Salivary agglutinin is the major component in human saliva that modulates the lectin pathway of the complement system

Collection of saliva

Human whole saliva was collected without stimulation from healthy donors, three secretors and three non-secretors (aged 25–63 yr). In addition, secretions of the SM and SL glands were separately collected from one donor (male, blood group B, secretor) using custom-fitted devices, as previously described. ¹⁶ From the same donor, parotid saliva was collected with a Lashley cup, under stimulating conditions (chewing on a sugar-free menthol chewing gum). All saliva samples were homogenized on a Vortex mixer (1 min) and centrifuged (10,000 g, 10 min) to remove cellular debris. The supernatant was stored at −20℃ until use. Whole saliva was subsequently mentioned clarified human whole saliva (CHWS).

Serum samples

Blood was withdrawn from MBL-sufficient (MBL⁺) and MBL-deficient (MBL⁻) healthy volunteers, allowed to clot for 1 h at room temperature (20–25℃) and centrifuged (2000 g, 10 min). Serum was stored at −80℃ until use. Informed consent was obtained from all donors.

Determination of saliva-mediated activation of complement

C4 deposition assays on saliva were conducted as previously described.^10,14 Wells of microtiter plates (NuncMaxiSorp; Nunc, Roskilde, Denmark) were coated [overnight (16 h), 4℃] with saliva, twofold serially diluted in 0.1 M Na₂CO₃, pH 9.6. Serum (1%) was added in veronal buffer supplemented with 10 mM CaCl₂, 2 mM MgCl₂ and 0.02% Tween-20 (VB⁺⁺) or in veronal buffer with 10 mM EDTA and 0.02% Tween-20 (VB/EDTA), and incubated for 1 h at room temperature, followed by incubation with biotinylated anti-C4-10 mouse mAb (Sanquin, Amsterdam, the Netherlands) 1:2000 diluted in PBS with 0.1% Tween-20 (PBST) supplemented with 1% BSA (blocking buffer). HRP-conjugated avidin (Abcam, Cambridge, UK) (0.5 µg/ml in blocking buffer) was added (30 min, room temperature), followed by 100 µl o-phenylenediamine (OPD) substrate (SIGMAFAST; Sigma-Aldrich, St. Louis, MO, USA). Reaction was stopped by the addition of 50 µl 1 M H₂SO₄ and the absorbance at 492 nm was measured using a Multiskan FC microplate photometer (Thermo Scientific, Rockford, IL, USA). After each incubation step, plates were washed three times with PBST.

Inhibition of complement activation by saliva

Microtiter plate wells were coated with 10 µg/ml mannan (Sigma-Aldrich) (in 0.1 M Na₂CO₃, pH 9.6). ¹⁰ Wells were incubated with serial dilutions of serum in VB⁺⁺ supplemented with 20% saliva or 20% saliva buffer (2 mM potassium phosphate, 50 mMKCl, 1 mM CaCl₂, 0.1 mM MgCl₂, pH 6.8) as control. Incubation steps and detection of C4 deposition were performed as described above. Binding of MBL was determined with biotinylated anti-MBL/1 (Sanquin), 1:1000 diluted in VB⁺⁺, followed by incubation with streptavidin poly-conjugated with HRP (1:5000; Sanquin). For MBL-binding assays plates were washed with H₂O between incubation steps.

Determination of secretor status

The secretor status of each donor was determined as earlier described, ¹⁷ with minor alterations. Briefly, microtiter plate wells were coated with saliva (1:800 diluted in 0.1 M Na₂CO₃, pH 9.6) and blocked with blocking buffer. After washing, biotinylated α-Fuc-(1→2)-Gal-binding Ulex europaeus agglutinin (UEA-1; Pierce, Rockford, Il, USA) (1:2000 diluted in blocking buffer) was added, followed by incubation for 1 h. Wells were washed and incubated with HRP-conjugated avidin (Abcam) (0.5 µg/ml in blocking buffer) for 30 min. Color was developed with SIGMAFAST OPD. Donors with a positive result for UEA-1 binding were defined as secretors.

Salivary proteins

Human amylase was from Sigma-Aldrich. Histatin 2 was synthesized by solid-phase peptide synthesis using Fmoc chemistry with a MilliGen 9050 peptide synthesizer (Milligen-Biosearch, Bedford, MA, USA) and purity was checked by mass spectrometry, as previously described. ¹⁸ SAG was purified from parotid saliva by applying a precipitate on a Sepharyl S-400 gel filtration column as described before.^19,20 SAG-enriched samples were obtained as previously described and dissolved in 150 mM NaCl. ¹⁴ MUC5B was isolated from human saliva as described by Veerman et al. ²¹

SDS-PAGE analysis of saliva

SDS-PAGE was performed on NuPAGE 4–12% BisTris gels (Thermo Scientific) under reducing conditions. Saliva samples were boiled for 10 min with LDS sample buffer and reducing agent (Thermo Scientific). Samples were loaded on the gel and run for 35 min at 120 V. Novex sharp pre-stained proteins standards (Thermo Scientific) were used as molecular mass markers. Subsequently, gels were incubated with Coomassie R 350 stain (GE Healthcare, Uppsala, Sweden) for 2 h at room temperature, followed by overnight de-staining with 10% acetic acid.

MALDI-TOF analysis

After electrophoresis, protein bands of interest were cut out of the gels with a razor blade, sliced into smaller pieces, washed with 25% acetonitrile and 0.1% trifluoroacetic acid (TFA) in water, and sonicated for 30 min in 25% acetonitrile and 0.1% TFA. The samples were extracted from the gel pieces with 500 µl 65% acetonitrile and 0.1% TFA in water overnight at room temperature while shaking. The samples were lyophilized in a Speed vac evaporator (RVC 2–25 CD plus; Martin Christ, Osterode am Harz, Germany), dissolved in 50 µl H₂O, vortexed and lyophilized again. Then, the samples were analyzed by MALDI-TOF-MS as described previously. ¹⁹

Adsorbed saliva activates complement in an MBL-dependent way

The complement-activating properties of whole and glandular human saliva were investigated by determining saliva-mediated C4 deposition. To this end, serum was incubated on saliva-coated microtiter plates (Figure 1). Serum diluted in a buffer containing 10 mM EDTA was included as a negative control. To explore whether the CP or the LP was involved, both MBL⁺ and MBL⁻ serum were used. C4 deposited only with MBL⁺ serum on all types of saliva, indicating that saliva activates the complement system via the LP. Most C4 deposition was found for CHWS, followed by SM and SL saliva, while the least C4 deposition was found for parotid saliva. The amount of C4 deposition corresponded with the amount of SAG present in the different samples that were coated on the microtiter plates as determined with anti-SAG (Figure 1e). Equal amounts of SAG were detected on microtiter plates coated with CHWS, SM and SL saliva, whereas SAG was hardly detectable on microtiter plates coated with parotid saliva. C4 deposition was also tested in CHWS samples of three secretor and three non-secretor donors, as defined by UEA-1 binding (Supplementary Figure 1). C4 deposited on saliva of secretors, but not on saliva of non-secretors (Figure 2), in line with a previous study of SAG. ¹⁴ OPEN IN VIEWER

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Figure 2.

The effect of the secretor status on saliva-mediated complement activation. CHWS of six donors (d1–d6) was coated in serial dilutions on microtiter plates. After incubation with MBL⁺ serum (1% in VB⁺⁺) C4 deposition was measured. These results show the mean ± SE of three experiments performed in duplicate. secr: secretor; non-secr: non-secretor.

Saliva inhibits complement activation and MBL binding in solution

To investigate the complement-inhibiting properties of saliva, MBL⁺ serum was twofold serially diluted in VB⁺⁺ in the presence of 20% saliva solution or saliva buffer, which simulates the ionic composition of saliva, and added to mannan-coated microtiter plate wells. After incubation, deposition of C4 was determined. CHWS and SM saliva of a secretor inhibited both the C4 deposition on mannan and the MBL binding to mannan for 85–90% (Figure 3a, b), while parotid saliva had little, if any, effect. In contrast to C4 activation by adsorbed saliva, which was only mediated by saliva from secretors, saliva of both secretors and non-secretors inhibited the MBL-mediated C4 deposition in solution (Figure 3c), as well as the binding of MBL to mannan (Figure 3d). These results suggest that different interactions are responsible for activation and inhibition of the LP, depending on whether saliva is in adsorbed phase or in solution. OPEN IN VIEWER

Effect of SAG depletion on the complement-activating and complement-inhibiting properties of saliva

To test if besides SAG, other salivary proteins modulate the LP, saliva samples of one secretor donor were depleted from SAG by incubation with S. mutans. ELISA revealed that by these treatments approximately 70% of SAG was depleted from CHWS, 90% from SL saliva and virtually 100% from SL saliva (Figure 4a). SDS-PAGE followed by Coomassie Blue staining, showed that other salivary proteins were not depleted, except for a peptide from parotid and SM saliva (Figure 4b). MALDI-TOF analysis of the band excised from the gel revealed that this was histatin 2 (Figure 4c). OPEN IN VIEWER

Next, the SAG-depleted samples were tested for C4 activation or inhibition in comparison with the SAG-containing samples. Less C4 deposited on SAG-depleted CHWS and SL saliva than on the SAG-containing samples (Figure 5a). This corresponded with the lower amount of SAG detected in the ELISA (Figure 4a). In line with this, little C4 deposited on SM saliva (Figure 5a). CHWS and SM saliva in solution, but not the SAG-depleted saliva samples, inhibited MBL-mediated deposition of C4 on mannan, partially (CHWS; Figure 5b) or completely (SM saliva; Figure 5c). The partial inhibition by SAG-depleted CHWS may be due to residual SAG in this sample (Figure 4a). OPEN IN VIEWER

In addition to SAG, saliva contains a number of other glycosylated proteins, such as MUC5B, MUC7 and amylase, which could potentially activate the LP by binding carbohydrate recognition domains of MBL. ¹⁵ Thus, we tested the complement-modulating properties of a number of these proteins. Furthermore, we tested the un-glycosylated peptide histatin 2 because, besides SAG, it was the only compound that was removed from saliva by treatment with S. mutans (Figure 4b). C4 deposition was only found on adsorbed SAG, but not on amylase, MUC5B or histatin 2 (Figure 6a). In line with this, MBL bound only to adsorbed SAG but not to amylase, MUC5B or histatin 2 (Figure 6b). In solution, only purified SAG inhibited the C4 deposition on mannan, suggesting that when saliva is in solution, SAG is the major inhibitor of the MBL pathway. As complement activation by saliva is dependent on the secretor status, while complement inhibition by saliva in solution is independent of the secretor status, we assessed the complement-inhibiting properties of SAG-enriched parotid saliva samples from a secretor and a non-secretor. In solution, both SAG samples inhibited C4 deposition on mannan to the same extent (Figure 7). Taken together, these results show that SAG is the protein in saliva involved in LP modulation and that LP inhibition by saliva or SAG in solution is independent of a donor’s secretor status. OPEN IN VIEWER

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Figure 7.

Inhibition of C4 deposition by SAG. MBL⁺ sera were serially diluted in VB⁺⁺ supplemented with 10 µg/ml SAG (20%) of a secretor (filled circle), a non-secretor (filled square) or 30 mM NaCl (open circle) (NaCl), C4 deposition on mannan was measured. Results represent mean ± SE of three experiments performed in duplicate.