MSMB Human

What is the molecular structure and biochemical characteristics of human β-microseminoprotein?

Human β-microseminoprotein (MSMB) is a cysteine-rich nonglycosylated protein with an apparent molecular mass of 15 kDa as determined by SDS-PAGE . It is among the most abundant proteins secreted by the prostate and present in human seminal plasma. MSMB has been identified in several species including human, baboon, rhesus monkey, rat, pig, and mouse, with variations in amino acid composition affecting protein charge and function . The protein's high cysteine content contributes to its structural stability, and it can be detected in Western immunoblot analysis as a distinct 15 kDa band in seminal plasma protein preparations . This protein is encoded by a gene mapped to chromosome 10q11.2 as confirmed through fluorescence in situ hybridization techniques .

How is MSMB expressed across different human tissues?

While MSMB is primarily expressed in the prostate, quantitative studies have detected variable expression across multiple tissues. Using TaqMan real-time RT-PCR assays with absolute quantitation methods, researchers have observed the highest expression in normal human prostate tissue compared to other tissues and cell lines . In prostate cancer cell lines, MSMB expression varies significantly, with some lines showing robust expression (including PCa2b, NCI H660, PC3, LNCaP, and RWPE1) while others exhibit minimal expression . The gene's expression appears to be regulated by hormones, as suggested by the presence of three glucocorticoid response elements and one estrogen response element in the promoter region of its first intron . This hormonal regulation has been confirmed in studies of rat lateral prostate and may explain tissue-specific expression patterns .

What are the proposed biological functions of MSMB in human reproduction?

Research has demonstrated several key functions of MSMB in human reproduction:

• Acrosome reaction regulation: Experimental studies show that MSMB inhibits spontaneous acrosome reaction in spermatozoa, potentially preventing premature sperm activation .

• Sperm surface binding: MSMB binds to the human sperm surface, primarily localizing to the acrosomal region of the head and neck, suggesting direct modulation of sperm function .

• Protein complex formation: MSMB forms a high-affinity complex with CRISP3 in human seminal plasma through the N-terminal 'sperm coating protein' (SCP) domain .

• Motility regulation: In macaques, MSMB appears to play a role in regulating hyperactivated motility during sperm capacitation, though human studies show different localization patterns .

• Potential immunomodulation: Some research suggests MSMB may function as an immunoglobulin binding factor, potentially affecting immune responses in the reproductive tract .

The varied distribution and functions of MSMB across tissues suggest it may have multiple physiological roles extending beyond reproduction.

What is the relationship between MSMB levels and male fertility parameters?

Quantitative analysis of MSMB in seminal plasma has revealed significant correlations with male fertility parameters. Research examining seminal plasma from fertile donors and subfertile patients found significantly increased MSMB levels in subfertile groups compared to fertile subjects (p<0.02) .

*p<0.02 compared to fertile controls

This elevation in subfertile men suggests either altered prostate synthesis/secretion of MSMB or reduced binding of MSMB molecules to spermatozoa. Notably, pathologic groups with elevated MSMB levels shared characteristics of low percentages of morphologically normal spermatozoa and reduced motility, establishing MSMB as a potential biomarker for semen quality assessment .

How does MSMB interact with human spermatozoa at the molecular level?

The interaction between MSMB and human spermatozoa has been characterized using various molecular techniques. Using indirect immunofluorescence with specific polyclonal antibodies, researchers have demonstrated that MSMB binds to specific sites on the human sperm surface, primarily localizing to the acrosomal region of the sperm head and the neck . This binding pattern differs from that observed in macaque sperm, where MSMB appears to be associated with the flagellum, highlighting species-specific differences in MSMB-sperm interactions .

The molecular mechanism of this binding likely involves specific receptor proteins on the sperm surface, though these receptors have not been fully characterized. The binding appears to be functionally significant, as experimental data shows that preincubation of spermatozoa with MSMB results in significant inhibition of spontaneous acrosome reaction . This inhibitory effect suggests that MSMB plays a regulatory role in maintaining sperm in an uncapacitated state until appropriate physiological signals are encountered, a crucial function for preserving fertilization potential .

What methodologies are most effective for measuring MSMB in reproductive research?

Several complementary methodologies have been developed for MSMB quantification in reproductive research:

• Double-site ELISA: A sandwich ELISA using rabbit IgG as capture antibody and rat anti-MSMB as detection antibody provides sensitive and specific quantification of MSMB in seminal plasma and other biological fluids . This assay can detect MSMB concentrations ranging from 0.579–10.751 mg/ml in seminal plasma .

• Western immunoblot analysis: This technique confirms MSMB identity in biological samples, showing a characteristic 15 kDa band in seminal plasma proteins .

• Indirect immunofluorescence: For localization studies, this technique visualizes MSMB binding to spermatozoa, revealing specific binding patterns on the sperm surface .

• Quantitative RT-PCR: TaqMan real-time RT-PCR assays enable absolute quantitation of MSMB transcript levels in tissues and cell lines, allowing correlation analysis with other gene expression patterns .

• Functional assays: To assess the biological activity of MSMB, acrosome reaction assays can measure the inhibitory effect of purified MSMB on spontaneous acrosome reaction in sperm models .

For optimal reliability, researchers typically normalize MSMB concentration to total protein concentration in seminal plasma, allowing for meaningful comparisons between subject groups .

What is known about the promoter structure and regulation of the MSMB gene?

The MSMB gene promoter has been extensively studied through functional analysis and deletion mapping techniques. Research has examined a 752 bp fragment (-716/+36) comprising the putative promoter region, using progressive 5' deletions to generate nine constructs for functional analysis . In silico promoter prediction using ElDorado and Gene2Promoter online programs has identified multiple regulatory elements immediately upstream of MSMB exon 1 that likely control gene expression .

Key regulatory features of the MSMB promoter include:

• Hormone response elements: Three glucocorticoid response elements and one estrogen response element in the promoter region of the first intron suggest hormonal regulation of gene expression .

• Transcription factor binding sites: Multiple cis-acting elements controlling MSMB promoter activity have been identified through transfection experiments in prostate cancer cell lines .

• Polymorphic sites: The rs10993994 polymorphism in the promoter region has been associated with altered MSMB expression and prostate cancer risk .

These regulatory elements collectively determine the tissue-specific expression pattern of MSMB and may be affected by genetic variations that alter disease risk.

How do genetic polymorphisms in MSMB affect expression and disease risk?

Genome-wide association studies have identified significant associations between polymorphisms in the MSMB gene and prostate cancer risk, particularly the rs10993994 polymorphism . A meta-analysis of 11 publications containing 13 case-control studies confirmed that this polymorphism increases prostate cancer risk, with stratification analyses showing stronger effects among Caucasians than Asians .

Fine mapping association studies and functional analyses implicate this single nucleotide polymorphism (SNP) in MSMB at 10q11 as a causal variant for prostate cancer risk . The mechanism likely involves alteration of MSMB expression levels, as the polymorphism is located in the promoter region where it could affect transcription factor binding or other regulatory processes .

Research suggests that altered MSMB expression due to this polymorphism may disrupt normal prostate biology, potentially through changes in protein interactions or signaling pathways that contribute to carcinogenesis . Understanding these genetic associations provides valuable insights for disease risk stratification and may contribute to personalized screening strategies.

What experimental approaches can identify novel regulatory mechanisms for MSMB expression?

Several advanced experimental approaches can elucidate novel regulatory mechanisms controlling MSMB expression:

• Chromatin immunoprecipitation (ChIP) assays: These can identify transcription factors binding to the MSMB promoter in different cellular contexts, revealing condition-specific regulatory mechanisms.

• Promoter-reporter constructs: Luciferase assays with truncated promoter constructs can map functional regulatory elements, as demonstrated in studies examining the 752 bp promoter fragment .

• CRISPR-Cas9 genome editing: This technique can introduce targeted mutations in regulatory elements to assess their functional importance in MSMB expression.

• DNA methylation analysis: Bisulfite sequencing can determine methylation patterns in the MSMB promoter region, potentially revealing epigenetic regulatory mechanisms.

• Chromosome conformation capture (3C): This approach can identify long-range interactions between the MSMB promoter and distant regulatory elements.

• Single-cell RNA sequencing: This technology can reveal cell-type-specific expression patterns and regulatory networks controlling MSMB expression within heterogeneous tissues.

These methodologies, applied across different tissue types and disease states, can comprehensively map the regulatory landscape controlling MSMB expression and identify potential therapeutic targets for MSMB-associated disorders.

What is the significance of the rs10993994 polymorphism in prostate cancer risk assessment?

The rs10993994 polymorphism in the MSMB gene has emerged as a significant genetic marker for prostate cancer risk through multiple genome-wide association studies . A comprehensive meta-analysis of 13 case-control studies confirmed that this polymorphism increases prostate cancer risk, with stratification analyses revealing ethnic variations in this association . Specifically, the polymorphism increases prostate cancer risk among Caucasians but shows less consistent effects among Asian populations, highlighting the importance of ethnic background in genetic risk assessment .

This single nucleotide polymorphism is located in the promoter region of the MSMB gene, potentially affecting transcription factor binding and consequently altering MSMB expression levels . Fine mapping association studies and functional analyses have implicated this SNP as a causal variant for prostate cancer risk, providing a molecular basis for the observed epidemiological associations .

The identification of this genetic marker advances prostate cancer risk stratification, potentially enabling more personalized screening approaches that consider genetic susceptibility alongside traditional risk factors .

How can researchers design experiments to elucidate the mechanistic link between MSMB variants and prostate cancer?

To establish mechanistic links between MSMB variants and prostate cancer development, researchers can implement several experimental strategies:

• Gene expression analysis: Compare MSMB expression levels in normal prostate tissue versus tumor samples, stratified by rs10993994 genotype, using quantitative RT-PCR or RNA sequencing.

• Promoter activity assays: Conduct luciferase reporter assays with wild-type and variant (rs10993994) MSMB promoter constructs in prostate cell lines to quantify differences in transcriptional activity.

• Electrophoretic mobility shift assays (EMSA): Determine whether the rs10993994 polymorphism alters transcription factor binding to the MSMB promoter region.

• Chromatin immunoprecipitation (ChIP): Identify differences in transcription factor recruitment to the MSMB promoter between wild-type and variant alleles in prostate tissue samples.

• CRISPR-Cas9 gene editing: Generate isogenic prostate cell lines differing only at the rs10993994 locus to isolate the effects of this polymorphism on cellular phenotypes.

• Xenograft models: Compare tumor formation and progression in mouse models implanted with prostate cells expressing different levels of MSMB or harboring different rs10993994 genotypes.

• Protein interaction studies: Identify MSMB binding partners in prostate cells and assess how altered MSMB expression affects these interactions and downstream signaling pathways.

These approaches collectively would provide comprehensive insights into the molecular mechanisms through which MSMB variants contribute to prostate carcinogenesis.

What are the challenges in translating MSMB research findings into clinical applications?

Translating MSMB research findings into clinical applications faces several significant challenges:

• Genetic heterogeneity: The rs10993994 polymorphism shows ethnic variations in its association with prostate cancer risk , requiring population-specific risk models.

• Multifactorial disease etiology: Prostate cancer development involves numerous genetic and environmental factors beyond MSMB variants, complicating risk prediction models.

• Biomarker validation: While altered MSMB levels have been associated with subfertility and potentially prostate cancer , large-scale validation studies are needed to establish clinical utility.

• Analytical standardization: Different methodologies for MSMB quantification (ELISA, RT-PCR, etc.) may yield varying results, necessitating standardized clinical assays .

• Biological complexity: MSMB interacts with multiple proteins (including CRISP3) and potentially affects numerous biological processes, making it challenging to target therapeutically.

• Longitudinal assessment: The relationship between MSMB variants, expression changes, and long-term cancer outcomes requires extensive longitudinal studies.

• Integration with existing biomarkers: Determining how MSMB measurements complement established prostate cancer biomarkers (e.g., PSA) requires comprehensive comparative studies.

Addressing these challenges requires multidisciplinary collaboration between basic scientists, clinical researchers, bioinformaticians, and healthcare providers to translate genetic and molecular findings into improved diagnostic, prognostic, and therapeutic approaches.

What is known about MSMB-NCOA4 fusion transcripts and their biological significance?

MSMB-NCOA4 fusion transcripts represent an emerging area of MSMB biology with potential implications for prostate function and disease. These fusion transcripts have been detected in various tissues and cancer cell lines using RT-PCR techniques, which identified a 2,027 bp product in several prostate tumor cell lines, including PCa2b, NCI H660, PC3, LNCaP, and RWPE1 .

More sensitive TaqMan real-time RT-PCR assays have detected the fusion transcript in 14 of 26 tumor cell lines and all eight human tissues tested, with highest expression in normal human prostate tissue compared to other tissues . Importantly, expression of the MSMB-NCOA4 fusion transcript shows a strong positive correlation with MSMB expression in tissues and cancer cell lines (Pearson correlation = 0.70, p-value ~6.6 × 10^-6) , suggesting co-regulation.

This correlation indicates that the fusion transcript is likely regulated by the MSMB promoter, sharing transcriptional control mechanisms with the MSMB gene itself . The biological function of these fusion transcripts remains under investigation, but their presence across multiple tissues and correlation with MSMB expression suggests potential physiological significance rather than random transcriptional events .

How can advanced proteomics approaches enhance our understanding of MSMB function?

Advanced proteomics approaches offer powerful tools for elucidating MSMB function at multiple levels:

• Protein interaction networks: Proximity-dependent biotin labeling (BioID) or affinity purification-mass spectrometry (AP-MS) can identify MSMB-interacting proteins in different cellular contexts, expanding beyond known interactions with CRISP3 .

• Post-translational modifications: Mass spectrometry-based phosphoproteomics, glycoproteomics, and other PTM analyses can identify modifications on MSMB that might regulate its function or interactions.

• Structural proteomics: Hydrogen-deuterium exchange mass spectrometry (HDX-MS) or crosslinking mass spectrometry (XL-MS) can provide insights into MSMB's structural dynamics and conformational changes upon binding to partners.

• Spatial proteomics: Imaging mass spectrometry or proximity ligation assays can map MSMB's subcellular localization and proximity to other proteins in tissue sections.

• Quantitative proteomics: SILAC, TMT, or label-free quantification approaches can measure changes in MSMB abundance across different physiological or pathological states.

• Targeted proteomics: Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) can provide highly sensitive and specific quantification of MSMB in complex biological samples.

• Proteogenomics: Integrating proteomic data with genomic and transcriptomic information can reveal how genetic variants affect MSMB protein expression and function.

These approaches collectively would provide a comprehensive understanding of MSMB's functional roles in normal physiology and disease states, potentially revealing novel therapeutic targets.

What are the emerging hypotheses about MSMB's role in non-reproductive tissues?

While MSMB is primarily known for its abundance in seminal plasma and association with reproductive function, emerging research suggests broader physiological roles across multiple tissues:

• Immunomodulatory functions: The proposed role of MSMB as an immunoglobulin binding factor suggests potential immunomodulatory functions that could extend to non-reproductive tissues.

• Epithelial barrier regulation: Given MSMB's expression in tissues with epithelial barriers (prostate, respiratory tract), it may play a role in maintaining epithelial integrity or regulating secretory functions.

• Tumor suppression: The association between MSMB variants and prostate cancer risk suggests potential tumor suppressor functions that might apply to other epithelial tissues.

• Protein quality control: MSMB's interaction with multiple proteins through its SCP domain suggests a potential role in protein folding, trafficking, or quality control pathways.

• Signaling modulation: The presence of MSMB-NCOA4 fusion transcripts hints at potential roles in nuclear receptor signaling, given NCOA4's function as a nuclear receptor coactivator.

• Hormone-responsive regulation: The presence of hormone response elements in the MSMB promoter suggests that MSMB may participate in hormone-regulated processes across multiple tissues.

Testing these hypotheses requires tissue-specific knockout models, comprehensive expression profiling across tissues and disease states, and detailed molecular studies of MSMB's interactions and activities in non-reproductive contexts.