Recombinant Human Motile sperm domain-containing protein 1 (MOSPD1)

What is the basic structure and cellular localization of MOSPD1?

MOSPD1 is a protein containing a Motile Sperm (MSP) domain, which is structurally and functionally similar to the MSP domains found in other VAP family proteins like VAPA and VAPB. The protein contains conserved residues that form hydrophobic pockets, including V51, T54, V61, N64, K94, F95, and K125, which are critical for its interaction with binding partners .

Unlike VAPA and VAPB which are primarily endoplasmic reticulum (ER)-resident proteins, MOSPD1 is found in various cellular compartments. Immunohistochemical staining has shown that MOSPD1 can be localized in both the cytoplasm and nucleus of cells, particularly in tumorous cells of colorectal cancer tissues . This dual localization suggests MOSPD1 may have multiple functions depending on its cellular context.

How does MOSPD1 differ from other members of the VAP protein family?

MOSPD1 represents a divergent member of the VAP protein family. While it shares the characteristic MSP domain with VAPA, VAPB, and MOSPD2, the MOSPD1 MSP domain has evolved to recognize different binding motifs. Specifically, MOSPD1 preferentially interacts with FFNT (two phenylalanines in a neutral tract) motifs, whereas VAPA, VAPB, and MOSPD2 primarily bind to FFAT (two phenylalanines in an acidic tract) motifs .

This difference in binding preference is attributed to variations in the amino acid residues that form electrostatic bridges with binding partners. The residues K52 and R62 in MOSPD1 are less conserved compared to other VAP family members, corresponding with the observation that FFNT motifs preferred by MOSPD1 have fewer acidic elements . This specialized binding preference suggests that MOSPD1 has evolved to interact with a unique set of partner proteins, potentially mediating distinct cellular functions.

What is the tissue expression pattern of MOSPD1?

According to the GTEx Portal database, MOSPD1 is expressed in a diverse range of normal human tissues including esophageal mucosa, adrenal gland, testis, skin, and uterus . This broad expression pattern suggests MOSPD1 likely plays physiological roles in multiple tissue types.

In mice, Mospd1 is abundantly expressed in mesenchymal tissues, and its expression increases during differentiation in osteoblastic, myoblastic, and adipocytic cell lines . This expression pattern aligns with findings that Mospd1 is involved in the differentiation and proliferation of mesenchymal cells, indicating its potential importance in tissue development and homeostasis.

How is MOSPD1 regulated by the Wnt/β-catenin signaling pathway?

MOSPD1 has been identified as a target gene of the Wnt/β-catenin signaling pathway, particularly in colorectal cancer. The regulatory mechanism involves a novel enhancer element located in the 3'-flanking region of the MOSPD1 gene. This enhancer element contains three putative TCF-binding motifs that interact with the β-catenin/TCF7L2 transcriptional complex .

Experimentally, this regulation has been demonstrated through several approaches:

• siRNA-mediated knockdown of β-catenin in SW480 and HCT116 colorectal cancer cells suppressed MOSPD1 expression.

• Induction of β-catenin in HeLa cells treated with LiCl (30 and 100 mM) increased MOSPD1 expression.

• Chromatin immunoprecipitation (ChIP) assays confirmed the interaction between TCF7L2 and the 3'-enhancer region of MOSPD1, with a 10.3-fold enrichment in TCF7L2 precipitants compared to normal IgG controls .

• Chromatin conformation capture assays revealed that this 3'-flanking region physically interacts with the MOSPD1 promoter, facilitating transcriptional activation .

These findings establish MOSPD1 as a novel downstream target of the Wnt/β-catenin pathway, suggesting its potential involvement in Wnt-dependent biological processes including development and tumorigenesis.

What evidence supports the correlation between MOSPD1 expression and colorectal cancer?

Multiple lines of evidence support the upregulation of MOSPD1 in colorectal cancer:

• Analysis of gene expression data from the GSE21510 dataset (containing 104 CRC tissues and 25 non-tumorous colonic tissues) revealed that MOSPD1 expression was 2.18-fold higher (q-value: 3.05E-13) in tumor tissues compared to non-tumorous tissues .

• MOSPD1 expression levels showed positive correlation with known Wnt target genes: RNF43 (r=0.63), AXIN2 (r=0.55), and MYC (r=0.54), further supporting its regulation by the Wnt signaling pathway .

• Immunohistochemical staining of 11 CRC tissues demonstrated positive MOSPD1 staining in the cytoplasm and/or nucleus of tumorous cells in all cases tested, coinciding with β-catenin staining patterns .

This correlation suggests that MOSPD1 upregulation may contribute to colorectal cancer development and progression, potentially through mechanisms related to abnormal Wnt pathway activation.

What experimental approaches can be used to identify novel MOSPD1 interaction partners?

To identify novel interaction partners of MOSPD1, researchers can employ several complementary approaches:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged recombinant MOSPD1 (e.g., with FLAG, HA, or His tag) in relevant cell lines

  • Perform pull-down assays using antibodies against the tag

  • Analyze co-precipitated proteins by mass spectrometry

  • Validate interactions through co-immunoprecipitation with antibodies against endogenous proteins

• Yeast two-hybrid screening:

  • Use the MSP domain or full-length MOSPD1 as bait

  • Screen against a library of prey constructs from relevant tissues

  • Validate positive interactions with alternative methods

• Proximity-labeling approaches:

  • Fuse MOSPD1 with BioID or APEX2 enzymes

  • Express the fusion protein in cells and provide biotin

  • Identify biotinylated proximal proteins by streptavidin pull-down and mass spectrometry

• In silico prediction:

  • Use the available FFAT/FFNT motif search algorithms to identify potential binding partners

  • Prioritize candidates based on biological relevance and subcellular localization

  • Validate predicted interactions experimentally

• Protein microarrays:

  • Screen recombinant MOSPD1 against protein microarrays containing thousands of human proteins

  • Identify binding partners through detection of protein-protein interactions

When validating interactions, researchers should consider using domain mapping to identify specific regions involved in the interaction and mutagenesis studies to confirm the importance of the FFNT motifs in the interaction partners.

What role does MOSPD1 play in mesenchymal cell differentiation?

MOSPD1 appears to play a critical role in mesenchymal cell differentiation, as evidenced by several experimental findings:

• In mice, Mospd1 is abundantly expressed in mesenchymal tissues, and its expression increases during differentiation in osteoblastic, myoblastic, and adipocytic cell lines .

• Mospd1-null embryonic stem cells, while able to proliferate, are unable to differentiate into osteoblasts, adipocytes, and hematopoietic progenitors . This finding strongly suggests that Mospd1 is essential for the differentiation of mesenchymal lineages.

• Knockdown of Mospd1 in MC3T3-E1 cells (established from mouse osteoblasts) resulted in downregulation of Runx2 and Osteocalcin , two key transcription factors required for osteoblastic differentiation. This indicates that MOSPD1 may regulate the expression of genes essential for osteoblast differentiation.

These observations collectively suggest that MOSPD1 functions as a regulator of mesenchymal cell differentiation, potentially by modulating the expression of lineage-specific transcription factors and differentiation markers.

How might MOSPD1 contribute to epithelial-mesenchymal transition (EMT)?

Evidence suggests MOSPD1 may play a role in epithelial-mesenchymal transition (EMT), a process crucial for cancer invasion and metastasis:

• Knockdown of Mospd1 in MC3T3-E1 cells induced the expression of epithelial cadherin Cdh1 while decreasing the expression of Snai1, Snai2, and mesenchymal cadherin Cdh11 . This expression pattern is consistent with a reversal of EMT, suggesting that MOSPD1 normally promotes EMT.

• Ovarian cancer cells with high-invasion phenotypes expressed significantly increased levels of MOSPD1 compared to cells with low-invasion phenotypes , supporting a potential role for MOSPD1 in promoting cancer cell invasion.

• The Wnt signaling pathway, which regulates MOSPD1 expression, is known to induce EMT in cancer . This suggests that MOSPD1 might be one of the downstream effectors through which Wnt signaling promotes EMT.

These findings indicate that MOSPD1 might contribute to EMT by regulating the expression of EMT-related transcription factors (Snai1, Snai2) and cadherins, potentially facilitating cancer invasion and metastasis. Further research is needed to elucidate the precise mechanisms by which MOSPD1 modulates the EMT program.

What are the optimal conditions for expressing and purifying recombinant human MOSPD1?

For optimal expression and purification of recombinant human MOSPD1, researchers should consider the following methodological guidelines:

Expression System Selection:

• Bacterial systems (E. coli): Suitable for expressing the MSP domain alone due to its relatively small size and lack of post-translational modifications

• Eukaryotic systems (mammalian cells, insect cells): Preferable for full-length MOSPD1 to ensure proper folding and potential post-translational modifications

Expression Optimization in E. coli:

• Vector: pET-based vectors with T7 promoter for high-level expression

• Host strain: BL21(DE3) or Rosetta(DE3) for rare codon usage

• Fusion tags: 6xHis or GST tags at the N-terminus to facilitate purification

• Induction conditions:

  • Temperature: Lower temperature (16-18°C) for overnight induction to enhance solubility

  • IPTG concentration: 0.1-0.5 mM depending on construct

  • OD600: Induce at 0.6-0.8 for optimal expression

Purification Strategy:

• Affinity chromatography: Ni-NTA for His-tagged proteins or glutathione sepharose for GST-tagged proteins

• Size exclusion chromatography: To remove aggregates and ensure monodisperse protein preparation

• Buffer optimization: PBS or Tris-based buffers (pH 7.4-8.0) with 150-300 mM NaCl and 5-10% glycerol for stability

Quality Control:

• SDS-PAGE and Western blot to confirm purity and identity

• Dynamic light scattering to assess monodispersity

• Circular dichroism to verify proper folding

• Functional assays: Binding studies with synthetic FFNT peptides to confirm activity

For studies requiring the interaction between MOSPD1 and its binding partners, co-expression systems or in vitro reconstitution approaches should be considered to form stable complexes.

What experimental approaches can be used to study the enhancer activity of the 3'-flanking region of MOSPD1?

To investigate the enhancer activity of the 3'-flanking region of MOSPD1, researchers can employ several complementary experimental approaches:

1. Reporter Assays:

• Clone the putative enhancer region upstream of a minimal promoter driving a reporter gene (luciferase or GFP)

• Transfect the construct into relevant cell lines (e.g., colorectal cancer cell lines)

• Measure reporter activity under basal conditions and following Wnt pathway modulation

• Create mutants of the TCF-binding motifs to confirm their functional importance

2. Chromatin Immunoprecipitation (ChIP):

• Perform ChIP using antibodies against TCF7L2, β-catenin, and histone marks associated with active enhancers (H3K27ac, H3K4me1)

• Design primers specific to the 3'-flanking region of MOSPD1

• Quantify enrichment using qPCR, as was done in the study showing 10.3-fold enrichment of the MOSPD1 3'-enhancer region in TCF7L2 precipitants

• Consider ChIP-seq for genome-wide analysis of binding sites

3. Chromosome Conformation Capture (3C) and Derivatives:

• Use 3C to confirm physical interaction between the enhancer and the MOSPD1 promoter

• Consider 4C or Hi-C for more comprehensive analysis of chromatin interactions

• Quantify interaction frequency under normal conditions and following Wnt pathway modulation

4. CRISPR-Based Approaches:

• Use CRISPR/Cas9 to delete the enhancer region or introduce specific mutations in the TCF-binding motifs

• Assess the impact on MOSPD1 expression using qRT-PCR or Western blotting

• For more precise analysis, use CRISPRi (dCas9-KRAB) to silence the enhancer activity without altering the DNA sequence

5. In Vivo Enhancer Testing:

• Generate transgenic models (e.g., zebrafish) with the enhancer region driving reporter gene expression

• Analyze spatial and temporal patterns of reporter expression

• Assess the response to Wnt pathway modulation

By combining these approaches, researchers can comprehensively characterize the enhancer activity of the 3'-flanking region of MOSPD1 and its regulation by the Wnt/β-catenin signaling pathway.

How might MOSPD1 dysregulation contribute to cancer progression?

MOSPD1 dysregulation may contribute to cancer progression through several potential mechanisms:

• Promotion of EMT: MOSPD1 appears to regulate EMT-related genes, including Snai1, Snai2, and cadherins . Overexpression of MOSPD1 might promote EMT, enhancing cancer cell invasion and metastasis. This is supported by the observation that ovarian cancer cells with high invasion phenotypes express increased levels of MOSPD1 .

• Wnt/β-catenin signaling amplification: As a target of the Wnt/β-catenin pathway, MOSPD1 upregulation in cancers with aberrant Wnt activation (such as colorectal cancer) might amplify oncogenic signaling through positive feedback mechanisms or by modulating other Wnt target genes.

• Mesenchymal differentiation modulation: MOSPD1's role in mesenchymal differentiation suggests it might influence tumor stroma formation or cancer stem cell properties, potentially affecting tumor microenvironment and therapy resistance.

• Membrane contact site (MCS) disruption: As a member of the VAP family involved in forming membrane contact sites, MOSPD1 dysregulation might alter lipid transfer between organelles, potentially affecting cancer cell metabolism or signaling.

Evidence supporting MOSPD1's role in cancer comes from gene expression data showing 2.18-fold higher expression in colorectal tumors compared to normal tissues, positive correlation with known Wnt targets (RNF43, AXIN2, MYC), and positive immunohistochemical staining in all tested CRC cases .

Are there any known genetic variants of MOSPD1 associated with human diseases?

Although research on MOSPD1 genetic variants is limited, there is at least one documented case involving MOSPD1 gene duplication associated with developmental abnormalities:

A duplication of the Xq26.1-26.3 region, which includes the MOSPD1 and GPC3 genes, was identified in a boy with short stature and double outlet right ventricle (a congenital heart defect) . This suggests that altered MOSPD1 dosage might contribute to developmental disorders, particularly those affecting growth and cardiac development.

• Mesenchymal cell differentiation

• EMT and cancer progression

• Wnt/β-catenin signaling

• Membrane contact site formation

It's reasonable to hypothesize that genetic variants affecting MOSPD1 expression or function might influence susceptibility to:

• Developmental disorders

• Cancer predisposition or progression

• Disorders of bone, muscle, or adipose tissue development

• Fibrotic conditions

Future genomic studies, particularly whole-exome or whole-genome sequencing of patients with relevant phenotypes, may uncover additional MOSPD1 variants with clinical significance.

What are the most promising approaches to elucidate the complete function of MOSPD1?

To comprehensively understand MOSPD1 function, several complementary research approaches should be pursued:

• Structural biology:

  • Determine the crystal or cryo-EM structure of MOSPD1, particularly its MSP domain

  • Compare with structures of other VAP family proteins to understand binding specificity

  • Co-crystallize with FFNT-containing peptides to elucidate interaction mechanisms

• Proteomics and interactomics:

  • Identify the complete interactome of MOSPD1 using proximity labeling approaches (BioID, APEX)

  • Characterize tissue-specific interactomes in normal and disease states

  • Validate key interactions through detailed biochemical and structural studies

• Genetic models:

  • Generate conditional knockout mouse models to assess tissue-specific functions

  • Use CRISPR/Cas9 to create cell lines with MOSPD1 mutations affecting specific domains

  • Develop zebrafish or Drosophila models for developmental studies

• Single-cell approaches:

  • Perform single-cell RNA-seq to identify cell populations with high MOSPD1 expression

  • Use spatial transcriptomics to map MOSPD1 expression patterns in tissues

  • Combine with lineage tracing to understand MOSPD1's role in differentiation

• Membrane contact site biology:

  • Characterize MOSPD1's role in forming membrane contact sites

  • Identify specific lipids or molecules transferred at MOSPD1-mediated contact sites

  • Compare with other VAP family members to understand functional specialization

• Clinical correlations:

  • Analyze MOSPD1 expression across various cancer types and stages

  • Correlate with clinical outcomes and response to therapies

  • Assess potential as a biomarker or therapeutic target

How might targeting MOSPD1 be exploited for therapeutic development?

Based on current understanding, several strategies for therapeutic targeting of MOSPD1 could be explored:

• Direct inhibition approaches:

  • Develop small molecules that bind to MOSPD1's MSP domain, preventing interaction with FFNT-containing proteins

  • Design peptide mimetics that compete for binding with natural FFNT motifs

  • Create aptamers that selectively bind and inhibit MOSPD1 function

• Transcriptional regulation:

  • Target the Wnt/β-catenin pathway to modulate MOSPD1 expression in cancers

  • Develop antisense oligonucleotides or siRNAs for selective MOSPD1 knockdown

  • Use CRISPR interference to suppress MOSPD1 enhancer activity

• Cancer applications:

  • In colorectal cancers with upregulated MOSPD1, inhibition might reduce EMT and metastatic potential

  • Combination with Wnt pathway inhibitors could provide synergistic effects

  • Monitoring MOSPD1 expression could serve as a biomarker for Wnt pathway activation

• Developmental disorders:

  • For conditions associated with MOSPD1 overexpression (as in the Xq26.1-26.3 duplication case ), targeted reduction might ameliorate symptoms

  • Gene therapy approaches could normalize MOSPD1 expression in affected tissues

• Differentiation therapy:

  • Modulation of MOSPD1 activity might influence mesenchymal differentiation

  • Potential applications in regenerative medicine, particularly for osteoblast, adipocyte, or hematopoietic lineages

Before pursuing therapeutic development, additional research is needed to:

• Fully characterize MOSPD1's physiological functions

• Identify tissue-specific roles to anticipate potential side effects

• Develop appropriate model systems for preclinical testing

• Establish clear disease contexts where MOSPD1 modulation would be beneficial