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The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes
Published on: May 25, 2018
Versatile substrates and probes for IgA1 protease activity
Santosh K Choudary1, Jiazhou Qiu, Andrew G Plaut
1Department of Chemistry, Tufts University, 62 Talbot Avenue, Medford MA 02155 (USA).
Abstract:
Bacterial meningitis is a severe infectious disease with high mortality. Gram-positive and Gram-negative bacteria that cause meningitis secrete immunoglobulin A1 (IgA1) proteases to assist in mucosal colonization, invasion, and immune evasion. IgA1 proteases have unique selectivity, with few reported substrates other than IgA1 from human tissue. Here we describe the design, characterization, and application of peptide substrates for diverse IgA1 proteases from Neisseria, Haemophilus, and Streptococcus bacteria. IgA1 proteases from diverse strains showed unexpected selectivity profiles among peptide substrates derived from autoproteolytic sites. A fluorescence probe derived from one of these peptides was used to quantitate IgA1 protease activity in buffer and in human cerebrospinal fluid; it was able to detect recombinant Haemophilus influenzae type 1 IgA1 protease at less than 1 μg mL(-1) . We also used the probe to establish the first high-throughput screen for IgA1 protease inhibitors. This work provides tools that will help investigate the roles of IgA1 proteases in bacterial colonization, immune evasion, and infection.
Insights
Researchers developed novel peptide substrates and a fluorescence probe to study bacterial immunoglobulin A1 (IgA1) proteases. This tool enables quantification of IgA1 protease activity and the screening for inhibitors, aiding infection research.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Bacterial meningitis is a life-threatening infection.
- Pathogenic bacteria like Neisseria, Haemophilus, and Streptococcus use immunoglobulin A1 (IgA1) proteases for colonization and immune evasion.
- Existing knowledge of IgA1 protease substrates is limited.
Purpose of the Study:
- To design and characterize novel peptide substrates for bacterial IgA1 proteases.
- To develop a sensitive assay for quantifying IgA1 protease activity.
- To establish a high-throughput screening method for IgA1 protease inhibitors.
Main Methods:
- Design and synthesis of diverse peptide substrates based on bacterial IgA1 proteases.
- Characterization of protease selectivity profiles using these peptide substrates.
- Development of a fluorescence probe assay for IgA1 protease activity measurement.
- Application of the probe in buffer and human cerebrospinal fluid.
- Establishment of a high-throughput screen for IgA1 protease inhibitors.
Main Results:
- Diverse IgA1 proteases exhibited unexpected selectivity profiles on the designed peptide substrates.
- A fluorescence probe accurately quantified IgA1 protease activity, detecting Haemophilus influenzae type 1 IgA1 protease at <1 μg/mL.
- The first high-throughput screen for IgA1 protease inhibitors was successfully established.
Conclusions:
- Novel peptide substrates and a fluorescence probe provide valuable tools for studying bacterial IgA1 proteases.
- These tools facilitate the investigation of IgA1 protease roles in bacterial pathogenesis.
- The developed screening platform enables the discovery of potential therapeutic inhibitors.
