Recombinant Rabbit Myocilin (MYOC)

Introduction to Myocilin

Myocilin (MYOC), also known as trabecular meshwork-induced glucocorticoid response protein (TIGR) or GLC1A, is a secreted glycoprotein that plays essential roles in cellular physiology. It regulates the activation of different signaling pathways in adjacent cells to control various processes including cell adhesion, cell-matrix adhesion, cytoskeleton organization, and cell migration . This multifunctional protein promotes substrate adhesion, spreading, and formation of focal contacts while negatively regulating cell-matrix adhesion and stress fiber assembly through Rho protein signal transduction .

Myocilin modulates the organization of actin cytoskeleton by stimulating the formation of stress fibers through interactions with components of Wnt signaling pathways. It also promotes cell migration through activation of PTK2 and the downstream phosphatidylinositol 3-kinase signaling pathway . Beyond its role in cellular mechanics, myocilin plays significant roles in bone formation, promoting osteoblast differentiation in a dose-dependent manner through mitogen-activated protein kinase signaling .

The importance of myocilin in ocular physiology cannot be overstated, as mutations in the MYOC gene have been directly linked to juvenile and adult-onset open-angle glaucoma . These mutations, including Pro370Leu (P370L) and Gln368stop (Q368X), have been identified in numerous patients with glaucoma .

Expression Systems

The production of recombinant rabbit myocilin involves several expression systems, with yeast being the most common for commercial production. The yeast protein expression system offers a balance between the economic efficiency of prokaryotic systems and the post-translational modification capabilities of eukaryotic systems .

The yeast expression system is particularly valuable for recombinant rabbit myocilin production as it allows for modifications such as glycosylation, acylation, and phosphorylation that ensure native protein conformation . These modifications are crucial for maintaining the functional properties of myocilin.

Purification Techniques

Recombinant rabbit myocilin with His tag is typically purified using nickel ion affinity chromatography, similar to the methods used for human recombinant myocilin . The purification process generally involves:

1. Cell lysis to release the expressed protein

2. Affinity chromatography using the His tag affinity for nickel ions

3. Elution using imidazole or pH changes

4. Further purification steps as needed (e.g., size exclusion chromatography)

5. Quality assessment including SDS-PAGE and Western blotting

The final product typically achieves >90% purity, making it suitable for research applications .

Functional Properties of Recombinant Rabbit Myocilin

Recombinant rabbit myocilin shares many functional properties with human myocilin due to high sequence homology. Key functional properties include:

Cell Adhesion and Migration Regulation

Myocilin promotes substrate adhesion and cell spreading while regulating cell migration through activation of PTK2 and downstream phosphatidylinositol 3-kinase signaling . Research has shown that myocilin influences cell migration, which may be relevant to its role in trabecular meshwork function.

Cytoskeletal Organization

Recombinant myocilin modulates the organization of the actin cytoskeleton by stimulating the formation of stress fibers through interactions with components of Wnt signaling pathways . This function is critical for maintaining cellular structural integrity.

Molecular Chaperone Function

Myocilin has been shown to function as a molecular chaperone in several assays. It protects citrate synthase activity against thermal inactivation in a concentration-dependent manner, with nearly full protection of 1.5 μM citrate synthase in the presence of 650 nM myocilin . It also reduces thermal aggregation of citrate synthase to levels 36% to 44% of control levels and protects GAPDH from thermal inactivation .

Regulation of Metalloprotease Activity

Myocilin regulates metalloprotease 2 (MMP2) activity through interaction with tissue inhibitor of metalloproteinases 3 (TIMP3). The olfactomedin domain of myocilin is essential for this interaction . Studies have shown that myocilin markedly enhances the inhibitory activity of TIMP3 toward MMP2, suggesting a role in extracellular matrix remodeling .

Antibody Development

Recombinant rabbit myocilin has been utilized in the development of antibodies for research purposes. Rabbit polyclonal and monoclonal antibodies against myocilin have been generated and characterized for their ability to detect myocilin in various applications including Western blot, immunohistochemistry, immunoprecipitation, and immunofluorescence .

The development of these antibodies typically involves:

1. Immunization of rabbits with purified recombinant myocilin

2. Screening for immune responsiveness using Western blot

3. Hybridoma generation for monoclonal antibodies

4. Purification and characterization of the resulting antibodies

These antibodies are crucial tools for studying myocilin expression, localization, and function in both normal and pathological conditions .

Functional Assays

Recombinant myocilin is used in various functional assays to study:

• Protein-protein interactions, particularly with TIMP3 and other extracellular matrix components

• Cellular processing and trafficking

• Effects on outflow resistance in ocular tissue

• Proteolytic processing mechanisms

Role in Glaucoma Studies

Recombinant rabbit myocilin has contributed significantly to understanding the molecular mechanisms of glaucoma. Studies using recombinant myocilin have revealed that:

Effects on Intraocular Pressure

Infusion of recombinant myocilin into human anterior segments causes an increase in intraocular pressure (IOP) over 12 hours, increasing outflow resistance by 94%, while control samples only increased by 12% . This effect is specific to functional myocilin, as heat-denatured myocilin, recombinant β-galactosidase, bovine serum albumin, and fetal calf serum did not cause similar increases in outflow resistance .

Proteolytic Processing

Recombinant myocilin undergoes proteolytic processing by calpain II, which cleaves the central region of the protein, releasing one N-terminal and one C-terminal fragment . This cleavage is culture time-dependent but independent of cell density, and is affected by extracellular bicarbonate concentration . The processing is reduced by glaucoma mutations, suggesting a potential mechanism for disease pathogenesis .

Cellular Processing in Glaucoma Models

Studies using recombinant myocilin with glaucoma-associated mutations (P370L and Q368X) have shown that these mutations affect the turnover rate and cellular processing of the protein . In normal homeostatic situations, endogenous myocilin turnover involves ubiquitin-proteasome and lysosomal pathways, but when myocilin is upregulated or mutated, the ubiquitin-proteasome function is compromised and autophagy is induced .

Comparative Analysis with Human and Other Species' Myocilin

The broad species reactivity of some anti-myocilin antibodies (recognizing human, mouse, rat, and rabbit myocilin) suggests significant conservation of epitopes across species . This conservation facilitates translational research using rabbit models for human diseases.

Future Research Directions

The study of recombinant rabbit myocilin opens several promising avenues for future research:

Therapeutic Applications

Understanding the molecular chaperone function of myocilin may lead to novel therapeutic strategies for glaucoma and other diseases where protein misfolding plays a role. The ability of myocilin to protect proteins from thermal inactivation and aggregation suggests potential applications in protein stabilization technologies.

Improved Production Methods

Development of more efficient expression systems for recombinant rabbit myocilin could enhance yield and purity while reducing production costs. Research into alternative host systems, including optimized E. coli strains, mammalian cells, or novel expression systems, could facilitate larger-scale production for therapeutic applications .

Structure-Function Relationships

Further elucidation of the structure-function relationships in rabbit myocilin, particularly in comparison with human myocilin, could provide insights into species-specific differences in glaucoma susceptibility and progression. High-resolution structural studies using X-ray crystallography or cryo-electron microscopy would be valuable for this purpose.

Novel Biomarkers

The unique processing characteristics of myocilin could potentially be exploited to develop novel biomarkers for early detection of glaucoma or for monitoring disease progression and treatment response.