title: Single residue determines the specificity of neutrophil elastase for Shigella virulence factors.
abstract: Human neutrophil elastase (NE) is a key host defense protease that cleaves virulence factors of Gram-negative bacteria. NE and cathepsin G (CG) are chymotrypsin-like serine proteases with sequence and structural similarities, and both are abundant in neutrophil granules. Unlike NE, CG does not cleave virulence factors of enteric bacteria. Through structure-function analysis, we identified regions in NE that are essential for cleaving Shigella virulence proteins. NE residues at eight different positions were replaced with analogous amino acids in CG or with alanine. Functional analysis of recombinant mutant proteins showed that a single residue at position 98 and multiple amino acid stretches in the three different regions 58A-61, 163-181, and 216-224 determine NE specificity. These NE mutants cleaved the CG-specific, but not the NE-specific, synthetic peptide substrate and did not degrade Shigella virulence factors. Interestingly, exchanging the amino acid at position 98 in CG for the NE equivalent enabled this CG mutant to cleave Shigella virulence factors. Analysis of the NE proteolytic products of the Shigella virulence factor IpaB shows that NE has specific cleavage sites. These results indicate that Shigella virulence factor specificity maps to a distinct region close to NE's active site.
authors.affiliation: Department of Cellular Microbiology, Max Planck Institute for Infection Biology, Chariteplatz 1, 10117 Berlin, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Averhoff
authors.forename: Michael
authors.initials: M
authors.lastname: Kolbe
authors.forename: Arturo
authors.initials: A
authors.lastname: Zychlinsky
authors.forename: Yvette
authors.initials: Y
authors.lastname: Weinrauch
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D056649:Cathepsin G; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D019272:Leukocyte Elastase / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000138:chemical synthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D012763:Shigella flexneri; D013379:Substrate Specificity; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; C087447:ipaB protein, Shigella; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D019272:Leukocyte Elastase