Purification and characterization of a 315 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats.
A keratinolytic protease, secreted as the major component by a feline clinical isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography on bacitracin agarose and gel filtration. The apparent molecular mass of the enzyme was 31.5 kDa and the pI was 11.8. The enzyme was not glycosylated and its first 15 N-terminal amino acids showed numerous similarities with other fungal subtilisins. The optimum pH was around 9 while inactivation of the enzyme was reversible at pH 4, but not at pH 11. The enzyme was stable at 37 degrees C with an apparent optimum temperature around 55 degrees C. PMSF, soybean trypsin inhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The highest affinity (Km of 0.37 mM) and physiological efficiency (k(cat)/Km) were obtained for the synthetic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide. These results indicate that the keratinase belongs to the subtilisin-like serine protease family. Purified rabbit immunoglobulins G prepared against the keratinase and used in an immunohistochemical test allowed the detection of the keratinase produced by the fungus invading hair structures in naturally infected cats. The in vitro keratinolytic activity of the enzyme and its production in vivo suggest that it may contribute to pathogenicity.
Mignon
B
B
Department of Parasitology & Parasitic Diseases, Faculty of Veterinary Medicine, University of Liège, Belgium. bmignon@ulg.ac.be
Swinnen
M
M
Bouchara
J P
JP
Hofinger
M
M
Nikkels
A
A
Pierard
G
G
Gerday
C
C
Losson
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002371:Cat Diseases / Q000382:microbiology*; D002415:Cats; D003881:Dermatomycoses / Q000382:microbiology / Q000662:veterinary*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D007700:Kinetics; D008864:Microsporum / Q000201:enzymology*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011480:Protease Inhibitors; D011817:Rabbits; D012867:Skin / Q000382:microbiology; D013696:Temperature
D011480:Protease Inhibitors; D010447:Peptide Hydrolases; C035855:keratinase