The Arg451Cys-neuroligin-3 mutation associated with autism reveals a defect in protein processing

Abstract

The neuroligins are a family of postsynaptic transmembrane proteins that associate with presynaptic partners, the beta-neurexins. Neurexins and neuroligins play a critical role in initiating formation and differentiation of synaptic junctions. A recent study reported that a mutation of neuroligin-3 (NL3), an X-linked gene, was found in siblings with autistic spectrum disorder in which two affected brothers had a point mutation that substituted a Cys for Arg451. To characterize the mutation at the biochemical level, we analyzed expression and activity of the mutated protein. Mass spectrometry comparison of the disulfide bonding pattern between the native and the mutated proteins indicates the absence of aberrant disulfide bonding, suggesting that the secondary structure of the mutated protein is conserved. However, the mutation separately affects protein expression and activity. The Cys mutation causes defective neuroligin trafficking, leading to retention of the protein in the endoplasmic reticulum. This, in turn, decreases the delivery of NL3 to the cell surface. Also, the small fraction of protein that reaches the cell membrane lacks or has markedly diminished beta-neurexin-1 (NX1beta) binding activity. Other substitutions for Arg451 allow for normal cellular expression but diminished affinity for NX1beta. Our findings reveal a cellular phenotype and loss of function for a congenital mutation associated with autistic spectrum disorders.

Figure 1.

Cellular secretion of wild-type and mutant NL1 and NL3. Immunoblots of the cell extract (CE) and medium (M) from transiently transfected FLAG-tagged NL3 and NL1 mutants truncated at positions 639 and 691, respectively. Sixty hours after transfection, cells and culture media proteins were separated by SDS-PAGE under reducing conditions, and neuroligin proteins were visualized with anti-neuroligin-1/3 monoclonal antibody. Molecular weight standards are shown on the left.

Figure 2.

Immunofluorescence detection of the cellular disposition of full-length NL1 and NL3 proteins. Stably transfected full-length NL3 and NL1, wild type and mutants, were used to investigate the cellular localization of the mature protein in HEK293 cells. Anti-FLAG antibody was used to visualize the neuroligin proteins in cells permeabilized with 0.5% saponin. a, NL1 wild type; b, R473C-NL1; c, CA/RC-NL1; d, R473T-NL1; e, NL3 wild type; f, R471C-NL3. Scale bar, 10 μm.

Figure 3.

A, Comparison of NL1 and NL3 proteins resolved on SDS-PAGE gel under nonreducing conditions. Immunoblots of the cell extract (CE) and medium (M) of transiently transfected NL1 and NL3 truncated mutants. The samples were prepared in the absence of β-mercaptoethanol. Other conditions were identical to those in Figure 1. B, Surface plasmon resonance binding analysis of soluble neuroligins. Top, NL3-639 and R471C-NL3-639 (3 μm) were injected on the same NX1β surface, and binding was measured over a 160 sec interval, followed by a wash period of 160 sec. Dashed line, NL3-639; continuous line, R471C-NL3. Bottom, NL1-691, R473T-NL1-691, and R473E-NL1-691 (111 nm) were injected on the same NX1β surface using the same parameters as for NL3. Dashed line, NL1-691 wild type; dotted line, R473T-NL1-691; continuous line, R473E-NL1-691. RU, Resonance units