Insights into cathepsin-B activity in mature dentin matrix
Introduction
Dentin matrix exhibits intrinsic host-derived gelatinolytic activity that has been mainly attributed to the presence of matrix metalloproteinases (MMPs) (Martin-De Las Heras, Valenzuela, & Overall, 2000; Mazzoni et al., 2007; Santos et al., 2009). Classified as matrixins, MMPs are zinc- and calcium-dependent enzymes capable of hydrolyzing almost all components of the extracellular matrix (ECM) (Visse & Nagase, 2003). In the dentin-pulp complex, MMP activity is associated with breakdown of collagen matrices in the pathogenesis of dentinal caries (Mazzoni et al., 2015; Tjäderhane et al., 1998), dentin erosion (Tjäderhane, Buzalaf, Carrilho, & Chaussain, 2015) and degradation of aged, poorly resin-infiltrated dentin-bonded interfaces (Breschi et al., 2018).
Other mammalian proteases potentially exhibiting collagenolytic/gelatinolytic activities include serine and cysteine proteases (Lecaille, Kaleta, & Brömme, 2002). Under certain conditions, lysosomal cysteine proteases have been shown to degrade several extracellular matrix components of connective tissues (Dickinson, 2002; Turk, Turk, & Turk, 1997). The co-expression of MMPs and cysteine proteases has been reported in human odontoblasts, pulp tissues (Nascimento et al., 2011; Tersariol et al., 2010) and dentin matrices (Scaffa et al., 2017). However, co-distribution of two distinct families of proteases (MMPs and Cathepsins) in a mature substrate like dentin is unanticipated, especially considering that the synthesizing cells (i.e. odontoblasts) are expected to exhibit low metabolic activity in post-development physiologic conditions. Thus, the observation of markedly higher activity of MMPs and cathepsins in carious versus dentin, indicates these two groups of proteases may likely play adjunctive roles in caries pathogenesis (Nascimento et al., 2011; Vidal et al., 2014).
While MMP collagenases (MMP-1, -8, -13) cleave the triple helical structure of native collagens at a neutral pH, and at specific a single site (Chung et al., 2004; Gioia et al., 2007), cathepsins primarily cleave the non-helical telopeptide extensions of tropocollagen at acidic pH (Garnero et al., 1998; Visse & Nagase, 2003). To date, the only cathepsin that has shown true triple helical collagenolytic activity is cathepsin K (Pungercar et al., 2009). Although several other cathepsins have been detected in the dentin-pulp complex, gene expression for cathepsin B in intact teeth has been shown to be expressed more than other cathepsins (Tersariol et al., 2010). Cathepsin B is the most abundant and thoroughly studied cysteine protease in human cells (Pungercar et al., 2009), and is a unique member of the papain family of enzymes that exhibits endo- and exopeptidase activities. This means that cathepsin B can exert its activity by mediating the breakdown of peptide bonds between non-terminal amino acids (i.e. within the molecule) or C-terminal amino acids (Mort, 2013).
The reason why cathepsin B is abundantly expressed in dentin-pulp complex (Tersariol et al., 2010) and co-exists with MMPs in intact dentin matrix (Scaffa et al., 2017) is presently unknown. A previous study by our group indicated that even if co-existing, the gelatinolytic activity of MMPs in intact mature dentin seems to be predominant over those of cathepsins (Scaffa et al., 2017). We suspect that the leading activity of one group of proteases over other in a given tissue is likely dependent on pH. Cathepsins (CTs) are auto-activated and functional in low pH, but they are shown to be unstable and inactive in neutral pH (Turk, Turk, & Turk, 2000). In order to shed light upon a potential interplay between MMPs and CTs in mature dentin, the present study assessed the host-derived proteolytic activity of cathepsin B in mature dentin matrix under conditions wherein collagen and its denatured form (i.e. gelatin) should be predominantly degraded by MMPs (i.e. in neutral pH). The tested hypothesis was that cathepsin B does not exhibit gelatinolytic activity at pH that favors dentinal MMP activity.
Section snippets
Material and methods
Reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
Western-blot
Three bands were detected at 95, 72 and 30 kDa for all dentin extracts, corresponding to the activated forms of the enzymes MMP-9, MMP-2 and cathepsin B, respectively (Fig. 1).
Hydrolysis of fluorogenic peptides and mass spectroscopy
Gelatinolytic activities in G1- and G2-dentin extracts were variably inhibited by the different protease inhibitors selected for this study (Fig. 2). A significant decrease in the hydrolysis of the peptide Abz-GPLGLWARG-EDDnp was observed only in the presence of o-phenanthroline (Fig. 2A) (p < 0.05), with the extract G1
Discussion
The use of fluorescence resonance energy transfer (FRET) peptide substrates has proven to be convenient approach to assess the proteolytic activity of dentinal proteases (Scaffa et al., 2017). It relies on the fact that the fluorescence of a donor group grafted into the intact peptide substrate may be easily monitored, and that the fluorescence is quenched by a distant group upon separation by proteolytic cleavage (Chagas, Juliano, & Prado, 1991; Knight, 1995). Donor/acceptor pairs frequently
Author contributions
Marcela R. Carrilho contributed to conception, design, data acquisition and interpretation, drafted and critically revised the manuscript, project administration and funding acquisition; Polliana Scaffa contributed to data acquisition and interpretation, drafted and critically revised the manuscript; Vitor Oliveira contributed to data interpretation and analyses, drafted and critically revised the manuscript. Leo Tjäderhane: contributed to conception, data curation and analyses, drafted and
Declaration of Competing Interest
We, undersigned authors of the manuscript titled “Insights into Cathepsin-B Activity in Mature Dentin Matrix”, declare no conflict of interest of any nature and that the present manuscript has been submitted solely to the Achieves Oral Biology and is not under consideration for publication in any other journal. This paper has been compiled with the knowledge, input and approval of all the named authors.
Acknowledgments
The authors report no conflict of interest. The study was funded with grants FAPESP (11/12226-8, 16/07996-2 P.I. Carrilho and 13/05822-9 P.I. Nascimento) and CNPq (453651/2011-9 and 312768/2013-3 P.I. Carrilho), Brazil. The fluorogenic peptides were generous gifts from Dr. L. Juliano (Federal University of São Paulo, Brazil).
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