Binding of salivary agglutinin to IgA

Abstract

SAG (salivary agglutinin), which is identical to gp-340 (glycoprotein-340) from the lung, is encoded by DMBT1 (deleted in malignant brain tumours 1). It is a member of the SRCR (scavenger receptor cysteine-rich) superfamily and contains 14 SRCR domains, 13 of which are highly similar. SAG in saliva is partially complexed with IgA, which may be necessary for bacterial binding. The goal of the present study was to characterize the binding of purified SAG to IgA. SAG binds to a variety of proteins, including serum and secretory IgA, alkaline phosphatase-conjugated IgGs originating from rabbit, goat, swine and mouse, and lactoferrin and albumin. Binding of IgA to SAG is calcium dependent and is inhibited by 0.5 M KCl, suggesting that electrostatic interactions are involved. Binding of IgA was destroyed after reduction of SAG, suggesting that the protein moiety is involved in binding. To pinpoint further the binding domain for IgA on SAG, a number of consensus-based peptides of the SRCR domains and SRCR interspersed domains were designed and synthesized. ELISA binding studies with IgA indicated that only one of the peptides tested, comprising amino acids 18-33 (QGRVEVLYRGSWGTVC) of the 109-amino-acid SRCR domain, exhibited binding to IgA. This domain is identical to the domain of SAG that is involved in binding to bacteria. Despite this similar binding site, IgA did not inhibit binding of Streptococcus mutans to SAG or peptide. These results show that the binding of IgA to SAG is specifically mediated by a peptide sequence on the SRCR domains.

Figure 1. Binding of SAG to various proteins

Microplates were coated with serum IgA, secretory IgA (S-IgA) (4 μg/ml), human albumin (10 μg/ml) (A), bovine lactoferrin (1 mg/ml), rabbit IgG antibodies against CEA-1 antigen (10 μg/ml) or gelatin (1 mg/ml) (B). After coating, microplates were incubated with parotid saliva serially diluted in PBST-Ca [1:1 (v/v) starting dilution]. Bound SAG was revealed with antibody 5E9. SAG bound to antibodies, albumin and lactoferrin, but only weakly to gelatin.

Figure 2. Effects of bivalent ions on SAG binding to IgA

Microplates coated with serum IgA (2 μg/ml) were incubated with serial dilutions of SAG in PBST containing different bivalent ions (0.5 mM). After washing, bound SAG was revealed with monoclonal antibody 143. SAG binding was Ca²⁺-dependent (♦). Binding completely disappeared in the presence of 5 mM of the calcium-chelating agent EDTA (▪). Ca²⁺ ions could not be replaced by other bivalent ions such as Mg²⁺ (▴), Ba²⁺ (×) or Zn²⁺ (✱).

Figure 3. Inhibition of SAG binding to IgA

IgA-coated microplates (2 μg/ml) were incubated with parotid saliva serially diluted in PBST-Ca containing inhibitory substances. After washing, bound SAG was revealed with antibody 143. (A) Binding of SAG to IgA was completely inhibited by amine-containing substances such as 0.1 M NH₄Cl (▪), but not by carbohydrates such as 0.1 M maltose (×), in the presence of which binding was comparable with that in the control (♦). (B) Negatively charged ions such as nitrate (•) and carbonate (×) (0.1 M) showed low or intermediate inhibition compared with the control (♦). KCl at 0.5 M (▴) completely inhibited binding.

Figure 4. Binding of antibodies to SAG and effect of chemical reduction

Parotid saliva was separated by SDS/PAGE under reducing (+) or non-reducing (−) conditions, transferred on to nitrocellulose and incubated with antibody 143 against SAG, UEA-1 lectin or alkaline phosphatase-conjugated goat anti-rabbit (goat) or rabbit anti-mouse (rabbit) IgG antibodies. After reduction, the binding of antibody 143 as well as of conjugated antibodies disappeared. With UEA-1 lectin it was observed that SAG migrated more slowly in the gel, suggesting a conformational change.

Figure 5. (A) Structure of the SAG molecule, (B) alignment of SRCR domains and (C) design of peptides

(A) SAG contains a unique N-terminal sequence, 13 SRCR domains (dark grey) separated by SIDs (dotted boxes), a serine-proline-threonine-rich sequence (striated black), two CUB domains (circles) separated by a 14th SRCR domain, a zona pellucida domain (hexamer) and a unique sequence. (B) The first 13 highly similar SRCR domains were aligned and a consensus sequence was developed. Grey-shaded sequences are identical in all 13 SRCR domains. (C) Seven peptides were synthesized based on the consensus sequence, representing loops in the SRCR domain between disulphide bridges.

Figure 6. Binding of IgA to SRCR and SID peptides

Peptides were coated at serial dilutions on to microplates and tested for IgA binding. Bound IgA was revealed using HRP-conjugated antibodies against IgA. Only SRCRP2 bound IgA. None of the other peptides, of which SRCRP1 and SRCRP6 are shown as typical examples, bound SAG.

Figure 7. Effect of IgA on binding of S. mutans to SAG and SRCRP2

Microplates, coated with SAG (▴, ▵; 10 μg/ml starting concentration) or SRCRP2 (♦, ⋄; 40 μg/ml starting concentration), were incubated with PBST-Ca (▴, ♦) or with 10 μg/ml IgA (▵, ⋄) in PBST-Ca. After washing, binding of S. mutans was analysed using the fluorescent probe SYTO-13, as described in the Experimental section. The binding of S. mutans either to SAG or to SRCRP2 peptide was not inhibited by IgA. Note that the lines for SRCRP2 (♦, ⋄) overlap.