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.

Figure 1

Comparison of protease activities in WSS and WS. Synthetic histatin 5 (100 µg) was added to 1 mL of WS (A) or WSS (B) and incubated at 37°C. Aliquots of 100 µL were removed after the indicated time intervals, boiled, dried, and analyzed by cationic-PAGE. Left lanes in (A) and (B): 10 µg histatin 5; right lanes in (A) and (B): 100 µL WS and WSS, respectively. (C) densitometric analysis of histatin 5 as a function of incubation time in WS and WSS. The intensity of the histatin 5 band immediately upon addition to WS or WSS (t = 0) was set to 100%.

Figure 2

Histatin zymography of WSS and WS. WSS and WS samples were analyzed on zymogram gels with incorporated histatin 1 (left panel), histatin 3 (middle panel) or histatin 5 (right panel). The volumes loaded of each of the saliva sample are indicated. The position of the molecular weight standards (pierced) are depicted in the far left and right lanes.

Figure 3

Assessment of histatin-degradating activity in FPLC fractions from WSS. A mixture of synthetic histatin 1, 3, and 5 (5 µg each) was added to 161 µL of individual FPLC fractions of WSS proteins. After 0, 30, and 90 min of incubation, 50 µL aliquots were boiled, dried, and analyzed by cationic PAGE. (A) example of a fraction displaying activity toward all histatins, (B) example of a fraction displaying activity toward histatin 3 only. (C) example of a fraction devoid of proteolytic activity toward any of the histatins.

Figure 4

Zymography of active WSS fractions. Proteins in fractions 20, 21, 25, 26, 31, 36, 48, and 49 were analyzed by histatin 5 zymography (left panel) and proteins in fractions 6, 9, 14, 15, and 17 were subjected to histatin 3 zymography (right panel). Left lanes, molecular weight standard. Sixteen protein bands displaying proteolytic activity were excised from the gel for subsequent trypsinization and processing by LC-ESI-MS/MS.