Insights into cathepsin-B activity in mature dentin matrix

Marcela R. Carrilho, Polliana Scaffa, Vitor Oliveira, Leo Tjäderhane, Ivarne L. Tersariol, David Henry Pashley, Franklin Tay, Fabio D. Nascimento

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Article number104830
JournalArchives of Oral Biology
Volume117
DOIs
StatePublished - Sep 2020

Keywords

  • Cathepsins
  • Collagen
  • Cysteine proteases
  • Degradation
  • Dentin
  • Proteolytic activity

ASJC Scopus subject areas

  • Otorhinolaryngology
  • Dentistry(all)
  • Cell Biology

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  • Cite this

    Carrilho, M. R., Scaffa, P., Oliveira, V., Tjäderhane, L., Tersariol, I. L., Pashley, D. H., Tay, F., & Nascimento, F. D. (2020). Insights into cathepsin-B activity in mature dentin matrix. Archives of Oral Biology, 117, [104830]. https://doi.org/10.1016/j.archoralbio.2020.104830