Abstract
Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.
Highlights
Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily
This 300 – 400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain
Based on analogies to mucins, we have proposed that these polymorphisms may lead serum albumin; SRCR, scavenger receptor cysteine-rich; SRCRP, SRCR peptide; SID, SRCR-interspersed domain; ZP, zona pellucida; Q-TOF MS, quadrupole-time of flight mass spectrometry; HPLC, high pressure liquid chromatography; CHAPS, 3-[ lsqb] (3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MARCO, macrophage receptor with collagenous structure
Summary
Purification of Agglutinin—Human parotid saliva was collected with a Lashley cup. Twenty-five ml of parotid saliva was kept on ice water for 30 min to promote the formation of a precipitate. Liquid-phase Binding Assay—Two hundred l of an S. mutans suspension (109 bacteria/ml in PBS) was centrifuged at 3,000 ϫ g for 10 min. After incubation at 4 °C for 16 h, plates were washed twice with Tris-buffered saline containing 0.1% Tween 20 and 1 mM Ca2ϩ (TTC). 100 l of an S. mutans suspension (5 ϫ 108 bacteria/ml TTC) were added to each well and incubated for 2 h at 37 °C. Agglutination Assays—150 l of an S. mutans suspension (5 ϫ 108 bacteria/ml TTC) were mixed with 150 l of peptide solution at final peptide concentrations of 0 –200 g/ml in a 96-well microtiter plate Bacterial suspensions (5 ϫ 108 bacteria/ml TTC) supplemented with either calcium chloride or EDTA were preincubated for 2 h at 37 °C before peptides were added.
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