Ponlatham Chaiyarit1,2, Janthima Jaresitthikunchai3, Narumon Phaonakrop3, Sittiruk Roytrakul3, Barbara Potempa4, Jan Potempa4,5. 1. Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen, 40002, Thailand. cponla@kku.ac.th. 2. Research Group of Chronic Inflammatory Oral Diseases and Systemic Diseases Associated with Oral Health, Faculty of Dentistry, Khon Kaen University, Khon Kaen, Thailand. cponla@kku.ac.th. 3. Proteomics Research Laboratory, National Center for Genetic Engineering and Biotechnology, Pathumthani, Thailand. 4. Department of Oral Immunology and Infectious Disease, University of Louisville School of Dentistry, Louisville, KY, USA. 5. Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Abstract
OBJECTIVES: The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis. MATERIALS AND METHODS: Recombinant human TFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides. RESULTS: MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin. CONCLUSIONS: Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides. CLINICAL RELEVANCE: The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitis patients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.
OBJECTIVES: The present study was aimed to determine whether trefoil factor family (TFF) peptides which were generally considered to be resistant to proteolysis could be digested by gingipains, a major proteinases produced by Porphyromonas gingivalis. MATERIALS AND METHODS: Recombinant humanTFF1, TFF2, and TFF3 peptides were used as substrates. Gingipains including arginine gingipain (RgpB) and lysine gingipain (Kgp) were used as enzymes. Trypsin was used as a control protease. Matrix-assisted laser desorption/ionization with time-of-flight / time-of-flight (MALDI-TOF/TOF) and liquid chromatography mass spectrometry (LC-MS) were used for analyzing peptide mass signals and amino acid sequences of digested TFF peptides. RESULTS: MALDI-TOF/TOF analyses demonstrated that Kgp, RgpB, and trypsin were able to cleave TFF1 and TFF2 peptides, resulting in different patterns of digested fragments. However, impurity in recombinant TFF3 peptide substrates affected the interpretations of enzymatic reaction by MALDI-TOF/TOF. LC-MS analyses demonstrated that identified fragments of TFF1, TFF2, and TFF3 from digestion by gingipains were similar to those by trypsin. CONCLUSIONS: Using MALDI-TOF/TOF and LC-MS, the present study provides new information that gingipains containing trypsin-like activities are able to digest TFF peptides. CLINICAL RELEVANCE: The proteolytic effects of gingipains on TFF peptides may be responsible for reduction of salivary TFF peptides in chronic periodontitispatients. Further investigations to determine the pathological effects of gingipains on TFF peptides in saliva and periodontal tissues of patients with chronic periodontitis would be of interest.
Entities:
Keywords:
Gingipains; Mass spectrometry; Proteolysis; Proteomic; Trefoil factor
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