The contribution of Tannerella forsythia dipeptidyl aminopeptidase IV in the breakdown of collagen

Abstract

In this study, we characterized a serine protease from Tannerella forsythia that degrades gelatin, type I, and III collagen. Tannerella forsythia is associated with periodontitis progression and severity. The primary goal of this research was to understand the mechanisms by which T. forsythia contributes to periodontitis progression. One of our previous metatranscriptomic analysis revealed that during periodontitis progression T. forsythia highly expressed the bfor_1659 ORF. The N-terminal end is homologous to dipeptidyl aminopeptidase IV (DPP IV). DPP IV is a serine protease that cleaves X-Pro or X-Ala dipeptide from the N-terminal end of proteins. Collagen type I is rich in X-Pro and X-Ala sequences, and it is the primary constituent of the periodontium. This work assessed the collagenolytic and gelatinolytic properties of BFOR_1659. To that end, the complete BFOR_1659 and its domains were purified as His-tagged recombinant proteins, and their collagenolytic activity was tested on collagen-like substrates, collagen type I and III combined, and on the extracellular matrix (ECM) formed on human gingival fibroblasts culture HGF-1. BFOR_1659 was only found in T. forsythia supernatants, highlighting its potential role on the pathogenicity of T. forsythia. We also found that BFOR_1659 efficiently degrades all tested substrates but the individual domains were inactive. Given that BFOR_1659 is highly expressed in the periodontal pocket, its clinical relevance is suggested to periodontitis progression.

Keywords: collagen; periodontitis; periodontium destruction; progression.

Fig. 1

Structural sequence alignment of human, P. gingivalis and T. forsythia DPP IV (BFoR_1659). Signal peptide in black bold at the N-terminus, with a cleavage site between two glutamines Q-Q at positions 21 and 22 in BFoR_1659 and between threonine and glutamine (Q-T) at position 17 and 18 in P. gingivalis DPPIV. In bold red is the active site on the three proteins located at Ser-593, Asp-668, and His-700 (adopting BFoR_1659 numbering). The S1-pocket, highlighted in yellow is hydrophobic, lipophilic residues Tyr-594, Tyr-629, Trp-622, and Val-619 are almost identical between the three proteins. BFoR_1659 harbors Phe instead of Tyr-594 and Pro instead of Val. The S2-pocket is highlighted in green. P. gingivalis DPP IV and BFoR_1659 lack the phenylalanine and Arginine present in human DPPIV. In the three enzymes, the positive charge of the amino terminus of the substrate peptide is neutralized by the negative charge from the two glutamate residues flanking S2-pocket at the Glu-195 and Glu-196.

Fig. 1

Structural sequence alignment of human, P. gingivalis and T. forsythia DPP IV (BFoR_1659). Signal peptide in black bold at the N-terminus, with a cleavage site between two glutamines Q-Q at positions 21 and 22 in BFoR_1659 and between threonine and glutamine (Q-T) at position 17 and 18 in P. gingivalis DPPIV. In bold red is the active site on the three proteins located at Ser-593, Asp-668, and His-700 (adopting BFoR_1659 numbering). The S1-pocket, highlighted in yellow is hydrophobic, lipophilic residues Tyr-594, Tyr-629, Trp-622, and Val-619 are almost identical between the three proteins. BFoR_1659 harbors Phe instead of Tyr-594 and Pro instead of Val. The S2-pocket is highlighted in green. P. gingivalis DPP IV and BFoR_1659 lack the phenylalanine and Arginine present in human DPPIV. In the three enzymes, the positive charge of the amino terminus of the substrate peptide is neutralized by the negative charge from the two glutamate residues flanking S2-pocket at the Glu-195 and Glu-196.

Fig. 2

Western blots and Coomassie-stained SDS-PAGE gel of His₆-tag recombinant semi-pure proteins A) Coomassie-stained gel and Western blot of 20 μg of rDPPIV (A) Protein ladder (B) SDS-PAGE gel (C) western blot using anti-His-tag antibody. B) Coomassie-stained gel and Western blot of 20 μg of rDppIV and rPepS_9. (A) Western blot using anti-His-tag antibody of (A) rDppIV (B) rPepS_9 (C) of rDPPIV and Western blot showing 82KDa expected size B) Coomassie-stained SDS-PAGE gel and Western blot of rDppIV-N and rPepS_9 showing 38KDa and 25 kDa, respectively.

Fig. 3

Cellular localization of BFoR_1659. A) Western blot to validate the use of the anti-human DPPIV antibody. The signal was present on N-terminus of BFoR_1659 where major homology exists. No detectable signal was present on rPepS_9. B) Western blot showing the cellular localization of BFoR_1659. 40 μg of total proteins of supernatant and its cell lysate were loaded into the SDS-PAGE gel. No detectable signal was seen on cell lysate samples, supernatant sample showed the expected size of 80KDa. (n=3). The left panel shows the Coomassie-stained gel.

Fig. 4

Collagen-like degradation assays. A) Time series experiment to determine the level for expression of BFoR_1659 in supernatants at different times of growth using EnzChek Gelatinase assay. 20 μg of total protein from each supernatant were tested (n=3) B) Gelatinolytic activity of recombinant proteins (EnzChek assay). In all cases, 20 μg of total protein from each sample were tested (n=3) supernatant 96h was taken as reference C) DPP IV-Glo Protease Assay measuring specific DPP IV activity on 20 μg of each protein tested (n=3) supernatant 96h was taken as reference D) Hydrolysis FALGPA peptide 20 μg of total protein from each sample was tested (n=3). Collagenase of C. hystolyticum was taken as a standard of hydrolysis. E) Degradation of collagen type I and III in FITC-labeled Bio-Gide bilayer collagen membranes 20 μg of each protein were tested (n=3). Collagenase of C. hystolyticum was taken as reference.

Fig. 5

Degradation of ECM on Human Gingival Fibroblast (HGF-1) cells grown on cover slides. 30 μg of protein from each sample were incubated for 12h with HGF-1 cells. The residual collagen was measured with Sirius Red assay on culture and after the cells were fixed and imaged. A,B positive control (10 U ml⁻¹ of C.histolyticum collagenase) C,D rDPPIV; E,F rDppIV-N; G,Hr-PepS_9.