Elevated levels of cysteine protease activity in saliva and salivary glands of the nonobese diabetic (NOD) mouse model for Sjögren syndrome

Abstract

Nonobese diabetic (NOD) mice develop an anti-exocrine gland pathology similar to human Sjögren syndrome. Recently, we demonstrated that NOD-scid mice develop severe loss of submandibular acinar cells with concomitant appearance of abnormal isoforms of salivary proteins suggesting de novo enzymatic cleavage. Because these changes may indicate activation of apoptotic proteases, we examined saliva and salivary tissue for cysteine protease activity. Cysteine protease activities were elevated in saliva and gland lysates from 20-week-old NOD and NOD-scid mice as compared with age- and sex-matched BALB/c or 8-week-old NOD mice. This activity appeared in the submandibular glands, but not in the parotid glands. Western blot analyses using antibodies directed against specific apoptotic proteases (interleukin 1beta converting enzyme, Nedd-2, and Apopain/CPP 32) confirmed these findings. Submandibular glands from NOD-scid mice exhibited the greatest increase in proteolytic activity, indicating that infiltrating leukocytes are not responsible for these changes. Western blot analyses also failed to reveal changes in the levels of cystatins (saliva proteins that inhibit protease activity). Thus, increased cysteine protease activity appears to be directly related to submandibular acinar cell loss in NOD-scid mice involving the apoptotic pathway. Additional protease activity in saliva and gland lysates of older NOD and NOD-scid mice, apparently mutually distinct from cysteine proteases, generated an enzymatically cleaved parotid secretory protein. We suggest, therefore, that proteolytic enzyme activity contributes to loss of exocrine gland tolerance by generating abnormally processed protein constituents.

Figure 1

Histogram of cysteine protease activity in salivary gland lysates. Parotid (PAR) and submandibular (SMX) gland lysates were incubated with chromagenic substrate, BAPNA, for 60 min at 37°C (P-DM and DM, prediabetic and diabetic, respectively). Standard curve was generated by linear regression analysis of papain digestion of BAPNA. All values represent the mean ± SE performed in duplicate on three separate occasions.

Figure 2

Western blot analysis of saliva for the presence of apoptotic proteases. Whole saliva (15 μg) was separated on a 12% SDS/PAGE and evaluated for the presence of murine ICE, Nedd-2, and Apopain/CPP 32 using rabbit polyclonal antibodies. The fidelity of the antibody reaction was established by preincubation of the antibodies with the peptide antigen (1 μg/ml) prior to reaction with the nitrocellulose membrane. Cleavage of the ICE p45 pro-enzyme generates p20 and p10 active subunits. Proteolytic cleavage of Nedd-2 the p50 pro-enzyme generates the p20 and p12 active subunits, cleavage of the Apopain/CPP 32 p35 pro-enzyme produces the p17 and p12 subunits of the mature protease. Prestained molecular weight standards (Bio-Rad) are as follows: ovalbumin, 50,600 Da; carbonic anhydrase 35,500 Da; soybean trypsin inhibitor, 29,100 Da; and lysozyme, 20,900 Da.

Figure 3

Western blot analysis of saliva for the presence of the cysteine protease inhibitor, cystatin. Whole saliva (15 μg) was examined on a 12% SDS/PAGE gel using a rabbit-polyclonal anti-rat cystatin. A chronically treated isoproterenol rat whole saliva was included as a positive control. Isoproterenol treatment of mice was twice i.p. daily for 3 days. P-DM, prediabetic NOD; DM, diabetic NOD. Molecular weight standards are as follows: bovine serum albumin, 84,000 Da; ovalbumin, 54,000 Da; carbonic anhydrase, 35,000 Da; and soybean trypsin inhibitor, 28,000 Da.

Figure 4

Autoradiogram of differential PSP migration following incubation with saliva or salivary gland lysates. [¹²⁵I]PSP (10⁴ cpm) purified from BALB/c saliva was incubated for 4–6 hr at 37°C with whole saliva, submandibular (SMX) lysates, or parotid (PAR) lysates from BALB/c or NOD mice. NOD P-DM, prediabetic NOD; NOD DM, diabetic NOD. Radiolabeled BALB/c PSP (lane 1) incubated in PBS served as control migration.