1887

Abstract

An important virulence factor of the opportunistic human pathogen is elastase, a secreted thermolysin-like neutral zinc-metalloprotease (TNP). Elastase is synthesized as a larger precursor with an amino-terminal 18 kDa propeptide, and was the first TNP shown to require its propeptide as an intramolecular chaperone (IMC) for activity and secretion. This paper reports the analysis of the elastase propeptide to identify residues conserved among other TNP precursors that may be critical for its IMC function. The prosequences of TNP precursors from both Gram-negative ( species and species) and Gram-positive ( species) bacteria were found to show homology to the elastase propeptide. Two regions of conserved residues were observed: a hydrophilic region (ProM) found in the middle of the elastase propeptide and a more hydrophobic region (ProC) located proximal to the propeptide-processing site. To test whether such conserved motifs were important to function, single residue substitutions at eight conserved amino acids were introduced within the full-length pre-proelastase precursor by site-specific mutagenesis of , the gene encoding elastase. Mutant alleles were expressed from plasmids within a -deleted strain, FRD740, and the effects of these propeptide alterations on elastase enzyme activity, processing, stability and accumulation inside and outside of the cell were examined. Within the ProM region, substitution at Arg74 resulted in a dramatic accumulation of proelastase in the cell, suggesting a secretion defect, and a dramatic reduction in supernatant elastolytic activity. Substitution at Asn68 adversely affected the amount of elastase in the culture supernatant, apparently as a result of the reduced stability of the mutated proelastase in the cell. Within the ProC region, mutations at Ile181 and Ala183 abolished the accumulation of a stable elastase molecule in the supernatant. Most mutations produced a phenotype consistent with a defect in protein folding and stability. Overall, the data from this preliminary study show that conserved residues within the elastase propeptide are essential for its function and begin to define the mechanisms of action of IMCs in the TNP family.

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2004-12-01
2019-10-18
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