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth

(CMT) disease. This protein, and its paralogue GDAP1L1, appear to be

structurally related to the cytosolic glutathione S-transferases

(GST) including an N-terminal thioredoxin fold domain with conserved

active site residues. The specific function, of GDAP1 remains

unknown.

To further characterise their structure and function we purified

recombinant human GDAP1 and GDAP1L1 proteins using bacterial

expression and immobilised metal affinity chromatography. Like other

cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the

full-length proteins were largely insoluble, the deletion of a

proposed C-terminal transmembrane domain allowed the preparation of

soluble protein. The purified proteins were assayed for glutathione-

dependent activity against a library of 'prototypic' GST substrates.

No evidence of glutathione-dependent activity or an ability to bind

glutathione immobilised on agarose was found.