Characterization of a streptococcal endopeptidase with homology to human endothelin-converting enzyme.
A gene encoding an endopeptidase from Streptococcus parasanguis FW213 has been cloned and shown to have high sequence homology to genes encoding mammalian metalloendopeptidases. The gene, designated S. parasanguis pepO, was cloned into the pET28a expression vector, resulting in a fusion of vector sequences encoding a hexahistidine tag at the carboxyl terminus. The recombinant PepO (rPepO) was expressed in Escherichia coli and purified using an Ni(2+) affinity column. Polyclonal antiserum to rPepO was raised in rabbits and used to localize FW213 PepO to the cytosol. Southern hybridization and immunoblot analysis revealed that other oral streptococci contain regions of DNA with homology to pepO and produce a protein with antigenic properties similar to that of FW213 PepO. Enzymatic activity assays indicated that only S. parasanguis species possess the ability to cleave metenkephalin, the natural substrate of the human neutral endopeptidase (NEP). Inhibition assays revealed that S. parasanguis PepO is a member of the M13 category of metalloendopeptidases, which includes NEP and endothelin-converting enzyme 1 (ECE-1), an enzyme involved in the maintenance of vascular tone. Thiorphan and phosphoramidon, two specific inhibitors of this category of endopeptidases, were used to determine that S. parasanguis PepO is more similar to ECE-1 than to NEP.
Oetjen
J
J
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405, USA.
Fives-Taylor
P
P
Froeliger
E
E
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics; D003600:Cytosol / Q000737:chemistry; D000072396:Endothelin-Converting Enzymes; D036781:Fimbriae Proteins; D006801:Humans; D008666:Metalloendopeptidases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification; D013291:Streptococcus / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C403708:fap1 protein, Streptococcus; C063055:fimbrillin; D036781:Fimbriae Proteins; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C080385:oligopeptidase PepO; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes