Recombinant Human Motile sperm domain-containing protein 3 (MOSPD3)

What is MOSPD3 and what is its role in cellular biology?

MOSPD3 (Motile Sperm Domain-Containing Protein 3) is a member of the expanded VAP (VAMP-associated protein) family that plays a role in membrane contact site (MCS) formation between organelles. Unlike VAPA, VAPB, and MOSPD2 which interact with FFAT motifs, MOSPD3 preferentially binds to proteins containing FFNT (two phenylalanines in a Neutral Tract) motifs . MOSPD3 contains an MSP (Major Sperm Protein) domain that mediates these protein-protein interactions. Current research suggests MOSPD3 functions in the MOSPD1-MOSPD3 complex segregated from the VAPA-VAPB-MOSPD2 complex, potentially serving distinct cellular functions in membrane tethering .

How does MOSPD3 differ structurally from other members of the VAP family?

MOSPD3 contains an MSP domain that diverges significantly from those in VAPA, VAPB, and MOSPD2. While the hydrophobic pocket residues that accommodate the F2 and A5 positions of interaction motifs are largely conserved (V51, T54, V61, N64, K94, F95, and K125 in MOSPD1/MOSPD3), the residues that form electrostatic bridges with acidic elements are less conserved (such as K52 and R62) . This explains MOSPD3's preference for FFNT motifs rather than the acidic FFAT motifs preferred by VAPA/B and MOSPD2. The similarity between human MOSPD1 and human MOSPD3 is approximately 32% at protein levels , indicating significant evolutionary divergence while maintaining core functional domains.

What are the recommended expression systems for recombinant MOSPD3 production?

Based on available research, recombinant MOSPD3 can be successfully expressed in several systems:

For structural studies requiring proper folding and post-translational modifications, mammalian expression systems like HEK-293 cells are recommended . For basic binding studies or epitope mapping, E. coli expression may be sufficient. The choice of expression system should be determined by the specific downstream application and required protein quality .

What purification strategies are most effective for recombinant MOSPD3?

A standard purification protocol for His-tagged MOSPD3 involves:

• Cell lysis in 50 mM Tris (pH 8), 300 mM NaCl, 1% Triton X-100, 10 mM 2-mercaptoethanol, 10 mM MgCl₂, with lysozyme and benzonase nuclease

• Sonication (30% amplitude, 10-second pulses on/15-second pulses off)

• IMAC NTA affinity chromatography

• Elution with 500 mM imidazole in TBK buffer (pH 8.5) with 50% glycerol

• Concentration using a 10 kDa cutoff device

• Desalting against 40 mM Tris (pH 8.0), 100 mM KCl, and 10% glycerol

Protein purity should be confirmed by SDS-PAGE under reducing conditions (50 mM DTT) . For Strep-tagged variants, Strep-Tactin columns can be used following similar principles. Concentration can be calculated from absorbance at 280 nm, with expected concentrations greater than 0.5 mg/ml for properly expressed and purified protein .

How does MOSPD3 contribute to membrane contact site formation and what methodologies can be used to study this function?

MOSPD3 appears to function alongside MOSPD1 in a distinct complex from the VAPA-VAPB-MOSPD2 complex that mediates membrane contact sites. To study MOSPD3's role in MCS formation, researchers can employ several approaches:

• Cryo-electron tomography (cryo-ET): This technique allows visualization of intact membrane structures and protein complexes at MCS. As demonstrated with VAP-A , cryo-ET can reveal the architecture of tethering complexes at nanoscale resolution.

• In vitro membrane tethering assays: Using a system similar to that employed for MOSPD2 , researchers can assess MOSPD3's tethering capabilities:

  • Prepare two liposome populations: one bearing FFNT-containing peptides (LA) and another covered by recombinant MOSPD3 (LB)

  • Monitor tethering by dynamic light scattering (DLS)

  • Measure increases in particle size as evidence of tethering

  • Compare wild-type MOSPD3 with predicted binding-deficient mutants

• Proximity labeling with BioID or TurboID: Fusing MOSPD3 with promiscuous biotin ligases can identify proteins in close proximity at MCS .

• FRET/BRET assays: These can measure real-time interactions between MOSPD3 and potential partners at MCS in live cells.

Current understanding suggests MOSPD3 may be involved in forming specific types of MCS, with a preference for FFNT motif-containing proteins rather than the FFAT motif interactions that characterize VAPA/B and MOSPD2 .

What methodologies are recommended for studying MOSPD3 interactions with other proteins in the context of membrane biology?

To systematically study MOSPD3's interactions with other proteins, particularly in the context of membrane biology, several methodologies are recommended:

• ChIP-quantitative PCR: This approach was successfully used to study protein-DNA interactions for related proteins and can be adapted to study MOSPD3 if it has any chromatin-associated functions.

• Co-immunoprecipitation coupled with mass spectrometry:

  • Express tagged MOSPD3 in relevant cell lines

  • Perform immunoprecipitation using tag-specific antibodies

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate specific interactions with Western blotting

• GFP-Trap and immunoprecipitation: For studying MOSPD3 interactions in living cells, GFP-tagged MOSPD3 can be expressed and pulled down using GFP-Trap beads .

• Chromatin conformation capture assay: If MOSPD3 has potential roles in nuclear functions like its related protein MOSPD1 .

• Reconstituted membrane systems:

  • Incorporate MOSPD3 into proteoliposomes

  • Mix with liposomes containing potential interacting proteins

  • Assess interaction through liposome tethering assays

  • Visualize using cryo-electron microscopy

• Biomolecular fluorescence complementation (BiFC): Split fluorescent proteins fused to MOSPD3 and potential partners can visualize interactions in live cells.

When interpreting results, consider that MOSPD3 belongs to a family of proteins with similar functions, so specificity controls using related proteins (VAPA, VAPB, MOSPD1, MOSPD2) are essential to differentiate specific from general interactions.

How can researchers determine if MOSPD3 is implicated in disease pathologies, particularly neurodegenerative disorders?

Given MOSPD3's potential role in membrane contact sites and its mention in Alzheimer's disease-related research , several methodologies can be employed to investigate its involvement in disease states:

• Expression analysis in pathological samples:

  • Perform immunohistochemical staining of MOSPD3 in disease versus control tissues

  • Conduct RT-qPCR to quantify MOSPD3 transcript levels

  • Analyze publicly available transcriptomic datasets for differential expression in disease conditions

• Meta-analysis of gene expression data:

  • Integrate existing microarray and RNA-Seq datasets from human disease samples

  • Compare MOSPD3 expression across different neurological disorders

  • Calculate correlation coefficients between MOSPD3 and known disease genes

• Genetic association studies:

  • Screen for MOSPD3 mutations or SNPs in patient cohorts

  • Perform case-control studies to identify potential associations

  • Use existing GWAS datasets to look for MOSPD3 locus associations

• Functional assays in disease models:

  • Develop MOSPD3 knockout or knockdown models in relevant cell types

  • Assess effects on disease-relevant phenotypes (protein aggregation, etc.)

  • Perform rescue experiments with wild-type versus mutant MOSPD3

• Interactome analysis in disease context:

  • Identify MOSPD3 interaction partners in healthy versus diseased states

  • Look for altered interactions with disease-associated proteins

  • Determine if disease-associated proteins contain FFNT motifs

In Alzheimer's disease research, MOSPD3 was identified as a gene perturbed in both asymptomatic AD brain and AD patient blood samples , suggesting it may serve as part of a biomarker panel for early disease detection.

What are the optimal experimental approaches for designing site-directed mutagenesis studies of MOSPD3?

Based on structure-function analyses of related proteins, a methodical approach to MOSPD3 mutagenesis includes:

• Sequence alignment and structural prediction:

  • Align MOSPD3 with VAPA, VAPB, MOSPD1, and MOSPD2

  • Identify conserved residues in the MSP domain

  • Focus on residues likely involved in FFNT binding based on homology

• Selection of mutation targets:

  • Key residues in the hydrophobic pocket (V51, T54, V61, N64, K94, F95, K125)

  • Residues forming electrostatic interactions (K52, R62)

  • The equivalent of RD/LD mutant sites identified in MOSPD2

• Mutagenesis protocol:

  • Use KOD-Plus-Neo polymerase for amplification

  • Design mutagenic primers with 15-20 base pair overlaps

  • Digest with DpnI to remove template DNA

  • Transform into E. coli

  • Confirm mutations by Sanger sequencing

• Functional validation:

  • Express wild-type and mutant proteins

  • Compare binding to FFNT-containing peptides using pull-down assays

  • Measure binding affinities using SPR

  • Assess membrane tethering capabilities in vitro

  • Evaluate cellular localization and function in vivo

What experimental design would best elucidate the distinct functions of MOSPD3 versus other VAP family members?

To distinguish MOSPD3's specific functions from other VAP family members, a comprehensive experimental approach is required:

• Comparative depletion studies:

  • Generate single and combinatorial knockdowns/knockouts of VAP family proteins

  • Assess effects on different MCS using quantitative electron microscopy

  • Measure changes in lipid transfer and calcium signaling at MCS

  • Evaluate cellular phenotypes specific to each depletion

• Rescue experiments:

  • Deplete endogenous MOSPD3 and attempt rescue with:

    • Wild-type MOSPD3

    • Chimeric proteins (MOSPD3 MSP domain with VAPA/B transmembrane domain)

    • MOSPD3 with mutations in key binding residues

  • Quantify restoration of MCS formation and function

• Proteomics approach:

  • Perform IP-MS on each VAP family member

  • Create Venn diagrams of interacting proteins

  • Identify MOSPD3-specific versus shared interactors

  • Validate key interactions with co-IP and functional assays

• Interactome mapping:

  • Use BioID or TurboID proximity labeling with each VAP family member

  • Compare proximity proteomes to identify distinct vs. overlapping networks

  • Validate with orthogonal methods (FRET, co-IP)

• Subcellular localization analysis:

  • Perform super-resolution microscopy to map precise localization of each VAP member

  • Identify unique vs. overlapping distribution patterns

  • Correlate with MCS markers and partner proteins

Based on current evidence, MOSPD3 operates alongside MOSPD1 in a distinct complex from the VAPA-VAPB-MOSPD2 complex, with different binding preferences (FFNT vs. FFAT motifs) . This experimental design would help clarify whether this translates to distinct cellular functions and unique sets of interaction partners.