MMP 3 Human, GST

What is MMP-3 and what are its primary biological functions?

Matrix metalloproteinase-3 (MMP-3), also known as stromelysin-1, transin-1, SL-1, STMY, STMY1, STR1, or progelatinase, is a zinc-dependent endopeptidase that plays crucial roles in extracellular matrix (ECM) remodeling. MMP-3 is involved in normal physiological processes including embryonic development, reproduction, and tissue remodeling, as well as disease processes such as arthritis and metastasis . As a secreted zinc-dependent endopeptidase, MMP-3 exerts its functions mainly in the extracellular matrix, where it degrades collagen types II, III, IV, IX, and X, proteoglycans, fibronectin, laminin, and elastin . Importantly, MMP-3 can also activate other MMPs such as MMP1, MMP7, and MMP9, rendering it a critical regulator in connective tissue remodeling cascades .

What is the molecular structure of recombinant human MMP-3 with GST tag?

Recombinant human MMP-3 with GST tag typically contains the sequence from Arg101 to Thr272 (based on the reference sequence P08254) with an N-terminal GST tag and frequently a C-terminal His tag for dual purification options . The protein has a calculated molecular weight of approximately 43.8 kDa, though it often migrates at approximately 45 kDa when analyzed by SDS-PAGE due to post-translational modifications . The protein consists of two key structural domains: the catalytic (Cat) domain and the hemopexin (Hpx) domain, both of which are required for effective triple-helical collagen binding . High-purity preparations (>90%) can be verified using reducing SDS-PAGE analysis .

What are the optimal storage and handling conditions for recombinant human MMP-3 with GST tag?

For maximum stability, lyophilized MMP-3 protein with GST tag should be stored at -20°C to -80°C, where it typically remains stable for up to 12 months . After reconstitution, the protein solution can be stored at 4-8°C for short-term use (2-7 days) . For longer-term storage of reconstituted protein, it is advisable to prepare aliquots and store them at temperatures below -20°C, where they remain stable for approximately 3 months . It is essential to avoid repeated freeze-thaw cycles to maintain protein integrity and enzymatic activity . The protein is typically shipped as a lyophilized powder with ice packs to preserve stability during transit .

How should recombinant human MMP-3 with GST tag be reconstituted for experimental use?

For optimal reconstitution, sterile water should be added to the lyophilized protein vial to prepare a stock solution of 0.5 mg/mL . The concentration should be verified using UV-Vis spectrophotometry to ensure accuracy . The reconstituted protein is typically formulated in PBS with stabilizers such as 5% trehalose and 5% mannitol that help maintain protein structure and function . For enzymatic activity assays, specific buffer considerations are important, particularly the inclusion of divalent cations like zinc and calcium that are essential for MMP-3 catalytic activity .

How does MMP-3 recognize and bind to triple-helical collagen structures?

MMP-3 exhibits a unique binding pattern to collagen compared to collagenases such as MMP-1 and MMP-13. While MMP-3 cannot cleave triple-helical collagens, it binds to specific sites on collagens II and III that share a glycine-phenylalanine-hydroxyproline/alanine (GFO/A) motif . This binding is highly context-dependent and requires cooperative interaction of both the catalytic (Cat) and hemopexin (Hpx) domains with the triple helix . Neither the MMP-3 zymogen (proMMP-3) nor the individual catalytic and hemopexin domains alone interact with collagen peptides, revealing the necessity of domain cooperation for binding .

Molecular modeling combined with experimental validation suggests that MMP-3 binds to collagen with all three collagen chains making contact with the enzyme in a valley running across both Cat and Hpx domains . The GFO/A motif is recognized in specific contexts, with certain neighboring amino acids enhancing or disrupting the interaction .

What specific collagen sequences are recognized by MMP-3, and how is this binding regulated?

MMP-3 recognizes five distinct binding sites on collagen II and three sites on collagen III, all sharing the critical GFO/A motif (where G is glycine, F is phenylalanine, and O/A is hydroxyproline or alanine) . The recognition of this motif is strictly context-dependent, with specific amino acids in the surrounding positions significantly affecting binding affinity . The table below summarizes key factors influencing MMP-3 binding to collagen sequences:

This complex pattern of favorable and unfavorable residues explains why the GFO/A motif is recognized at only specific sites within collagens, despite its more frequent occurrence in collagen sequences .

How can researchers experimentally validate proposed MMP-3-collagen binding models?

To validate MMP-3-collagen binding models, researchers should employ a multi-faceted approach combining computational and experimental methods:

• Solid-phase binding assays with triple-helical peptides (THPs) containing systematic variations of the GFO/A motif and flanking sequences can identify critical determinants of binding specificity .

• Site-directed mutagenesis of key residues predicted to be important for binding, followed by binding affinity measurements, can test specific molecular interactions .

• Domain deletion studies comparing full-length MMP-3, proMMP-3, and isolated Cat and Hpx domains can confirm the cooperative binding mechanism .

• Molecular modeling with experimental restraints, including the generation of MMP-3-collagen complex models and evaluation of model quality through Rosetta refinement and NACCESS interface analysis .

• Testing disruptive mutations, such as replacing critical residues with bulky amino acids that would create steric clashes in the correct binding mode .

The integration of these approaches allows researchers to distinguish between alternative binding models and identify the most biologically relevant interaction mechanism .

What are optimal expression and purification protocols for high-quality recombinant human MMP-3 with GST tag?

For optimal expression and purification of recombinant human MMP-3 with GST tag, researchers should consider the following protocol:

• Expression system: E. coli is the preferred expression host for recombinant MMP-3 with GST tag, providing good yields of functional protein .

• Protein sequence: The optimal construct contains the human MMP-3 sequence from Arg101 to Thr272 (based on accession P08254), with an N-terminal GST tag and potentially a C-terminal His tag for dual purification options .

• Purification strategy: A two-step purification approach is recommended:

  • Affinity purification using glutathione-Sepharose for the GST tag

  • Optional secondary purification using nickel affinity chromatography for constructs with His tag

• Quality control assessments:

  • SDS-PAGE analysis to confirm purity (>90% is typically achievable)

  • Endotoxin testing (<10 EU/mg of protein using the LAL method)

  • Molecular weight verification (calculated: 43.8 kDa; observed on SDS-PAGE: approximately 45 kDa)

• Formulation: The final protein is typically lyophilized from a 0.2 μm filtered solution in PBS with 5% trehalose and 5% mannitol as stabilizers .

This systematic approach yields high-purity, functional MMP-3 protein suitable for a wide range of research applications.

How can researchers distinguish between specific and non-specific binding in MMP-3-collagen interaction studies?

To differentiate between specific and non-specific binding in MMP-3-collagen interaction studies, researchers should implement these methodological approaches:

• Control proteins: Include GST tag alone as a negative control to account for tag-mediated binding effects. The proMMP-3 form and individual domains (Cat, Hpx) serve as important structural controls that should show minimal binding .

• Competition assays: Perform competition experiments with unlabeled MMP-3 to demonstrate displacement of labeled MMP-3-GST binding in a concentration-dependent manner.

• Sequence specificity analysis: Test binding to triple-helical peptides containing the GFO/A motif in various sequence contexts, including mutated sequences where key residues are substituted .

• Background correction: In solid-phase binding assays, include appropriate blanks and subtract background signals to account for non-specific interactions. A 450 values ≥ 0.1 (except for peptide 1 with high background) can be considered specific binding .

• Multiple detection methods: Use complementary techniques such as solid-phase binding assays, surface plasmon resonance, and isothermal titration calorimetry to confirm binding specificity.

This comprehensive approach provides robust evidence to distinguish specific MMP-3-collagen interactions from non-specific binding artifacts.

What methods are available for assessing MMP-3 enzymatic activity in experimental systems?

For accurate assessment of MMP-3 enzymatic activity, researchers can employ the following methodological approaches:

• Fluorogenic substrate assays: Use selective fluorogenic peptide substrates that are preferentially cleaved by MMP-3, with FRET-based detection providing high sensitivity.

• Zymography: Perform casein zymography, which is particularly suitable for MMP-3 detection due to its substrate specificity.

• Activity controls:

  • Positive control: Active recombinant MMP-3 with known activity

  • Negative control: Heat-inactivated MMP-3 or samples treated with EDTA

  • Specificity control: MMP-3(E200A) mutant with abolished catalytic activity but retained binding capability

• Optimization parameters:

  • Buffer composition: Typically 25 mM MES, 150 mM NaCl, pH 6.0 for optimal MMP-3 activity

  • Temperature: 37°C to mimic physiological conditions

  • Cofactors: Include Zn²⁺ and Ca²⁺ ions essential for MMP activity

  • Protectants: Consider adding glycerol as a stabilizing agent

• Data analysis:

  • Calculate initial velocities in the linear range of the assay

  • Determine enzyme kinetic parameters (K​m, k​cat) if applicable

  • Normalize activity to protein concentration for comparative studies

These methods provide complementary approaches to accurately measure MMP-3 enzymatic activity across different experimental systems.

How can researchers effectively differentiate between the catalytic and non-catalytic functions of MMP-3?

To distinguish between catalytic and non-catalytic functions of MMP-3, researchers should implement these strategic approaches:

• Use of catalytically inactive mutants: The MMP-3(E200A) mutant provides an excellent tool for this purpose as it maintains structural integrity and binding capability while lacking proteolytic activity . This allows researchers to isolate binding-mediated effects from enzymatic functions.

• Domain-specific constructs: Compare the activities and binding properties of:

  • Full-length active MMP-3

  • Full-length MMP-3(E200A) (catalytically inactive)

  • Isolated catalytic domain

  • Isolated hemopexin domain

  • ProMMP-3 (zymogen form)

• Functional assay design:

  • For catalytic activity: Monitor substrate degradation using specific substrates

  • For binding functions: Evaluate protein-protein interactions using solid-phase binding assays with triple-helical peptides or collagen

• Comparative analysis: Systematically analyze which biological effects persist when using the catalytically inactive MMP-3(E200A) versus active MMP-3, thus revealing non-catalytic, binding-dependent functions.

This methodological approach has successfully revealed that MMP-3 binding to collagen requires both catalytic and hemopexin domains working cooperatively, despite not resulting in collagen cleavage .

What techniques can researchers use to study the structural dynamics of MMP-3 interaction with triple-helical collagens?

To investigate the structural dynamics of MMP-3 interactions with triple-helical collagens, researchers should consider these advanced methodological approaches:

• Integrative modeling: Combine computational methods with experimental restraints to develop comprehensive models of MMP-3-collagen complexes . This approach has successfully identified that MMP-3 binds in a valley running across both Cat and Hpx domains, with all three collagen chains making contacts with the enzyme .

• Molecular docking and refinement: Use sophisticated docking algorithms followed by Rosetta refinement to generate and evaluate candidate binding models . This can be performed using:

  • Homology models based on related MMP structures

  • Triple-helical peptide models based on X-ray structures

  • Systematic evaluation of different binding orientations

• Validation through mutagenesis: Test the effects of strategic mutations that are predicted to be either disruptive or beneficial for binding based on the computational model . For example, replacing key residues with bulky amino acids can create steric clashes that disrupt binding in the correct model.

• Interface analysis: Employ tools like NACCESS to analyze the protein-protein interfaces in proposed complexes, evaluating buried surface areas, hydrogen bonding networks, and complementary surface features .

• Structure-activity relationship studies: Systematically analyze the effects of sequence variations in collagen-like peptides on MMP-3 binding to refine understanding of the structural determinants of specificity .

These complementary approaches provide a robust framework for elucidating the complex structural basis of MMP-3-collagen interactions.

How can recombinant MMP-3 with GST tag be utilized as a tool in developing therapeutics for matrix-related diseases?

Recombinant MMP-3 with GST tag offers several valuable applications in therapeutic development for matrix-related diseases:

• Target validation: Recombinant MMP-3 can be used to validate the role of specific matrix interactions in disease models, helping identify which MMP-3 functions (catalytic vs. binding) contribute to pathology.

• Inhibitor screening: Pure, active MMP-3 with GST tag provides an excellent tool for high-throughput screening of potential inhibitors, allowing discrimination between compounds that block:

  • Catalytic activity

  • Collagen binding

  • Activation of other MMPs

  • Interaction with tissue inhibitors of metalloproteinases (TIMPs)

• Structural studies: The detailed understanding of MMP-3 binding to collagen via the GFO/A motif in specific contexts can guide structure-based drug design targeting these interactions .

• Biomarker development: Pure recombinant MMP-3 serves as an essential standard for developing quantitative assays to measure MMP-3 levels and activity in patient samples.

• Disease-specific targeting: Knowledge of MMP-3's binding preferences for specific collagen sequences can inform the development of targeted therapies that modulate MMP-3 activity only in disease-relevant contexts .

These applications demonstrate how recombinant MMP-3 with GST tag can serve as both a research tool and a foundation for therapeutic development in conditions involving aberrant matrix remodeling.

What methodological considerations are important when studying MMP-3 in the context of extracellular matrix remodeling?

When investigating MMP-3 in extracellular matrix remodeling contexts, researchers should consider these critical methodological aspects:

• Protein form selection: Carefully choose between different forms of MMP-3 based on the specific research question:

  • ProMMP-3: To study activation mechanisms

  • Active MMP-3: For enzymatic activity studies

  • MMP-3(E200A): To isolate binding effects from catalytic functions

  • Domain-specific constructs: To dissect domain-specific contributions

• Physiological relevance:

  • Use appropriate buffer conditions mimicking the extracellular environment (pH, ion concentrations)

  • Consider the presence of natural inhibitors (TIMPs, α2-macroglobulin) in complex systems

  • Account for interactions with other matrix components and cell-surface proteins

• Activation considerations: MMP-3 is secreted as a zymogen and requires activation, which can be achieved by:

  • Chemical means (organomercurials)

  • Enzymatic activation (other proteases)

  • Physiological activators in cell-based systems

• Experimental context complexity:

  • Simple systems: Purified MMP-3 with individual substrates for mechanistic studies

  • Complex systems: Cell cultures or tissue explants for integrated functional analysis

  • In vivo models: For physiological and pathological relevance

• Downstream effects analysis:

  • Direct effects: Substrate cleavage, binding competition

  • Indirect effects: Activation of other MMPs (MMP1, MMP7, MMP9)

  • Cellular responses: Migration, differentiation, gene expression changes

This comprehensive methodological framework ensures that MMP-3 studies yield physiologically relevant insights into extracellular matrix remodeling processes.