GLU-C S.aureus

What is the cleavage specificity of GLU-C S.aureus and how can it be modified?

GLU-C S.aureus preferentially cleaves peptide bonds on the carboxyl-terminal side of glutamic acid and, less frequently, aspartic acid residues (Glu-|-Xaa, Asp-|-Xaa) . This specificity can be modified by buffer composition:

• In ammonium bicarbonate and ammonium acetate buffers, the enzyme exhibits higher specificity for glutamic acid residues

• In phosphate buffers, cleavage occurs at both aspartic and glutamic acid residues

N-terminomics studies have revealed subtle preferences beyond the P1 position (directly upstream of the scissile bond). While glutamic acid (E) is the primary preference at P1, there are slight preferences for glycine (G) at P1′, threonine (T) at P2′, and valine (V) at P4′ .

What are the optimal conditions for GLU-C activity in research applications?

The enzyme maintains optimal activity between pH 4.0-9.0, with activity peaks at approximately pH 4.0 and 7.8 when using hemoglobin as a substrate . The enzyme is most stable when:

• Stored as a lyophilized powder below -18°C

• Once reconstituted, stored at 4°C for 2-7 days

• For long-term storage, stored below -18°C with a carrier protein (0.1% HSA or BSA)

• Freeze-thaw cycles should be avoided

Inhibitors of GLU-C activity include diisopropyl fluorophosphate (DFP) and monovalent anions such as F⁻, Cl⁻, Br⁻, CH₃COO⁻, and NO₃⁻, which should be excluded from reaction buffers .

How does GLU-C differ from other commonly used proteases in proteomics research?

GLU-C provides complementary coverage to trypsin, the most commonly used protease in proteomics:

This complementary specificity makes GLU-C valuable for protein identification by peptide mass fingerprinting or MS/MS spectral matching, particularly when used in combination with other proteases to increase sequence coverage .

What is the recommended protocol for in-solution protein digestion using GLU-C?

For optimal in-solution protein digestion:

• Reconstitute lyophilized GLU-C in sterile water (18MΩ-cm) at a concentration not less than 100μg/ml

• Prepare proteins in appropriate buffer based on desired specificity:

  • Ammonium bicarbonate (25-100mM) for glutamic acid specificity

  • Phosphate buffer (25-100mM) for both glutamic and aspartic acid specificity

• Use enzyme-to-substrate ratios between 1:20 to 1:50 (w/w)

• Incubate at 25-37°C for 2-18 hours depending on application

• Stop the reaction by acidification (0.1% TFA or formic acid) or heating at 90°C for 5 minutes

Note: GLU-C is not recommended for in-gel digestion applications, which is an important methodological consideration when designing proteomics workflows .

How can N-terminomics approaches be used to study GLU-C cleavage products?

Two primary N-terminomics methods have been successfully applied to study GLU-C cleavage specificity and identify its substrates:

• Aminobiotinylation Method:

  • Labels α-amines at protein N-termini and lysine side chains

  • Followed by digestion and enrichment of N-terminal peptides

  • Provides good coverage of cleavage events

• Terminal Amine Isotopic Labeling of Substrates (TAILS):

  • Involves differential isotopic labeling of N-termini

  • Negative selection approach where non-N-terminal peptides are removed

  • Identified 280 putative V8 cleavage sites in 85 unique human serum proteins

These methods are especially valuable for discovering proteolytic targets in complex biological matrices like human serum, providing insight into the role of GLU-C in S. aureus pathogenesis .

What troubleshooting approaches can be used when GLU-C digestion efficiency is low?

When GLU-C digestion yields suboptimal results, consider these methodological adjustments:

• Inefficient protein denaturation:

  • Ensure complete protein unfolding using higher concentrations of denaturants (8M urea or 6M guanidine HCl)

  • Add mild detergents compatible with downstream applications

• Buffer interference:

  • Check for presence of monovalent anions that inhibit GLU-C activity

  • Verify pH is within optimal range (4.0-9.0)

• Enzyme activity issues:

  • Verify enzyme hasn't undergone multiple freeze-thaw cycles

  • Consider adding carrier proteins to stabilize the enzyme

  • Increase enzyme-to-substrate ratio to 1:10 for difficult-to-digest proteins

• Substrate accessibility:

  • Ensure reduction and alkylation of disulfide bonds is complete

  • Extend digestion time to 24 hours for resistant proteins

How can GLU-C be incorporated into multi-protease digestion strategies to improve proteome coverage?

Multi-protease strategies incorporating GLU-C can significantly enhance proteome coverage:

• Sequential digestion approach:

  • Begin with trypsin digestion under standard conditions

  • Followed by GLU-C digestion in ammonium bicarbonate buffer

  • Analyze samples separately or combine prior to LC-MS/MS

• Parallel digestion approach:

  • Split sample into aliquots for separate digestion with trypsin and GLU-C

  • Analyze individually and combine results bioinformatically

  • Provides complementary peptide coverage due to orthogonal specificities

• Protease cocktail approach:

  • Simultaneous digestion with multiple proteases including GLU-C

  • Requires careful optimization of buffer conditions

  • Best for limited sample amounts where multiple digestions aren't feasible

The complementary specificity of GLU-C (C-terminal to Glu/Asp) to trypsin (C-terminal to Lys/Arg) ensures improved sequence coverage, especially for proteins with low basic amino acid content .

What bioinformatic considerations are necessary when interpreting GLU-C digestion results?

Proper bioinformatic analysis of GLU-C digestion data requires:

• Search parameter optimization:

  • Configure search engines with correct enzyme specificity (C-term Glu or C-term Glu/Asp based on buffer)

  • Allow for missed cleavages (typically 2-3)

  • Consider semi-specific search for unexpected cleavages

• Data analysis adjustments:

  • GLU-C typically generates longer peptides than trypsin

  • Higher charge states may be observed (3+, 4+, 5+)

  • More complicated fragmentation patterns may require additional validation

• Combining multi-protease datasets:

  • Use protein inference algorithms that properly combine evidence from different proteases

  • Avoid counting the same protein region multiple times in quantitative analyses

  • Consider specialized software designed for multi-protease proteomics

• Peptide-spectrum match validation:

  • GLU-C peptides may have different fragmentation efficiency

  • Adjust false discovery rate calculations accordingly

  • Validate unexpected cleavage patterns manually

What host proteins are targeted by GLU-C during S. aureus infection?

N-terminomics studies have identified approximately 85 human serum proteins as targets of GLU-C, with particular relevance to pathogenesis . Key target categories include:

• Complement system components:

  • Cleaved within peptidase and sushi domains

  • Disrupts host complement cascade

• Immunoglobulins:

  • Cleaves IgG, IgA, and IgM heavy chains

  • Protects bacteria against host defense mechanisms

• Coagulation cascade proteins:

  • Disrupts normal blood clotting processes

  • Contributes to invasive infection ability

• Host protease inhibitors:

  • Cleaved outside their protease-trapping motifs

  • Compromises host protease regulation

• Nutrient sequestration proteins:

  • Including iron-binding proteins

  • Facilitates bacterial nutrient acquisition

This extensive "pathodegradome" highlights the multifaceted role of GLU-C in S. aureus virulence and host immune evasion strategies .

How do mutations in S. aureus affect GLU-C expression and function?

Research has identified that stress conditions, including elevated temperatures, can induce mutations in S. aureus regulatory systems that indirectly affect virulence factor expression . While the provided search results don't directly address GLU-C expression under these conditions, related findings suggest:

• Mutations in the purine biosynthesis repressor (purR) enhance S. aureus pathogenic potential

• These mutations can arise in response to environmental stressors, including host immune pressures

• Such regulatory changes often affect multiple virulence factors simultaneously

Understanding the regulatory networks controlling GLU-C expression under various stress conditions represents an important area for future research, as modulation of this protease could contribute significantly to S. aureus adaptation during infection .

What methodologies can be used to study GLU-C activity in infection models?

To investigate GLU-C activity in infection contexts, researchers can employ several approaches:

• Ex vivo proteolysis assays:

  • Incubate purified GLU-C with human serum or tissue extracts

  • Apply N-terminomics approaches to identify cleavage events

  • Compare with control samples to identify GLU-C-specific cleavage sites

• Comparative proteomics of wild-type vs. GLU-C-deficient strains:

  • Generate isogenic S. aureus strains lacking functional GLU-C

  • Compare secretome profiles between wild-type and mutant strains

  • Identify substrates through differential abundance analysis

• In vivo infection models:

  • Utilize animal models infected with wild-type vs. GLU-C-deficient S. aureus

  • Collect samples from infection sites for proteomic analysis

  • Correlate GLU-C activity with pathology and disease progression

• Immunohistochemical approaches:

  • Develop antibodies against GLU-C cleavage-specific neo-epitopes

  • Apply to infected tissues to visualize proteolytic activity in situ

  • Correlate with bacterial localization and tissue damage

These methodologies can provide complementary insights into how GLU-C contributes to S. aureus pathogenesis across different host niches and infection stages.

How does GLU-C activity interact with other S. aureus virulence factors?

GLU-C functions within a complex network of S. aureus virulence mechanisms:

• Regulatory relationships:

  • GLU-C is required for proteolytic maturation of thiol protease SspB

  • It inactivates SspC, an inhibitor of SspB

  • Creates a protease activation cascade similar to host complement systems

• Modulation of bacterial surface proteins:

  • Degrades fibronectin-binding protein (FnBP)

  • Cleaves surface protein A

  • May regulate bacterial adhesion and host cell interactions

• Host-pathogen interface manipulation:

  • Degrades multiple host immune factors

  • Likely works in concert with other immune evasion factors

  • May have temporally regulated expression during different infection phases

Understanding these interactions is crucial for developing comprehensive models of S. aureus pathogenesis and identifying potential intervention points.

What structural features of GLU-C determine its substrate specificity?

The structural basis of GLU-C specificity involves several key features:

Detailed structural studies coupled with substrate profiling represent an important frontier in GLU-C research.

How might GLU-C be exploited as a target for anti-virulence therapeutics?

Given its role in S. aureus pathogenesis, GLU-C presents opportunities for therapeutic intervention:

• Direct inhibition approaches:

  • Development of specific GLU-C inhibitors based on structural information

  • Peptide-based inhibitors mimicking natural substrates

  • Small molecule inhibitors targeting the active site

• Anti-virulence vaccination:

  • GLU-C as a vaccine antigen to generate neutralizing antibodies

  • Similar approaches targeting other S. aureus proteases have shown promise

  • Studies suggest anti-Fnb antibodies can protect against hypervirulence in S. aureus

• Combination approaches:

  • Target multiple S. aureus proteases simultaneously

  • Combine protease inhibition with conventional antibiotics

  • Exploit regulatory networks controlling GLU-C expression

• Diagnostic applications:

  • Detect GLU-C activity as a biomarker of virulent S. aureus infection

  • Develop rapid tests based on specific GLU-C cleavage patterns

  • Use to guide therapeutic decision-making

These approaches could provide alternatives to conventional antibiotics in an era of increasing antimicrobial resistance.