Recombinant Mouse Protein-glutamine gamma-glutamyltransferase 4 (Tgm4)

What is Mouse Protein-glutamine gamma-glutamyltransferase 4 (Tgm4) and how does it compare to human TGM4?

Mouse Protein-glutamine gamma-glutamyltransferase 4 (Tgm4) is a member of the transglutaminase family that, like its human counterpart, is predominantly expressed in the prostate. Transglutaminases catalyze the calcium-dependent formation of isopeptide bonds between lysine and glutamine residues in proteins . Based on studies of human TGM4, mouse Tgm4 likely plays a role in rodent reproduction, specifically in forming the copulatory plug and affecting sperm antigenicity . While human and mouse TGM4/Tgm4 share significant homology, species-specific differences in their regulation and function may exist. In rodents, TGM4 is responsible for the formation of the copulatory plug and sperm antigenicity, but the function in humans is largely unknown .

What is the tissue-specific expression pattern of mouse Tgm4?

Mouse Tgm4 shows a highly tissue-specific expression pattern, predominantly in the prostate gland, similar to human TGM4 which is exclusively expressed and secreted from the prostate . The expression of TGM4 is primarily regulated by retinoic acid and secondarily modulated by androgens . For experimental design, this tissue specificity means that prostate tissue or prostate-derived cell lines are the most relevant biological systems for studying endogenous Tgm4 in mice.

What expression systems are most effective for producing recombinant mouse Tgm4?

Based on experience with human TGM4, several expression systems can be considered for mouse Tgm4:

• Insect cell expression systems: Spodoptera frugiperda Sf21 cells with baculovirus have been successfully used for human TGM4 production and likely suitable for mouse Tgm4 . These systems provide proper folding and post-translational modifications.

• E. coli expression systems: Several transglutaminases, including human TGM4, have been successfully expressed in E. coli . While simpler to use, bacterial systems may yield proteins with different properties compared to insect cell systems.

For optimal results, consider using a 6-His tag for purification and expressing the full-length protein (similar to human TGM4's Met2-Leu684) .

What purification methods yield the highest purity and activity for recombinant mouse Tgm4?

For high-purity recombinant mouse Tgm4:

• Affinity chromatography: Use Ni-NTA or similar columns if the protein contains a His-tag

• Size exclusion chromatography: To remove aggregates and provide high purity

• Consider carrier-free formulations for certain applications where the presence of bovine serum albumin (BSA) might interfere

Optimal buffer conditions based on human TGM4 preparations include Tris, NaCl, DTT, and glycerol . The protein should be filtered through a 0.2 μm filter and stored at low temperatures to avoid repeated freeze-thaw cycles . Typically, purity >95% can be achieved, which can be verified by SDS-PAGE and mass spectrometry of tryptic fragments .

What assays can be used to measure the transamidase activity of recombinant mouse Tgm4?

Several assays are suitable for measuring mouse Tgm4 transamidase activity:

• Biotin-pentylamine incorporation assay:

  • Substrate proteins are coated on microplate wells

  • Tgm4 catalyzes the incorporation of biotin-pentylamine

  • Detection via streptavidin-conjugated reporters

• Z-Gln-Gly hydroxylamine assay:

  • Follow the assay procedure:

    • Prepare substrate mixture containing Z-Gln-Gly, sodium acetate, DTT, CaCl₂, hydroxylamine hydrochloride, and NaCl

    • Dilute Tgm4 to 0.2 mg/mL in assay buffer

    • Mix diluted Tgm4 with substrate mixture

    • Incubate at 37°C for 2 hours

    • Stop reaction with stop solution and measure absorbance at 525 nm

• Dansyl cadaverine incorporation assay:

  • Kinetic assay used by commercial providers to compare transglutaminase activities

Note: Based on data from human TGM4, mouse Tgm4 may also have relatively low transamidase activity compared to other transglutaminases .

How does calcium concentration affect mouse Tgm4 activity?

Calcium dependency is a critical feature of transglutaminase activity:

• Mouse Tgm4, like human TGM4, requires Ca²⁺ for its transamidase activity

• Optimal Ca²⁺ concentration is likely around 5 mM, based on human TGM4 data

• Higher concentrations (e.g., 10 mM) may inhibit activity due to protein aggregation

For human TGM4, activity demonstrates an optimal calcium concentration at 5 mM, with higher levels (10 mM) showing an inhibitory effect, probably due to protein aggregation . When designing experiments, include calcium in reaction buffers and use EDTA as a negative control to chelate calcium.

What is the optimal pH and reducing environment for mouse Tgm4 activity?

Based on human TGM4 characterization:

• pH preference:

  • Human TGM4 prefers slightly acidic pH, which is likely true for mouse Tgm4 as well

  • The transamidase activity is optimal at pH 6.0-6.5, with higher activity in acidic conditions than in alkaline environment

• Reducing environment:

  • Reducing conditions enhance activity

  • GSH (reduced glutathione) improves activity

  • Higher GSH:GSSG ratios favor higher activity

Human TGM4 prefers reducing conditions to express increased transamidase activity, as demonstrated in experiments with various ratios of reduced and oxidized glutathione . This characteristic is likely shared by mouse Tgm4.

How do detergents like SDS affect mouse Tgm4 activity in experimental settings?

Detergents can significantly impact transglutaminase activity:

• SDS effects on human TGM4:

  • Low concentrations (0.1-0.5 mM) enhance activity 3-4 fold

  • Higher SDS concentrations are inhibitory

  • This enhancing effect of SDS was not observed with human TGM2, where increasing SDS resulted in enzyme inactivation

For experimental design, consider including low SDS concentrations to enhance Tgm4 activity. Human TGM4 shows approximately three to four times increase in transamidase activity with SDS in the range of 0.1–0.5 mM, while higher concentrations are rather inhibitory . This characteristic may be utilized to enhance the relatively low activity of mouse Tgm4 in experimental settings.

How does the transamidase activity of Tgm4 compare to other transglutaminases?

Human TGM4 has remarkably low transamidase activity compared to other transglutaminases, a characteristic likely shared by mouse Tgm4:

The transamidase activity of 0.5 μg human TGM4 was measured at 0.62 ± 0.29 mAbs/min, which is extremely low compared to human TGM2 (29.25 ± 5.93 mAbs/min at 5 mM Ca²⁺) . This significant difference in activity levels suggests specialized functions for TGM4 compared to other transglutaminases.

Does mouse Tgm4 bind GTP, and how does this differ from other transglutaminases?

Unlike some other transglutaminases (especially TGM2), human TGM4 does not bind GTP, and mouse Tgm4 likely shares this characteristic:

• GTP binding tests with human TGM4:

  • No binding observed with the fluorescent BODIPY-FL-GTPγS reagent

  • GTP-agarose resin cannot bind human TGM4 protein, while the positive control human TGM2 showed binding

This lack of GTP binding suggests that mouse Tgm4 activity is not regulated by guanine nucleotides, unlike TGM2. This has important implications when designing experiments to study regulatory mechanisms of Tgm4 compared to other transglutaminases.

How can I identify potential protein substrates of mouse Tgm4?

To identify physiological substrates of mouse Tgm4:

• Biotin-pentylamine (BPA) incorporation approach:

  • Incubate cell/tissue extracts with recombinant Tgm4, Ca²⁺, and BPA

  • Isolate BPA-labeled proteins using streptavidin pull-down

  • Identify substrates by mass spectrometry

Human TGM4 has been shown to incorporate biotin-pentylamine into several proteins in cell extracts. In one study, mass spectrometry analysis revealed eight proteins from the cytoplasm and 230 proteins from the nucleus as potential human TGM4 substrates or interacting proteins . Cytoplasmic substrates included keratins, while nuclear fractions contained numerous potential substrates. Similar approaches can be applied to identify mouse Tgm4 substrates, focusing on prostate-derived samples.

How does proteolytic processing potentially affect mouse Tgm4 activity?

Proteolytic processing can regulate transglutaminase activity:

• Limited proteolysis experiments with human TGM4:

  • Dispase I shows higher proteolytic activity than dispase II

  • Thrombin does not cleave human TGM4 (no recognition sites)

For mouse Tgm4 research, analyzing the sequence for potential protease cleavage sites (using tools like ExPASy-PeptideCutter) would be valuable . Testing the effect of prostate-specific proteases on activity could reveal physiologically relevant regulatory mechanisms. When designing experiments to study proteolytic activation, use pure recombinant proteases at physiological concentrations and monitor both protein integrity and activity.

What strategies can enhance the relatively low transamidase activity of Tgm4 in experimental settings?

Given the observed low transamidase activity of human TGM4 compared to other transglutaminases, several strategies may enhance mouse Tgm4 activity:

• Optimization of reaction conditions:

  • Use slightly acidic pH (optimally pH 6.0-6.5)

  • Ensure reducing conditions with DTT or GSH

  • Include optimal calcium concentration (around 5 mM)

  • Add low concentrations of SDS (0.1-0.5 mM) which can enhance activity 3-4 fold

• Substrate selection:

  • The low activity might be due to using non-preferred substrates in standard assays

  • Consider using prostate-specific proteins as potential substrates

  • Test different amine donors beyond standard options like biotin-pentylamine

• Enzyme concentration:

  • Using higher enzyme concentrations may be necessary, as human TGM4 shows a linear correlation between enzyme amount and activity

Even with these optimizations, mouse Tgm4 may still exhibit lower activity than other transglutaminases, which might reflect its specialized biological function.

What considerations are important when selecting antibodies for mouse Tgm4 detection?

While no specific information about mouse Tgm4 antibodies is provided in the search results, lessons from antibody characterization for human TGM2 can be applied:

• Validation criteria:

  • Compare signal in wild-type versus knockout samples

  • Test in multiple applications (Western blot, immunoprecipitation, immunofluorescence)

  • Verify specificity using recombinant protein

• Application-specific considerations:

  • For Western blotting: Select antibodies that recognize denatured epitopes

  • For immunoprecipitation: Choose antibodies that recognize native conformations

  • For immunofluorescence: Consider antibodies validated for fixed cell preparations

• Host species and antibody format:

  • For mouse Tgm4, avoid mouse-derived antibodies to prevent background in mouse tissues

  • Consider rabbit monoclonal antibodies for high specificity and low background

  • Recombinant antibodies may offer better reproducibility than polyclonal options

When selecting commercial antibodies for mouse Tgm4 research, check the manufacturer's validation data and predicted cross-reactivity with mouse Tgm4 based on epitope conservation.