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Isolation, characterization and regulation of moonlighting proteases from Candida glabrata cell wall.

Microbial pathogenesis (2020-10-04)
Pirjo Pärnänen, Timo Sorsa, Taina Tervahartiala, Pirjo Nikula-Ijäs
ABSTRACT

Candida glabrata (C. glabrata) cell wall proteins play a role in virulence and in initial host immune recognition and responses. We isolated and characterized C. glabrata cell wall proteases from a clinical hospital C. glabrata T-1638 blood isolate and estimated the enzymatic activities and their ability to degrade gelatin and processing proMMP-8 and assess the regulation of these proteases with salt treatment, mercaptoethanol and fermented lingonberry juice from Vaccinium vitis idaea L. The cell wall proteases were enzymatically released from the cell wall and beta- 1,3- bonded proteases were fractioned into 10-50 kDa and >50 kDa fractions with anionic DEAE-sepharose ion-exchange chromatography and gel filtration. Proteins were monitored and analyzed with MDPF- zymography, and five gelatinolytic bands were cut out from a parallel silver-stained gel for the LC- MS/MS analysis. The proteases lacked a signal sequence, indicating that they are moonlighting proteases. Human proMMP-8 activation assays were performed with both fractions and verified by western-immunoblot using aMMP-8 specific antibody. Inhibition of proMMP-8 conversion to the lower molecular active enzyme species were demonstrated with fermented lingonberry juice. The results indicate that moonlighting proteases may play a role in the virulence of C. glabrata.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
DL-Dithiothreitol, ≥98% (HPLC), ≥99.0% (titration)