NLGN4X Antibody

What is NLGN4X and why is it significant in neuroscience research?

NLGN4X (Neuroligin-4 X-linked) is a 110 kDa type I transmembrane glycoprotein belonging to the type B carboxyesterase/lipase family of proteins . It is postsynaptically expressed on neurons and plays a critical role in initiating excitatory presynapse maturation through its binding with specific isoforms of beta-neurexin . The human NLGN4X gene is located on the X-chromosome and encodes a protein with an 816 amino acid sequence comprising a 41 aa signal sequence, a 635 aa extracellular domain (ECD), a 21 aa transmembrane domain, and a 119 aa cytoplasmic tail .

NLGN4X has gained significant research attention because mutations in this gene have been associated with autism spectrum disorders (ASD), Asperger syndrome, and Tourette syndrome . Understanding the structure and function of NLGN4X is therefore critical for elucidating the neurobiological basis of these conditions.

How does NLGN4X differ from NLGN4Y at the molecular and functional levels?

Despite sharing 97% sequence homology, NLGN4X and NLGN4Y exhibit profound functional differences . NLGN4Y displays severe deficits in maturation, surface expression, and synaptogenesis compared to NLGN4X . Biochemical analyses reveal that NLGN4Y primarily exists in its immature form, whereas NLGN4X is detected in both mature and immature forms .

This functional difference is regulated by a single critical amino acid substitution in the extracellular domain - specifically, a proline at position 93 in NLGN4X corresponds to a serine in NLGN4Y (P93S) . When this amino acid is mutated in NLGN4X to match NLGN4Y (P93S), it results in decreased expression of the mature form, mimicking the NLGN4Y phenotype . The inability of NLGN4Y to compensate for NLGN4X functional deficits has implications for the male bias observed in NLGN4X-associated neurodevelopmental disorders .

What are the primary applications for NLGN4X antibodies in neuroscience research?

NLGN4X antibodies serve multiple critical applications in neuroscience research:

• Western Blotting: Used to detect NLGN4X in cell lysates, including those from neuronal cultures, demonstrating specificity for the ~110 kDa protein .

• Immunohistochemistry (IHC): Applied to detect NLGN4X in fixed tissue sections, such as human brain cortex, where specific staining is localized to neuronal cell bodies and their processes .

• Immunocytochemistry (ICC): Used to visualize NLGN4X expression and localization in cultured neurons .

• Flow Cytometry: Employed to detect NLGN4X expression in cell populations, including human-induced pluripotent stem cells (hiPSCs) .

• ELISA: Used for quantitative detection of NLGN4X in various sample types .

These applications enable researchers to investigate NLGN4X expression, localization, and function in various experimental contexts relevant to neurodevelopmental disorders.

How should I design experiments to distinguish between NLGN4X and NLGN4Y expression?

When designing experiments to distinguish between NLGN4X and NLGN4Y, consider these methodological approaches:

• Antibody Selection: Use antibodies specifically developed against unique epitopes of NLGN4X or NLGN4Y. Validate these antibodies by testing them against both proteins expressed in a heterologous system like HEK293T cells .

• Western Blot Analysis: Exploit the different migration patterns - NLGN4X typically shows both mature and immature bands, while NLGN4Y predominantly shows the lower molecular weight immature band .

• RT-PCR with Specific Primers: Design primers that target non-homologous regions between NLGN4X and NLGN4Y transcripts. Sequence verification of amplicons may be necessary for confirmation .

• Sex-specific Controls: Include both male and female samples in your experimental design. NLGN4Y should only be detected in male-derived samples, providing an internal control for antibody specificity .

• Surface Biotinylation Assays: To differentiate their trafficking properties, use surface biotinylation to compare the surface expression of NLGN4X versus NLGN4Y .

When reporting results, clearly document the antibody clone, epitope, and validation experiments performed to substantiate your findings.

What are the optimal cell models for studying NLGN4X function and trafficking?

Several cell models offer distinct advantages for investigating NLGN4X function and trafficking:

• Human-derived Neuronal Cultures: Differentiated neurons from human induced pluripotent stem cells (hiPSCs) represent a physiologically relevant model that expresses endogenous NLGN4X . Male-derived lines additionally express NLGN4Y, enabling comparative studies .

• Primary Rat/Mouse Hippocampal Neurons: These provide a well-established system for studying synaptogenesis. When using these models, consider microRNA-mediated knockdown of endogenous neuroligins (NLmiRs) to avoid potential heterodimerization with endogenous neuroligins when expressing human NLGN4X .

• HEK293T Cells: Though non-neuronal, these cells are valuable for biochemical characterization, protein interaction studies, and trafficking experiments due to their high transfection efficiency and lack of endogenous neuroligin expression .

• NTera-2 Cells: This human testicular embryonic carcinoma cell line has been validated for NLGN4X expression and can be useful for antibody validation and basic expression studies .

When selecting a model system, consider whether you need to study endogenous protein or if heterologous expression is sufficient, and whether you aim to investigate neuronal-specific functions like synaptogenesis.

What controls should be included when studying NLGN4X in autism research?

For rigorous NLGN4X studies in autism research, implement these essential controls:

• Genetic Controls:

  • Include both wild-type NLGN4X and NLGN4Y for comparison with autism-associated variants

  • Use the P93S mutation as a functional control that phenocopies NLGN4Y

  • Include other neuroligin family members (NLGN1-3) as specificity controls

• Experimental Validation Controls:

  • For surface expression studies, include transferrin receptor (TfR) as a surface protein control

  • For protein processing experiments, include BiP/GRP78 detection, as this ER chaperone differentially interacts with NLGN4Y compared to NLGN4X

• Sample Controls:

  • Where possible, include matched samples from typically developing individuals alongside autism samples

  • For sex-linked analyses, include both male and female samples to account for NLGN4Y expression in males

• Technical Controls:

  • Validate antibody specificity using overexpression systems and knockout models

  • For mRNA expression studies, perform RT-PCR with primers that can detect both NLGN4X and NLGN4Y to ensure comparable expression levels when comparing function

These controls enhance experimental rigor and facilitate proper interpretation of results in the context of autism research.

What are the optimal conditions for detecting NLGN4X using Western blotting?

For optimal NLGN4X detection via Western blotting, consider these methodological details:

• Sample Preparation:

  • Lyse cells in a buffer containing protease inhibitors to prevent degradation

  • For neuronal samples, NTera-2 human testicular embryonic carcinoma cells can serve as a positive control

• Gel Conditions:

  • Use PVDF membrane for protein transfer

  • Run samples under reducing conditions

  • Use Immunoblot Buffer Group 3 for optimal results as demonstrated in R&D Systems protocols

• Antibody Parameters:

  • Primary antibody concentration: 1 μg/mL has been validated for the R&D Systems Anti-Human Neuroligin 4X/NLGN4X Antigen Affinity-purified Polyclonal Antibody (Catalog # AF5158)

  • Secondary antibody: HRP-conjugated Anti-Sheep IgG for the R&D Systems antibody

• Detection Considerations:

  • Look for a specific band at approximately 110 kDa

  • Be aware that NLGN4X typically appears as two bands - the mature (higher molecular weight) and immature (lower molecular weight) forms

  • The ratio of mature to immature bands can provide important information about protein processing

These optimized conditions should enable reliable detection of NLGN4X in Western blot applications while minimizing non-specific background.

How can I accurately perform immunostaining for NLGN4X in brain tissue sections?

For successful immunostaining of NLGN4X in brain tissue sections, follow this validated protocol:

This protocol has been validated for human cortical tissue and should yield specific staining of NLGN4X with minimal background.

What methods can reliably distinguish mature from immature forms of NLGN4X?

Several complementary methods can reliably distinguish mature from immature forms of NLGN4X:

• SDS-PAGE Mobility Analysis:

  • The mature glycosylated form of NLGN4X migrates at a higher molecular weight (~110 kDa) compared to the immature form

  • Use gradient gels (e.g., 4-15%) for optimal separation of these bands

• Glycosidase Treatment:

  • Treat protein samples with EndoH or PNGaseF enzymes

  • Immature ER-resident forms are typically EndoH-sensitive, while mature forms that have passed through the Golgi are EndoH-resistant but PNGaseF-sensitive

• Subcellular Fractionation:

  • Separate ER, Golgi, and plasma membrane fractions

  • Immature NLGN4X predominantly localizes to ER fractions, while mature forms are found in Golgi and plasma membrane fractions

• Surface Biotinylation Assays:

  • Label surface proteins in live cells with sulfo-NHS-SS-biotin

  • Isolate with streptavidin pulldown

  • Only mature, properly processed NLGN4X will be detected in the surface fraction

• Immunofluorescence Microscopy:

  • Use antibodies against NLGN4X along with organelle markers

  • Co-localization with ER markers indicates immature forms, while surface or synaptic staining indicates mature forms

The ratio of mature to immature forms provides valuable information about NLGN4X processing efficiency, which is particularly relevant when studying autism-associated variants.

How do autism-associated NLGN4X mutations affect protein trafficking and function?

Autism-associated NLGN4X mutations profoundly impact protein trafficking and function through several mechanisms:

• Impaired Protein Maturation:

  • A cluster of autism-associated variants surrounds the critical amino acid position 93 in NLGN4X

  • These mutations phenocopy NLGN4Y by showing deficits in protein maturation, with reduced expression of the higher molecular weight mature form

• Deficient Surface Trafficking:

  • Autism-associated NLGN4X variants demonstrate significantly reduced surface expression compared to wild-type

  • This trafficking defect correlates with increased association with ER chaperones like BiP, suggesting impaired protein folding

• Compromised Synaptogenic Activity:

  • Mutations that affect surface trafficking consequently impair the synaptogenic function of NLGN4X

  • The inability to properly localize to the postsynaptic membrane prevents interaction with presynaptic neurexins, disrupting synaptogenesis

• Loss of Compensation Mechanism:

  • NLGN4Y cannot compensate for the functional deficits of autism-associated NLGN4X mutations

  • This may partly explain the male bias observed in NLGN4X-associated autism spectrum disorders

• Altered Protein-Protein Interactions:

  • Some mutations affect the hydrophilic interface through which NLGN4X interacts with beta-neurexin in a calcium-dependent manner

  • Others may impact intracellular interactions with proteins like syntrophin-gamma 2

These molecular consequences provide critical insights into the pathogenic mechanisms underlying NLGN4X-associated neurodevelopmental disorders.

What post-translational modifications of NLGN4X are important for its function?

Several post-translational modifications (PTMs) critically regulate NLGN4X function:

• N-Glycosylation:

  • Essential for proper folding and trafficking through the secretory pathway

  • Contributes to the molecular weight difference between mature and immature forms

  • Specific glycosylation patterns may influence binding to neurexins

• Phosphorylation:

  • The search result indicates phosphorylation of NLGN4X regulates spinogenesis, suggesting this PTM plays a role in dendritic spine formation and synaptic plasticity

  • Specific phosphorylation sites likely modulate protein-protein interactions in the postsynaptic density

• Proteolytic Processing:

  • Neuroligins can undergo regulated proteolysis that may modulate their synaptic functions

  • The extracellular domain containing the esterase homology domain can be cleaved, potentially regulating neurexin binding

• Dimerization:

  • NLGN4X forms homodimers via a hydrophobic interface, which is distinct from the hydrophilic, calcium-dependent interface used for neurexin binding

  • This dimerization is critical for proper function and may be regulated by intracellular signals

• S-Palmitoylation:

  • While not specifically mentioned in the search results, S-palmitoylation has been documented for other neuroligins and may influence NLGN4X membrane localization and lateral mobility

Understanding these PTMs provides critical insights for researchers investigating NLGN4X function in normal development and disease states, and may inform therapeutic strategies targeting specific modifications.

What experimental approaches can measure NLGN4X-mediated synaptic effects?

To quantify NLGN4X-mediated synaptic effects, researchers can employ these sophisticated approaches:

• Electrophysiological Recordings:

  • Whole-cell patch-clamp recordings to measure changes in excitatory postsynaptic currents (EPSCs) or inhibitory postsynaptic currents (IPSCs)

  • Paired recordings can assess the strength of specific synaptic connections

  • These techniques can directly measure functional consequences of NLGN4X expression or mutation

• High-Content Imaging of Synaptogenesis:

  • Co-culture assays where NLGN4X-expressing non-neuronal cells are cultured with neurons to quantify induced presynaptic differentiation

  • Automated microscopy with synaptic marker quantification in primary neurons expressing wild-type or mutant NLGN4X

  • Use of synapse-specific markers like vGLUT1 (excitatory) or vGAT (inhibitory) to determine synapse type specificity

• Live Imaging of Surface Trafficking:

  • pH-sensitive GFP tags (pHluorin) fused to NLGN4X to visualize surface insertion events in real-time

  • FRAP (Fluorescence Recovery After Photobleaching) to measure lateral mobility and clustering dynamics

• Super-Resolution Microscopy:

  • STORM/PALM imaging to visualize nanoscale organization of NLGN4X at synapses

  • Dual-color super-resolution to measure co-localization with other synaptic proteins at nanometer resolution

• Biochemical Assays:

  • Proximity ligation assays to detect protein-protein interactions in situ

  • Co-immunoprecipitation followed by mass spectrometry to identify the NLGN4X interactome under different conditions

These methodologies provide complementary data on how NLGN4X influences synaptic development, function, and plasticity in both physiological and pathological contexts.

How can I investigate the differential interaction of NLGN4X and NLGN4Y with neurexins?

To investigate differential interactions between NLGN4X/Y and neurexins, implement these specialized approaches:

• In vitro Binding Assays:

  • Purify the extracellular domains of NLGN4X, NLGN4Y, and neurexin variants

  • Perform surface plasmon resonance (SPR) or bio-layer interferometry to measure binding kinetics and calcium dependence

  • Compare association/dissociation rates and binding affinities quantitatively

• Structural Studies:

  • X-ray crystallography of NLGN4X and NLGN4Y ECDs with and without neurexin binding

  • Focus on the hydrophilic, calcium-dependent interface through which NLGN4X interacts with β-neurexin

  • Molecular modeling of the critical P93S difference and its effects on protein structure

• Cell-Based Interaction Assays:

  • Cell adhesion assays with cells expressing neurexins adhering to NLGN4X or NLGN4Y-expressing cells

  • Use split GFP or FRET-based reporters to visualize interactions in living cells

  • Manipulate calcium levels to assess the calcium-dependence of interactions

• Proteomic Analysis:

  • Utilize label-free LC-MS/MS to compare the interactomes of NLGN4X and NLGN4Y

  • Focus on neurexin binding partners and associated scaffolding proteins

  • Quantitative analysis can reveal differences in protein complex formation

• Mutagenesis Studies:

  • Generate chimeric proteins swapping domains between NLGN4X and NLGN4Y

  • Create point mutations at the critical P93S position and surrounding residues

  • Assess the effect of mutations on neurexin binding and downstream signaling

These approaches provide complementary data on the molecular determinants of differential neurexin binding and signaling between NLGN4X and NLGN4Y.

What factors might affect the detection of NLGN4X in experimental samples?

Several factors can significantly influence NLGN4X detection:

• Sample Preparation Issues:

  • Protein degradation due to inadequate protease inhibition

  • Insufficient denaturation for Western blotting, particularly important as NLGN4X forms dimers via hydrophobic interfaces

  • Incomplete extraction from membrane fractions due to its transmembrane nature

• Antibody-Related Factors:

  • Cross-reactivity with other neuroligins (particularly NLGN3, which shares significant homology with NLGN4X)

  • Epitope masking due to protein-protein interactions or conformational changes

  • Batch-to-batch variability in antibody production affecting sensitivity and specificity

• Expression Level Variations:

  • Cell type-specific expression patterns

  • Developmental timing effects on expression

  • Sex differences (male samples may show confounding NLGN4Y signals)

• Post-translational Modifications:

  • Variable glycosylation affecting antibody recognition

  • Phosphorylation states altering protein mobility or epitope accessibility

  • Proteolytic processing generating fragments with different detection properties

• Technical Variables:

  • For IHC/ICC: fixation methods affecting epitope preservation

  • For flow cytometry: cell permeabilization efficiency influencing access to intracellular epitopes

  • For Western blotting: transfer efficiency for this high molecular weight protein

Understanding these variables is crucial for experimental design and troubleshooting when inconsistent results occur.

How should I interpret differences in mature versus immature NLGN4X band patterns?

The interpretation of mature versus immature NLGN4X band patterns provides valuable insights into protein processing:

This interpretation framework allows researchers to extract mechanistic insights from what might otherwise appear to be simple differences in band patterns.

What are common pitfalls in NLGN4X research and how can they be avoided?

Researchers studying NLGN4X should be aware of these common pitfalls and their solutions:

• Antibody Cross-Reactivity Issues:

  • Pitfall: Antibodies may cross-react with other neuroligin family members, particularly NLGN3 which shares significant homology .

  • Solution: Validate antibody specificity using overexpression systems with all neuroligin family members. The search results indicate that R&D Systems antibody showed <5% cross-reactivity with recombinant human NLGN3 in direct ELISAs .

• Confusing NLGN4X and NLGN4Y Signals:

  • Pitfall: In male-derived samples, NLGN4Y signals may be misinterpreted as NLGN4X.

  • Solution: Use sex-specific controls and NLGN4X/Y-specific antibodies. Validate findings using RT-PCR with specific primers .

• Improper Controls in Mutation Studies:

  • Pitfall: Comparing mutant NLGN4X directly to wild-type without accounting for expression level differences.

  • Solution: Normalize protein levels, perform mRNA quantification, and include NLGN4Y as a biological control for processing deficiency .

• Overlooking Surface Expression:

  • Pitfall: Focusing only on total protein levels without assessing functional surface expression.

  • Solution: Always complement Western blot analysis with surface biotinylation assays or live-cell surface labeling techniques .

• Species Differences:

  • Pitfall: Applying findings from mouse models directly to human NLGN4X function.

  • Solution: Note that human NLGN4X ECD shares only 62% amino acid identity with mouse NLGN4, indicating potential functional differences. Use human neurons derived from iPSCs for more translational studies .

• Expression System Artifacts:

  • Pitfall: Overexpression systems may saturate cellular machinery, creating artificial trafficking bottlenecks.

  • Solution: Include dose-response experiments and validate key findings in systems with endogenous expression levels when possible.

Awareness of these pitfalls and implementing appropriate controls will significantly enhance the rigor and reproducibility of NLGN4X research.

What emerging technologies could enhance NLGN4X functional studies?

Several cutting-edge technologies offer promising avenues for advancing NLGN4X research:

• CRISPR-Based Approaches:

  • CRISPR/Cas9 genome editing to generate isogenic human iPSC lines with NLGN4X mutations

  • CRISPRa/CRISPRi for endogenous gene modulation without overexpression artifacts

  • CRISPR base editors for precise introduction of autism-associated point mutations

• Advanced Imaging Technologies:

  • Live super-resolution microscopy to track NLGN4X dynamics at synapses in real-time

  • Expansion microscopy for enhanced visualization of synaptic proteins

  • Lattice light-sheet microscopy for long-term imaging of NLGN4X trafficking with minimal phototoxicity

• Organoid and 3D Culture Systems:

  • Brain organoids to study NLGN4X function in complex human neural networks

  • Microfluidic devices to examine NLGN4X's role in circuit formation

  • 3D bioprinting of neural tissues with controlled NLGN4X expression patterns

• Single-Cell Approaches:

  • Single-cell transcriptomics to profile cell type-specific NLGN4X expression

  • Single-cell proteomics to examine NLGN4X interactome heterogeneity

  • Patch-seq to correlate NLGN4X expression with electrophysiological properties

• In Situ Structural Biology:

  • Cryo-electron tomography to visualize NLGN4X-neurexin complexes in their native environment

  • In-cell NMR to probe NLGN4X structural dynamics and interactions

  • Mass photometry for single-molecule characterization of NLGN4X complexes

These emerging technologies promise to overcome current limitations in understanding NLGN4X biology and pathology, potentially leading to therapeutic strategies for associated neurodevelopmental disorders.

How might NLGN4X research inform therapeutic approaches for autism spectrum disorders?

NLGN4X research has significant potential to inform novel therapeutic strategies for autism spectrum disorders (ASD):

• Protein Trafficking Enhancement:

  • Small molecules that facilitate proper folding of mutant NLGN4X proteins

  • Pharmacological chaperones that stabilize specific conformations to promote ER exit

  • These approaches could target the processing deficits observed in autism-associated NLGN4X variants that phenocopy NLGN4Y

• Post-translational Modification Modulation:

  • Targeting specific phosphorylation sites that regulate spinogenesis

  • Modulating glycosylation pathways to enhance NLGN4X maturation

  • These interventions could potentially rescue functional deficits in mutant proteins

• Synaptic Function Modulation:

  • Compounds that enhance or mimic NLGN4X-neurexin interactions

  • Modulation of downstream signaling pathways to compensate for NLGN4X dysfunction

  • These approaches address the fundamental synaptic abnormalities in ASD

• Gene Therapy Approaches:

  • CRISPR-based repair of specific NLGN4X mutations

  • Viral delivery of wild-type NLGN4X to overcome haploinsufficiency

  • Antisense oligonucleotides to modulate NLGN4X splicing or expression

• Compensatory Mechanisms:

  • Targeting other neuroligin family members to compensate for NLGN4X dysfunction

  • Enhancing NLGN4Y function in males could provide sex-specific therapeutic strategies

  • This approach acknowledges the redundancy and compensation within the neuroligin family