Identification of the enzyme activity of human Demodex aspartic protease and its function to hydrolyse host...

Aspartic protease (ASP), a common proteolytic enzyme, plays an important role in the pathogenesis of numerous parasites. However, its role in Demodex remains unclear. Herein, we studied the expression, purification, enzymatic activity detection, and hydrolysis function of human Demodex ASP. The findings showed that recombinant ASP (rASP) possessed aspartic protease activity, which reached optimum levels at pH 2.5-3.0 and 35 °C. Furthermore, the activity of Demodex folliculorum rASP (Df.ASP) was considerably higher than that of Demodex brevis rASP (Db.rASP). Df.rASP also exhibited a more potent hydrolytic ability than Db.rASP. Df.rASP hydrolysed IgG, IgM, and fibronectin, whereas Db.rASP only slightly hydrolysed IgG. Mass spectrometry analysis revealed that Df.rASP exerted hydrolytic effects on 38 HaCaT proteins, more than the 23 proteins hydrolysed by Db.rASP. Sequence alignment and structure modelling of the substrate binding cleft identified three distinct amino acids between Df.ASP and Db.ASP, which should be the molecular basis for their difference in enzymatic activity and hydrolytic function. These results imply that Df.rASP may play a more critical role in the pathogenesis of human Demodex, and molecular data will provide a scientific basis for future analyses of their molecular pathogenesis.