Activity‐based mass spectrometric characterization of proteases and inhibitors in human saliva

Abstract

Proteases present in oral fluid effectively modulate the structure and function of some salivary proteins and have been implicated in tissue destruction in oral disease. To identify the proteases operating in the oral environment, proteins in pooled whole saliva supernatant were separated by anion-exchange chromatography and individual fractions were analyzed for proteolytic activity by zymography using salivary histatins as the enzyme substrates. Protein bands displaying proteolytic activity were particularly prominent in the 50-75 kDa region. Individual bands were excised, in-gel trypsinized and subjected to LC/ESI-MS/MS. The data obtained were searched against human, oral microbial and protease databases. A total of 13 proteases were identified all of which were of mammalian origin. Proteases detected in multiple fractions with cleavage specificities toward arginine and lysine residues, were lactotransferrin, kallikrein-1, and human airway trypsin-like protease. Unexpectedly, ten protease inhibitors were co-identified suggesting they were associated with the proteases in the same fractions. The inhibitors found most frequently were alpha-2-macroglobulin-like protein 1, alpha-1-antitrypsin, and leukocyte elastase inhibitor. Regulation of oral fluid proteolysis is highly important given that an inbalance in such activities has been correlated to a variety of pathological conditions including oral cancer.

Changes in the salivary protein profile of morbidly obese women either previously subjected to bariatric surgery or not

“…This protease has been identified as a constituent of human saliva which has a role to play in histatin proteolysis [43]. Although it is associated with the human phenotype linked to starvation [3], its role in the composition of saliva is not well understood.…”

Salivary Gluten Degradation and Oral Microbial Profiles in Healthy Individuals and Celiac Disease Patients

“…Donors refrained from eating, smoking, drinking (except water), use of a mouth rinse, and tooth brushing for at least 1 h prior to sample collection. They were then asked to rinse their mouth with water three times and were given a 20-cm by 20-cm Parafilm (Sigma, St. Louis, MO) for masticatory stimulation of salivary flow (44).…”

endoProteoFASP: A novel FASP approach to profile salivary peptidome and disclose salivary proteases

“…Protein fragments at T0 were attributed to the activity of CTSL1 and MEP1A. To date, CTSL1, but not MEP1A, was already identified in saliva through the combination of histatin 5 zymography and LC-ESI-MS/ MS [11]. The presence of MEP1A in saliva remains to be confirmed, though it seems a relevant protease in saliva autolysis, as confirmed by its prediction in all incubation time points.…”

“…For instance, through the analysis of cleavage sites on the main salivary peptide classes, generation of small peptides has been sugested to emerge from cathepsin D activity [9] or from enzymes with activity resembling those of the gastrointestinal tract, namely trypsin, chymotrypsin or elastase [10]. A different strategy using histatin 5 as a substrate in zymography allowed the identification of a total of 13 proteases with cleavage specificities towards arginine and lysine residues [11]. Neverthless, the identification of glutamine endoproteinase that recognizes KPQ↓ as the main consensus sequence remains unconclusive, though being suggested that it probably derives from dental plaque and it is likely of microbial origin [12].…”