TY - JOUR
T1 - Insights into cathepsin-B activity in mature dentin matrix
AU - Carrilho, Marcela R.
AU - Scaffa, Polliana
AU - Oliveira, Vitor
AU - Tjäderhane, Leo
AU - Tersariol, Ivarne L.
AU - Pashley, David H.
AU - Tay, Franklin
AU - Nascimento, Fabio D.
N1 - Publisher Copyright:
© 2020
PY - 2020/9
Y1 - 2020/9
N2 - Objective: Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH. Design: Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates’ cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B. Results: Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity. Conclusions: Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH.
AB - Objective: Cysteine proteases are lysosomal enzymes that, under specific circumstances, may be secreted into the extracellular space and participate in protein turnover. This study investigated the involvement of cathepsin B in the gelatinolytic activity of mature dentin matrices at neutral pH. Design: Human dentin fragments were made into powder and enzymes were extracted using guanidine-HCl/EDTA. Host-derived dentin proteases (cathepsin B, MMP-2 and MMP-9) were identified by immunoblotting, and their activities were evaluated spectrofluorimetrically using fluorogenic substrates. Proteases activities were monitored by measuring the rate of hydrolysis of substrates in the presence/absence of MMP- or cysteine cathepsin inhibitors, at neutral pH (7.4). Mass spectroscopy was used to determine the substrates’ cleavage points. Reverse zymography was performed to examine the gelatinolytic activity of cathepsin B. Results: Western-blots of dentin extracts yielded strong bands at 95, 72 and 30 kDa, corresponding respectively to MMP-9, MMP-2 and Cathepsin B. Greater fluorogenic substrates hydrolysis occurred in the absence of MMP and cysteine cathepsin inhibitors than in their presence. Cathepsin B exhibited significant gelatinolytic activity. Conclusions: Together with MMP-2 and MMP-9, cathepsin B also account for the host-derived gelatinolytic activity and matrix turnover of mature dentin at physiological, neutral pH.
KW - Cathepsins
KW - Collagen
KW - Cysteine proteases
KW - Degradation
KW - Dentin
KW - Proteolytic activity
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U2 - 10.1016/j.archoralbio.2020.104830
DO - 10.1016/j.archoralbio.2020.104830
M3 - Article
C2 - 32673819
AN - SCOPUS:85087788620
SN - 0003-9969
VL - 117
JO - Archives of Oral Biology
JF - Archives of Oral Biology
M1 - 104830
ER -