Bacteria Binding by DMBT1/SAG/gp-340 Is Confined to the VEVLXXXXW Motif in Its Scavenger Receptor Cysteine-rich Domains

Floris J Bikker,Antoon J.M Ligtenberg,Caroline End,Marcus Renner,Stephanie Blaich,Stefan Lyer,Rainer Wittig,Wim Van'T Hof,Enno C.I Veerman,Kamran Nazmi,Jolanda M.A De Blieck-Hogervorst,Petra Kioschis,Arie V Nieuw Amerongen,Annemarie Poustka,Jan Mollenhauer

doi:10.1074/jbc.m406095200

Abstract

The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.

Highlights

The scavenger receptor cysteine-rich (SRCR)1 proteins form an archaic group of metazoan proteins [1,2,3,4,5]
With SRCRP2, the agglutination of S. mutans was observed after ϳ30 min, whereas with the corresponding concentrations of DMBT1pbs1 the agglutination of S. mutans was already observed after 2–3 min (Fig. 2)
Analysis of the Bacteria Binding Features of Motifs Present within Other Members of the SRCR Superfamily—Because SRCR domains are highly conserved across species, we investigated whether DMBT1pbs1-corresponding sequences in other SRCR proteins exhibited bacteria binding

Summary

EXPERIMENTAL PROCEDURES

Purification of DMBT1SAG—DMBT1SAG was purified by gel filtration as described previously [29]. Peptide Design, Synthesis, and Purification—All synthetic peptides were SRCR domain consensus-based and designed as described previously using alignment software (DNASTAR, Lasergene Inc., Madison, WI) [17]. Fluotrac 600 microtiter plates (Greiner, Recklinghausen, Germany) were coated with various amounts of either synthetic peptides or purified DMBT1SAG. Plates were incubated in the dark at the ambient temperature for 15 min and washed three times with the same buffer. Fluorescence was measured in a Fluostar Galaxy microtiter plate fluorescence reader (BMG Laboratories, Offenburg, Germany) at 488-nm excitation and 509-nm emission wavelengths These experiments were repeated at least three times. Agglutination Assays—100 ␮l of a bacterial suspension (5 ϫ 108 bacteria/ml) were mixed with 20 ␮l of peptide solution at final peptide concentrations of 0 –200 ␮g/ml in 48-well microtiter plates (Falcon, Piscataway, NJ) and incubated at 37 °C for 5–15 min.

RESULTS

DISCUSSION

IF Xenopus laevis