CST3 Mouse, sf9

What are the optimal storage conditions for maintaining CST3 stability?

For optimal stability, recombinant mouse CST3 should be stored at 4°C if the entire vial will be used within 2-4 weeks. For longer periods, it should be stored frozen at -20°C. For long-term storage, it is recommended to add a carrier protein (0.1% HSA or BSA). The recombinant protein is stable for up to 1 year from date of receipt when stored at -70°C. It's important to avoid repeated freeze-thaw cycles to maintain protein integrity .

What is the standard formulation of recombinant mouse CST3?

The standard formulation consists of CST3 protein solution (1mg/ml) in Phosphate Buffered Saline (pH 7.4) containing 10% glycerol. This formulation helps maintain protein stability during storage and handling .

What are the key heparan sulfate binding regions in CST3?

NMR studies and site-directed mutagenesis have identified two distinct heparan sulfate (HS) binding regions in CST3:

• The highly dynamic N-terminal segment (K4 to E14), which is flexible and can adopt multiple conformations

• A flexible region located between residues 70-94, particularly involving residues R70, H90, R93, and K94

These residues form a cluster of basic amino acids typical of HS-binding sites in most HS-binding proteins. Notably, the composition of the HS-binding site by two highly dynamic halves is unique among known HS-binding proteins. All identified HS-binding residues are conserved between murine and human CST3 .

How does pH affect CST3's interaction with heparan sulfate?

CST3 exhibits pH-dependent interaction with heparan sulfate, binding only at acidic pH (pH 5.5). This pH-dependent binding has significant functional implications, as the interaction with HS severely impairs CST3's inhibitory activity towards papain, suggesting that interaction with extracellular matrix components could actively regulate CST3 activity in vivo. The interaction has been demonstrated in murine bone tissues at low pH, highlighting its physiological relevance .

How can mutations in key residues affect CST3's binding properties?

Site-directed mutagenesis studies reveal the critical importance of specific residues for HS binding:

• N-terminal truncation (∆N-CST3, lacking Ser1-Val10) resulted in complete loss of HS binding

• H90A mutation eliminated binding to heparin column

• R93Q-K94Q double mutation also eliminated heparin binding

• R70A mutation reduced binding (50 mM reduction in salt concentration required for elution)

• K75A and H86A mutations had no significant effect on binding

These findings confirm the two-region model of HS binding, with both the N-terminal segment and the basic residue cluster (R70, H90, R93, K94) being essential for the interaction .

What techniques are most effective for analyzing CST3-heparan sulfate interactions?

Several complementary techniques provide comprehensive analysis of CST3-HS interactions:

• NMR Spectroscopy:

  • 1H, 15N-HSQC experiments with HS hexasaccharide titration reveal chemical shift perturbations

  • 3D triple resonance experiments for peak assignment

  • Provides atomic-level resolution of binding interfaces

• Heparin Affinity Chromatography:

  • Quantitative assessment of binding strength through salt gradient elution

  • Wild-type CST3 requires high salt (>1M NaCl) for elution at pH 5.5

  • Mutant proteins show altered elution profiles corresponding to binding capacity

• Site-Directed Mutagenesis:

  • Systematic mutation of basic residues followed by binding assays

  • N-terminal truncation to assess contribution of this region

• Functional Assays:

  • Papain inhibition assays in the presence/absence of HS

  • pH-dependent activity measurements

What are the critical differences between E. coli and Sf9-expressed mouse CST3?

The expression system significantly impacts recombinant CST3 properties:

Despite these differences, studies have shown that the heparin binding properties of E. coli-expressed and mammalian cell-expressed CST3 are similar, suggesting that glycosylation may not significantly affect this particular function .

What are the optimal experimental conditions for studying CST3-HS interactions?

Based on published research, the following conditions are optimal:

• pH: Acidic pH (5.5) is essential for observing the interaction

• Buffer: Phosphate buffer with controlled ionic strength

• Temperature: 25°C for NMR studies, room temperature for chromatography

• Heparan sulfate: Well-characterized HS hexasaccharides with defined sulfation patterns

• Controls: Include parallel experiments at neutral pH (7.4) as negative controls

• Protein preparation: Highly purified recombinant CST3 (>95% purity)

• Analytical methods: NMR spectroscopy at pH 5.5 with gradual HS titration, heparin affinity chromatography with salt gradient elution

How does the unique two-region binding site of CST3 contribute to its regulatory function?

The unusual composition of CST3's HS-binding site, involving two highly dynamic regions (N-terminal segment and residues 70-94), represents a novel binding mechanism among HS-binding proteins. This structural arrangement allows for:

• Conditional regulation: The dynamic nature of these regions suggests conformational changes may influence binding affinity

• pH-dependent control: The binding only at acidic pH indicates microenvironment-specific regulation

• Functional switching: When bound to HS, CST3's inhibitory activity against papain is severely impaired, suggesting a mechanism for spatiotemporal control of protease inhibition

• Tissue-specific effects: Demonstrated interaction with bone matrix HS at low pH indicates tissue-specific regulatory roles

This unique binding mechanism may explain how CST3 activity is precisely regulated in different tissue microenvironments, particularly those with acidic pH such as inflammatory sites, tumor microenvironments, or bone resorption zones .

What is the current evidence for CST3 genetic associations with neurological disorders?

Despite CST3's biological importance, genetic evidence for its association with neurological disorders remains limited:

A comprehensive study in the Finnish population (568 Alzheimer's disease patients and 688 controls) examined two informative flanking SNPs of the CST3 gene (rs2424577 and rs3827143) and found:

• No significant differences in Alzheimer's disease risk in single SNP and haplotype analyses

• A nominally significant difference (p = 0.04) for AG-genotype carriers of rs3827143 compared to AA-genotype carriers, but this finding was not significant in the adjusted model

• The study concluded that the CST3 gene is not associated with Alzheimer's disease risk in the Finnish population despite strong linkage disequilibrium across the gene region

This suggests that while CST3 may have biological roles in neurological processes, genetic variation in CST3 may not significantly contribute to Alzheimer's disease susceptibility, at least in the population studied .

How might the pH-dependent HS binding mechanism of CST3 be exploited for therapeutic applications?

The unique pH-dependent interaction between CST3 and heparan sulfate presents several potential therapeutic opportunities:

• Targeted drug delivery: Designing drug carriers that release CST3 or CST3-derived peptides in acidic microenvironments (e.g., tumors, inflammatory sites)

• Engineered variants: Creating CST3 mutants with modified pH sensitivity to regulate protease activity in specific tissue contexts

• Synthetic peptide inhibitors: Developing peptides based on the CST3 HS-binding regions that could compete with native CST3-HS interactions

• Diagnostic applications: Using CST3-HS binding properties to detect changes in tissue pH or extracellular matrix composition

• Bone-targeting therapeutics: Exploiting the interaction with bone matrix HS at low pH for targeted delivery of osteoporosis treatments

These approaches would require detailed understanding of the molecular basis of the interaction and careful design to ensure specificity and efficacy .

How conserved are the CST3 HS-binding regions across species?

The key HS-binding residues in CST3 show remarkable evolutionary conservation:

• All identified HS-binding residues (including those in the N-terminal region and the R70, H90, R93, K94 cluster) are conserved between murine and human CST3

• Mouse CST3 has an orthologue on chromosome 3, while the rat orthologue is located on chromosome 2

• Human CST3 has two paralogues on chromosomes 4 and 10, suggesting evolutionary divergence and potential functional specialization

This high degree of conservation suggests that the pH-dependent HS-binding mechanism is likely an ancient and fundamentally important aspect of CST3 function across mammalian species .

What are the implications of CST3 orthologues and paralogues for functional studies?

The existence of mouse (chromosome 3) and rat (chromosome 2) orthologues, as well as two human paralogues on chromosomes 4 and 10, has important implications for research:

• Model system selection: When using mouse models to study CST3 function, researchers should be aware of potential species-specific differences

• Paralogous functions: The human paralogues may have evolved specialized functions that should be considered when translating findings from animal models

• Evolutionary insights: Comparative studies of CST3 across species can reveal conserved functional domains versus species-specific adaptations

• Experimental design: When targeting CST3 in animal models, researchers should consider cross-reactivity with paralogues and potential compensatory mechanisms

Understanding these evolutionary relationships is crucial for properly interpreting experimental results and translating findings across species .