Abstract
A mutation linked to autistic spectrum disorders encodes an Arg to Cys replacement in the C-terminal portion of the extracellular domain of neuroligin-3. The solvent-exposed Cys causes virtually complete retention of the protein in the endoplasmic reticulum when the protein is expressed in transfected cells. An identical Cys substitution was reported for butyrylcholinesterase through genotyping patients with post-succinylcholine apnea. Neuroligin, butyrylcholinesterase, and acetylcholinesterase are members of the alpha,beta-hydrolase fold family of proteins sharing sequence similarity and common tertiary structures. Although these proteins have distinct oligomeric assemblies and cellular dispositions, homologous Arg residues in neuroligin-3 (Arg-451), in butyrylcholinesterase (Arg-386), and in acetylcholinesterase (Arg-395) are conserved in all studied mammalian species. To examine whether an homologous Arg to Cys mutation affects related proteins similarly despite their differing capacities to oligomerize, we inserted homologous mutations in the acetylcholinesterase and butyrylcholinesterase cDNAs. Using confocal fluorescence microscopy and analysis of oligosaccharide processing, we find that the homologous Arg to Cys mutation also results in endoplasmic reticulum retention of the two cholinesterases. Small quantities of mutated acetylcholinesterase exported from the cell retain activity but show a greater K(m), a much smaller k(cat), and altered substrate inhibition. The nascent proteins associate with chaperones during processing, but the mutation presumably restricts processing through the endoplasmic reticulum and Golgi apparatus, because of local protein misfolding and inability to oligomerize. The mutation may alter the capacity of these proteins to dissociate from their chaperone prior to oligomerization and processing for export.
Highlights
Several mutations that were found in human neuroligin 3 and 4 genes appear to be associated with the autistic spectrum disorders [1, 2]
We demonstrate here that single point mutation of a conserved Arg found in the neuroligin gene of an autistic twin set [1] and in patients with post-succinylcholine apnea [5,6,7] causes a similar defect in protein expression for both NL3 and BChE
Demonstration of processing deficiencies in AChE and BChE, as ␣,-hydrolase fold family members related to the neuroligins, carries the advantage that catalytic parameters of the folded protein can be monitored during the intracellular processing and extracellular secretion steps of protein biosynthesis
Summary
Plasmids and Mutagenesis—A cDNA encoding mouse GPI-AChE was subcloned into a FLAG-tagged vector (Sigma) for detection and purification. The proteasome inhibitor, lactacystin (10 M, Sigma), was applied to the cells 24 h after transient transfection, and the altered cellular disposition of NL-3, AChE, and BChE was assessed after treatment for 24 h. Total RNA Extraction, cDNA Preparation, and Real Time RTPCR—Total RNA was extracted with TRIzol reagents (Invitrogen) from one 100-mm tissue culture dish of HEK-293 cells stably transfected with wild type and mutant AChE constructs. Analysis of Glycosylation—Cell lysates (10 g) from HEK-293 cells expressing NL1 and AChE wild type and mutant proteins were incubated with 1500 units of endoglycosidase H (Endo H) or 250 units of N-glycosidase F (PNGase F) (New England Biolabs, Beverly, MA) at 37 °C for 3 h at pH 5.5 and 7.5, respectively. Determination of Kinetic Parameters for the AChE Catalytic Activity—AChE activity was measured over acetylthiocholine concentrations of 0.010 to 100 mM, and individual kinetic constants were
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