Recombinant Mouse Mucin-19 (Muc19), partial

What is Mouse Mucin-19 (Muc19) and where is it expressed?

Mouse Mucin-19 (Muc19) is a gel-forming mucin that plays critical roles in mucous secretions across multiple organ systems. Muc19 expression demonstrates a highly tissue-specific pattern, with preferential expression in salivary major and minor mucous glands, submucosal glands of the tracheolarynx, and male accessory bulbourethral glands . The expression is primarily restricted to glandular structures under normal physiological conditions rather than surface epithelia, distinguishing it from some other mucin family members .

Interestingly, while both Muc5b and Muc19 are expressed in minor salivary glands, the major glands (sublingual and submandibular) appear to exclusively express Muc19 but not Muc5b . Beyond the respiratory and oral systems, Muc19 has been detected in bulbourethral glands (Cowper's glands) in the male reproductive system, indicating its diverse physiological roles across multiple secretory systems .

What is the genomic organization of the mouse Muc19 gene?

The mouse Muc19/Smgc gene exhibits a complex genomic organization containing 60 exons and spanning approximately 105 kb of genomic sequence . The gene structure demonstrates a unique arrangement where Smgc (submandibular gland protein C) is encoded by exons 1-18, whereas Muc19 transcripts incorporate exon 1 and exons 19-60 . This shared exon 1 between the two transcripts encodes most of the predicted signal peptide that directs each translation product to the secretory pathway .

The Muc19 gene structure follows the characteristic pattern of gel-forming mucins with evolutionarily conserved features: a 5′ end unique sequence, a large undisrupted central exon containing repetitive sequences, and a 3′ end unique sequence . Notably, mouse Muc19 has been completely sequenced and shown to have a cDNA length of 22,795 bp encoded by 43 exons and spanning 106 kb of genomic DNA . The central exon is particularly significant as it contains highly repetitive sequences and has no intron interruption, functioning as a single giant open reading frame (ORF) .

How does mouse Muc19 compare structurally to human MUC19?

Mouse Muc19 and human MUC19 share key structural characteristics typical of gel-forming mucins, though with some species-specific variations. Human MUC19 consists of 182 exons with a transcript of approximately 25 kb, producing a protein with the characteristic gel-forming mucin structure: VWD-VWD-VWD-"threonine/serine-rich repeats"-VWC-CT . Mouse Muc19 similarly demonstrates a gel-forming mucin structure with signal peptide, a large central exon containing tandem repeats, VWC, VWD, and C-terminal CT domains .

A distinctive structural feature of human MUC19, which is not found in other gel-forming mucins, is its long amino terminus upstream of the first VWD domain . This long amino terminus is mostly translated from sequences in the highly variable region (HVR) and contains serine-rich repetitive sequences that may contribute to its functional properties . While the search results don't explicitly compare this feature between species, the evolutionary conservation among gel-forming mucin orthologs suggests potential structural similarities across species.

Interestingly, pig MUC19 (also called porcine submaxillary gland mucin) is identified as the closest ortholog of human MUC19, providing additional insights into the structural conservation of this mucin family across mammalian species .

What are the common methods for detecting Muc19 expression in mouse tissues?

Several complementary methodological approaches have been established for detecting Muc19 expression in mouse tissues:

• RT-PCR: This technique enables detection of Muc19 transcripts using gene-specific primers designed to target unique regions of the Muc19 gene . The search results describe multiple RT-PCR reactions using various primer pairs to verify sequences from both 5′ and 3′ ends of Muc19 .

• Immunohistochemistry: For protein-level detection, antibodies developed against either the amino (N) or carboxy (C) terminus of MUC19 can be used . Specifically, chicken anti-human MUC19 antibodies targeting the C-terminus (CREENYELRDIVLD) and N-terminus (CGSYNNKAEDDFMSSQNILEKTSQ) have been developed, affinity purified, and ELISA tested for detection purposes .

• RACE (Rapid Amplification of cDNA Ends): This specialized PCR technique has proven valuable for obtaining complete cDNA sequences of Muc19 . The search results describe multiple rounds of RACE to extend sequence information from both 5′ and 3′ directions until reaching the central exon .

• Genetically modified reporter systems: The Muc19-EGFP knockin mouse model represents an innovative approach where mice express a fusion protein containing the first 69 residues of Muc19 followed by enhanced green fluorescent protein (EGFP) . This model provides a powerful tool for visualizing Muc19 expression patterns across tissues without relying on antibody detection methods .

These complementary approaches provide researchers with multiple options for detecting Muc19 at both transcript and protein levels, enabling comprehensive characterization of expression patterns.

What are the best approaches for producing recombinant partial Muc19 proteins?

Production of recombinant partial Muc19 proteins requires strategic approaches that address the structural complexity of this mucin:

• Domain-specific targeting: Rather than attempting full-length expression, researchers should focus on specific domains of interest within Muc19. The gel-forming mucin structure includes VWD domains, threonine/serine-rich repeats, VWC domain, and C-terminal domains that can be targeted individually . For partial recombinant expression, selecting non-repetitive domains like VWD or CT domains generally results in more successful expression compared to the highly repetitive central regions.

• Expression system selection: While the search results don't explicitly recommend specific expression systems, the complex post-translational modifications required for mucins suggest mammalian expression systems would be most appropriate for domains requiring glycosylation. For structural domains like VWD or CT that may not require extensive glycosylation, bacterial or yeast systems might offer higher yields and easier purification.

• Cloning strategy optimization: The search results describe various cloning approaches used to characterize Muc19, including RACE and RT-PCR with specific primers . Similar strategies could be adapted for generating expression constructs, with careful attention to incorporating appropriate secretion signals and purification tags.

• Sequence verification: Due to the repetitive nature of mucin sequences, rigorous sequence verification is essential. The search results describe multiple validation approaches including sequencing of TA-cloned PCR products , which would be applicable to verifying recombinant expression constructs.

• Expression verification: Antibodies against either the amino (N) or carboxy (C) terminus of MUC19 have been developed and could be utilized to verify successful expression of recombinant proteins through Western blotting or immunostaining approaches.

Given the complex structure and large size of full-length Muc19, domain-focused approaches represent the most practical strategy for recombinant protein production.

How can Muc19-EGFP knockin mice be utilized for studying mucin expression patterns?

The Muc19-EGFP knockin mouse model described in the search results represents a powerful tool for investigating mucin expression patterns in vivo . This model, in which mice express a fusion protein containing the first 69 residues of Muc19 followed by enhanced green fluorescent protein (EGFP), offers several methodological advantages:

• Direct visualization of expression: The EGFP fluorescent reporter enables straightforward visualization of tissues expressing Muc19 without requiring antibody staining procedures . This allows for comprehensive mapping of expression patterns across different tissues, developmental stages, and in response to various stimuli.

• Cellular resolution analysis: The fluorescent marker permits identification of specific cell types expressing Muc19 within heterogeneous tissues, enabling precise characterization of expression at the cellular level. This approach has confirmed Muc19 expression in salivary mucous glands, glands of the tracheolarynx, and male accessory bulbourethral glands .

• Live tissue and ex vivo studies: Unlike fixed-tissue immunohistochemistry, the EGFP reporter enables visualization in live tissues, allowing for dynamic studies of Muc19 expression in organ cultures or ex vivo preparations.

• Cross-validation with other methods: The search results indicate that findings from the Muc19-EGFP mouse model were consistent with results obtained through traditional RT-PCR and immunohistochemistry approaches , providing an important validation of this genetic model for studying Muc19 expression.

• Disease model integration: The Muc19-EGFP mice can be crossed with various disease model mice to investigate alterations in Muc19 expression under pathological conditions, providing insights into regulatory mechanisms controlling mucin expression.

The genetic strategy used to create these mice involved careful design of homologous arms and appropriate selection markers, with the EGFP coding region positioned in-frame within the 5′ end of the Muc19 coding sequence . This approach ensures that EGFP expression accurately reflects endogenous Muc19 promoter activity.

What are the challenges in purifying recombinant Muc19 due to its structural properties?

Purification of recombinant Muc19 presents several significant challenges attributable to its unique structural properties:

• Extreme size and complexity: Human MUC19 consists of 182 exons with a transcript of approximately 25 kb , translating to an extraordinarily large protein. Mouse Muc19 similarly has a cDNA length of 22,795 bp . This exceptional size creates fundamental challenges for expression system capacity and purification column exclusion limits.

• Repetitive sequence regions: Muc19 contains a central exon of approximately 12 kb with highly repetitive sequences . These repetitive regions not only complicate cloning procedures but also create potential instability in expression constructs and challenges in protein folding during recombinant expression.

• Extensive post-translational modifications: As a gel-forming mucin, Muc19 contains threonine/serine-rich repeat regions that typically undergo extensive O-linked glycosylation. These modifications are essential for mucin function but introduce heterogeneity that complicates purification and structural characterization.

• Domain organization complexity: The presence of multiple VWD domains, VWC domain, and CT domain creates a complex multi-domain protein structure. Ensuring proper folding of all domains simultaneously during recombinant expression represents a significant challenge.

• Unique structural features: The long amino terminus upstream of the first VWD domain that distinguishes MUC19 from other gel-forming mucins may present additional conformational challenges during expression and purification.

These challenges explain why researchers often focus on partial fragments or specific domains rather than attempting full-length expression of large mucins like Muc19. Domain-specific approaches, coupled with appropriate expression systems, represent the most practical strategy for obtaining purified recombinant protein for functional and structural studies.

How does Muc19 expression change in disease models affecting mucosal surfaces?

While the search results provide limited information about Muc19 expression in disease states, several significant observations have been reported:

• Altered expression in autoimmune conditions: Regulation and potential functional implications of MUC19/Muc19 have been reported in patients with Sjogren syndrome , an autoimmune condition affecting salivary and lacrimal glands. This suggests potential roles for Muc19 in the pathophysiology of autoimmune-mediated glandular dysfunction.

• Inflammatory response in middle ear: Increased MUC19 expression has been observed in cytokine-challenged middle ear epithelium , indicating inflammatory regulation of this mucin gene. This observation suggests potential involvement in middle ear diseases like otitis media.

• Allergic response modulation: Changes in Muc19 expression have been documented in an allergic mouse model , pointing to potential roles in the mucous barrier response to allergen exposure and subsequent inflammatory cascades.

• Developmental and differentiation disorders: Alterations in Muc19 have been noted in a mouse model of mucous cell deficiency in salivary glands , highlighting its potential involvement in developmental disorders affecting secretory cell differentiation.

• Epithelial expression under pathological conditions: While normal MUC19/Muc19 expression appears restricted to glandular structures, under certain disease conditions, it can be expressed in the epithelium . This ectopic expression pattern may represent a pathological response mechanism or altered cellular differentiation programs.

What controls should be included when validating Muc19 antibodies?

Rigorous validation of Muc19 antibodies requires comprehensive controls to ensure specificity and reliability:

• Tissue-specific positive and negative controls: Researchers should include known Muc19-expressing tissues such as salivary glands, tracheal submucosal glands, and bulbourethral glands as positive controls . Conversely, tissues documented not to express Muc19 should be included as negative controls to evaluate potential cross-reactivity.

• Peptide blocking controls: The search results describe specific peptides used to generate anti-human MUC19 antibodies (C-terminus antigen: CREENYELRDIVLD and N-terminus antigen: CGSYNNKAEDDFMSSQNILEKTSQ) . Pre-incubation of antibodies with these immunizing peptides should abolish specific staining in a peptide blocking experiment, providing critical evidence for antibody specificity.

• Multiple antibody approach: The search results mention antibodies developed against both the amino (N) and carboxy (C) terminus of MUC19 . Using multiple antibodies targeting different epitopes and comparing their staining patterns provides valuable cross-validation. Similar antibody staining patterns observed in both salivary and tracheal submucosal glands with N- and C-terminal antibodies supports specificity .

• Genetic model validation: The Muc19-EGFP knockin mouse model represents an excellent resource for antibody validation. Co-localization of antibody staining with EGFP fluorescence would provide strong evidence for antibody specificity.

• Methodological controls: Include appropriate isotype controls matching the primary antibody's host species and immunoglobulin class to evaluate potential non-specific binding. Additionally, secondary antibody-only controls should be included to assess background staining.

• Cross-species reactivity assessment: If the antibody is designed to detect Muc19 across multiple species, validation should include tissues from each target species alongside appropriate negative controls.

These comprehensive validation approaches are essential given the complex nature of mucin proteins and potential for cross-reactivity with other heavily glycosylated proteins.

Which expression systems are optimal for producing functional recombinant Muc19?

Selection of appropriate expression systems for recombinant Muc19 production must consider the protein's complex structural requirements:

• Mammalian expression systems: For producing glycosylated domains of Muc19, particularly those containing threonine/serine-rich repeat regions , mammalian expression systems represent the optimal choice. CHO, HEK293, or mucus-producing cell lines can provide the complex glycosylation machinery required for proper post-translational modifications. These systems would be essential for studies focusing on Muc19's gel-forming properties or interactions dependent on glycosylation patterns.

• Domain-specific expression strategies: For non-glycosylated domains such as the VWD, VWC, or CT domains described in the Muc19 structure , simpler expression systems may be appropriate. Bacterial (E. coli) systems could be suitable for structural studies of these domains, offering higher yields and simplified purification processes.

• Specialized secretion considerations: The search results highlight that Muc19 contains a signal peptide directing it to the secretory pathway . Expression constructs should preserve this feature or incorporate an alternative secretion signal to facilitate proper processing and potential secretion of the recombinant protein.

• Splicing variant considerations: The search results mention that a total of 20 different splicing variants were detected in the highly variable region (HVR) of human MUC19, with 18 variants able to translate into proteins . This suggests that expression of specific splice variants might be considered depending on the research question.

• Verification strategies: Regardless of the expression system selected, verification of successful expression should incorporate both the antibodies against the amino (N) or carboxy (C) terminus of MUC19 mentioned in the search results and appropriate analyses of post-translational modifications.

The optimal expression system ultimately depends on which aspects of Muc19 biology are being investigated and whether glycosylation is critical for the specific research questions being addressed.

How should researchers interpret differences in Muc19 molecular weight observed in different tissues?

Interpretation of tissue-specific variations in Muc19 molecular weight requires consideration of several biological and technical factors:

• Alternative splicing contributions: The search results reveal that a total of 20 different splicing variants were detected in the highly variable region (HVR) of human MUC19, with 18 variants able to translate into proteins . Similar splicing diversity likely exists in mouse Muc19, potentially resulting in tissue-specific expression of different variants with varying molecular weights.

• Post-translational modification differences: The threonine/serine-rich repeat regions in Muc19 represent primary sites for extensive O-linked glycosylation. Tissue-specific glycosylation machinery can produce dramatically different glycosylation patterns and densities, significantly altering the apparent molecular weight of Muc19 across different tissues.

• Proteolytic processing variation: The search results mention that the longest variant of MUC19 consists of 182 exons , producing an extremely large protein that may undergo tissue-specific proteolytic processing. Different tissues may express distinct proteases that process Muc19 differently.

• Technical analysis considerations: Standard SDS-PAGE methods often inadequately resolve very high molecular weight glycoproteins like mucins. Researchers should employ specialized electrophoresis methods such as agarose-acrylamide composite gels or gradient gels for more accurate molecular weight assessment of Muc19.

• Verification approaches: To distinguish between these possibilities, researchers should consider:

  • Deglycosylation experiments using enzymes that remove O-linked glycans

  • Western blotting with both N- and C-terminal antibodies to identify potential proteolytic processing

  • RT-PCR with primer sets targeting different regions to identify possible splice variants

  • Mass spectrometry for detailed characterization of tissue-specific forms

The unusual structural feature of MUC19, its long amino terminus upstream of the first VWD domain , may be particularly susceptible to tissue-specific processing or modification, potentially contributing to observed molecular weight differences.

What are the best approaches for studying Muc19 interactions with other proteins?

Investigation of Muc19 protein interactions requires specialized approaches that account for its complex structure and post-translational modifications:

• Domain-specific interaction analyses: The search results indicate that Muc19 contains multiple distinct domains including VWD domains, VWC domain, and CT domain . Each domain may mediate specific protein interactions and should be investigated individually using recombinant fragments. This reductionist approach can identify domain-specific binding partners before attempting to validate in the context of full-length Muc19.

• Immunoprecipitation strategies: The antibodies developed against the amino (N) and carboxy (C) terminus of MUC19 provide valuable tools for co-immunoprecipitation studies. Using these antibodies to isolate Muc19 from tissue lysates followed by mass spectrometry can identify physiologically relevant binding partners.

• Glycan-dependent interaction studies: Given the extensive glycosylation expected in the threonine/serine-rich regions , lectin affinity approaches can help identify interactions mediated by specific glycan structures. This is particularly important as many mucin interactions are glycan-dependent rather than protein backbone-dependent.

• Tissue-specific interaction mapping: The search results indicate tissue-specific expression of Muc19 in salivary glands, tracheal submucosal glands, and bulbourethral glands . Comparing interaction partners across these different tissues can provide insights into tissue-specific functions of Muc19.

• Muc19-EGFP model utilization: The Muc19-EGFP knockin mouse model offers opportunities for live-cell imaging approaches to study dynamic interactions, fluorescence resonance energy transfer (FRET) with suspected binding partners, or fluorescence recovery after photobleaching (FRAP) to study mobility and interactions within cellular compartments.

• Comparative analysis with other gel-forming mucins: The search results mention that Muc19 is expressed alongside other gel-forming mucins in some tissues . Comparative interaction studies can reveal shared versus unique binding partners among mucin family members.

The unique structural features of Muc19, particularly its long amino terminus upstream of the first VWD domain , may mediate novel protein interactions not observed with other gel-forming mucins and warrant specific investigation.

How can inconsistencies in Muc19 detection between RT-PCR and immunohistochemistry be resolved?

Resolving detection discrepancies between transcript and protein-level Muc19 analyses requires systematic troubleshooting:

• Technical validation approaches:

  • For RT-PCR: Evaluate primer design for potential secondary structures, verify amplicon sizes on agarose gels, and sequence PCR products to confirm target specificity. The search results describe multiple primer pairs used for Muc19 detection , providing options for cross-validation.

  • For immunohistochemistry: Verify antibody specificity using peptide blocking experiments with the specific peptides described (CREENYELRDIVLD for C-terminus and CGSYNNKAEDDFMSSQNILEKTSQ for N-terminus) . Optimize antigen retrieval methods, as mucin epitopes may be masked by extensive glycosylation.

• Biological interpretation considerations:

  • Post-transcriptional regulation: Discrepancies may reflect legitimate biological processes including mRNA stability differences, translational regulation, or protein half-life variations rather than technical artifacts.

  • Sensitivity thresholds: RT-PCR typically offers greater sensitivity than immunohistochemistry, potentially detecting low-level transcripts in tissues where protein remains below detection thresholds.

  • Splice variant specificity: The search results mention multiple splicing variants in the highly variable region (HVR) . Primer locations and antibody epitopes may differentially detect specific variants.

• Complementary validation approaches:

  • In situ hybridization to localize transcripts with spatial resolution comparable to immunohistochemistry

  • Quantitative RT-PCR for more precise transcript quantification

  • Western blotting as an alternative protein detection method

  • Utilization of the Muc19-EGFP mouse model as an independent reporter of gene expression

• Multi-tissue comparison: The search results indicate that primers from both the 3′ and 5′ end were used to demonstrate similar tissue expression patterns of MUC19 in trachea and salivary glands . Similar approaches comparing multiple primer sets targeting different regions can help resolve inconsistencies.

The search results mention that similar antibody staining patterns were observed with both N- and C-terminal antibodies in salivary and tracheal submucosal glands , providing an example of successful cross-validation that could be applied to resolve inconsistencies.

What statistical approaches are recommended for analyzing Muc19 expression data across different tissues?

Analysis of Muc19 expression across multiple tissues requires rigorous statistical approaches tailored to the specific experimental methods:

• RT-PCR data analysis:

  • Reference gene normalization: Select stable reference genes validated across all tissues being compared to normalize expression data. This is particularly important when comparing functionally distinct tissues that may have different baseline expression profiles.

  • Appropriate statistical tests: For multi-tissue comparisons, ANOVA with post-hoc tests (with appropriate corrections for multiple comparisons) should be employed. For non-normally distributed data, non-parametric alternatives such as Kruskal-Wallis with Dunn's post-test may be preferable.

  • Data transformation: Consider log transformation of RT-PCR data, particularly for comparative CT (ΔΔCT) methods, to address the exponential nature of PCR amplification.

• Immunohistochemistry quantification:

  • Systematic sampling methods: Establish consistent rules for field selection and cell counting to minimize selection bias.

  • Quantitative metrics: Consider multiple parameters including staining intensity, percentage of positive cells, and staining pattern (e.g., apical vs. cytoplasmic).

  • Blinded analysis: Implement observer blinding to tissue identity to prevent unconscious bias in quantification.

• Reporter model analysis (e.g., Muc19-EGFP) :

  • Fluorescence intensity quantification: Standardize exposure settings and implement background subtraction methods.

  • Cell-specific vs. tissue-level measurements: Determine whether single-cell or whole-tissue measurements are most appropriate for the research question.

• Multi-method integration:

  • Correlation analysis between different detection methods

  • Standardized scoring systems that can be applied across methods

  • Meta-analytic approaches to combine evidence from multiple experimental techniques

• Visualization approaches:

  • Tissue expression heat maps with hierarchical clustering

  • Principal component analysis to identify patterns across tissues

  • Radar plots for comparing expression profiles across multiple tissues simultaneously

The experimental approaches described in the search results, including RT-PCR, immunohistochemistry, and the reporter mouse model , would each require specific statistical approaches tailored to their unique data characteristics.