Elsevier

Chemico-Biological Interactions

Volumes 157–158, 15 December 2005, Pages 371-372
Chemico-Biological Interactions

(12) A single mutation near the C-terminus in (/(-fold protein family causes a defect in protein processing

https://doi.org/10.1016/j.cbi.2005.10.057Get rights and content

Abstract

An Arg to Cys mutation in the extracellular domain of neuroligin-3 (NL3) was recently found in a twin set with autism [S. Jamain, H. Quach, C. Betancur, M. Rastam, C. Colineaux, I.C. Gillberg, H. Soderstrom, B. Giros, M. Leboyer, C. Gillberg, T. Bourgeron, Paris Autism Research International Sibpair Study, mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism, Nat. Genet. 34 (2003) 27–29]. The Cys substitution in NL3 causes altered intracellular protein trafficking, intracellular retention and diminished association with its cognate partner, β-neurexin [D. Comoletti, A. De Jaco, L.L. Jennings, R.E. Flynn, G. Gaietta, I. Tsigelny, M.H. Ellisman, P. Taylor, The R451C-neuroligin-3 mutation associated with autism reveals a defect in protein processing, J. Neurosci. 24 (2004) 4889–4893]. NL3, butyrylcholinesterase (BuChE), and acetylcholinesterase (AChE), as members of the (/(-hydrolase fold family of proteins, share over 30% of amino acid identity in their extracellular domains. In particular, Arg451 in NL3 is conserved in the α/β-hydrolase fold family being homologous to Arg386 in BuChE and Arg395 in AChE. A Cys substitution at the homologous Arg in the BuChE was found studying post-succinylcholine apnea in an Australian population [T. Yen, B.N. Nightingale, J.C. Burns, D.R. Sullivan, P.M. Stewart, Butyrylcholinesterase (BCHE) genotyping for post-succinylcholine apnea in an Australian population, Clin. Chem. 49 (2003) 1297–308]. We have made the homologous mutation in the mouse AChE and BuChE genes and showed that the Arg to Cys mutations resulted in identical alterations in the cellular phenotype for the various members of the α/β-hydrolase fold family proteins.

Introduction

The α/β hydrolase fold family is a superfamily of proteins all sharing a common fold motif. Their functions extend from hormonal signaling (thyroglobulin) to intercellular communication and synapse formation (neuroligins NLs), and hydrolytic activity (cholinesterases ChEs). Amino acid sequences of closely related members such as the ChEs and NLs are highly conserved, both among family members and among animal species. Of particular interest is the arginine near the C-teminus because Arg to Cys mutation from both NL-3 and BuChE is a naturally occurring human mutation: R451C in NL-3 appeared in a twin set with autism spectrum disorders [1] and the homologous mutation R386C in BuChE is seen in a patient with post-succinylcholine apnea [3]. The mutation R395C in AChE has not been observed in the natural population. To study whether the introduction of a cysteine may interfere, by a common mechanism, with the processing of the α/β hydrolase fold proteins, we analyzed the cellular consequences of the RC mutation in the trans-membrane spanning dimer of NL-1 and in two forms, soluble and GPI-anchored, of AChE.

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Results and discussion

The RC mutation was constructed by site-directed mutagenesis in the T548 soluble monomeric form, containing a stop codon at amino acid 548, and in the GPI-anchored dimer. Expression of the AChE constructs in HEK cells was analyzed by Ellman assay [4]. The R395C form of T548 is undetectable in the medium and GPI R395C shows low activity when measured in the intact cell, indicating that it is not transported to the cell surface. Western blot analysis confirms that protein expression of both T548

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