Folding anomalies of neuroligin3 caused by a mutation in the alpha/beta-hydrolase fold domain

Abstract

Proteins of the alpha/beta-hydrolase fold family share a common structural fold, but perform a diverse set of functions. We have been studying natural mutations occurring in association with congenital disorders in the alpha/beta-hydrolase fold domain of neuroligin (NLGN), butyrylcholinesterase (BChE), acetylcholinesterase (AChE). Starting from the autism-related R451C mutation in the alpha/beta-hydrolase fold domain of NLGN3, we had previously shown that the Arg to Cys substitution is responsible for endoplasmic reticulum (ER) retention of the mutant protein and that a similar trafficking defect is observed when the mutation is inserted at the homologous positions in AChE and BChE. Herein we show further characterization of the R451C mutation in NLGN3 when expressed in HEK-293, and by protease digestion sensitivity, we reveal that the phenotype results from protein misfolding. However, the presence of an extra Cys does not interfere with the formation of disulfide bonds as shown by reaction with PEG-maleimide and estimation of the molecular mass changes. These findings highlight the role of proper protein folding in protein processing and localization.

Figure 1. Trypsin sensitivity and PEG-maleimide reaction of NLGN3 wild type and R451C mutant protein

A) Immunoprecipitated NLGN3 wild type and R451C were treated for 5 min with trypsin at the indicated concentrations and analyzed by SDS PAGE in reducing conditions followed by immunoblotting with an anti-NLGN antibody. Mutant protein is more sensitive to the digestion with trypsin compared to wild type. B) PEG-mal conjugation with free cysteines in NLGN3 wild type and R451C proteins. NLGN3 immunoprecipitated proteins are treated with 1mM PEG_5,000-maleimide in denaturing conditions and band shifts are observed on a SDS PAGE followed by immunoblotting with an anti-NLGN antibody. R451C NLGN3 shows a band shift likely corresponding to the alkylation of three cysteine thiols (arrows).

Figure 2. Schematic representation of an experimental approach to study a mutation in the α/β-hydrolase fold proteins

Determination of mutant protein location with immunofluorescence staining; protein function studies by surface plasmon resonance (NLGNs) or determination of enzyme catalytic constants (ChEs); protein folding by proteolytic digestion with trypsin, and exposure of free cysteine by treatment with PEG-maleimide. Possible alteration of glycosylation processing is studied by sensitivity to glycosidases, pulse-chase metabolic labelling and differential association of wild type and mutant proteins with molecular chaperones during protein biosynthesis. Protein trafficking in the cellular context of the nervous system can be approached using imaging techniques.