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

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 bacteriabinding protein loop (peptide SRCRP2; QGRVEVLYRG-SWGTVC) 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.The scavenger receptor cysteine-rich (SRCR) 1 proteins form an archaic group of metazoan proteins (1-5). This group of glycoproteins comprises cell surface molecules as well as secreted proteins that are characterized by the presence of one or more SRCR domains. SRCR domains consist of ϳ110 amino acids and are divided into groups A and B based on the number of conserved cysteine residues, namely six for group A and eight for group B.The best studied members of the group A SRCR proteins are the macrophage scavenger receptor (MSR1), the Mac 2-binding protein (Mac-2bp), and MARCO. Both MSR1 and MARCO are known to interact with bacteria (6, 7). In contrast to MARCO (8), the SRCR domain of MSR1 does not seem to be involved in bacteria binding (9, 10). Bacteria binding by MARCO involves an RXR motif within the SRCR domain, indicating that ionic interactions play a crucial role in the interaction with its negatively charged ligands (6).Group B SRCR proteins are generally involved in the regulation of cellular immune responses. In vertebrates, the group B SRCR proteins can be divided, on the basis of their structure and sequence homology, into three subgroups (11). The first subgroup includes CD5 (12), CD6 (13), and SP␣ (14). CD5 and CD6 are composed of an extracellular region of three SRCR domains, a transmembrane region, and a cytoplasmic region. SP␣ lacks the latter two regions but contains three SRCR domains that are highly homologous to those of CD5 and CD6. These three proteins are mainly expressed by T-cells and Bcells (12, 13). The second subgroup of SRCR group B molecules is the workshop cluster 1 (WC1) family, which includes WC1, ...

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340

“…Also, the AgI/II adhesins of viridans (oral) streptococci mediate binding to gp340 (17,18,46,47). The peptide core of SRCR domains of gp340, and especially the amino acid sequence VEVLXXXXW, has been identified to be important in these interactions (19). Our peptide inhibition experiments showed that the peptides RCRGRVEVL and RCQGRVEVL, devoid of the XXXXW motif, were good inhibitors for Spy0843 and other Lrr protein binding, which suggested unique interaction of these proteins with the SRCR domains.…”

“…A 16-mer peptide, SRCRP2, and especially the amino acids VEVLXXXXW in it, derived from the consensus sequence of the SRCR domains of gp340 has been previously reported to inhibit many microbial interactions of gp340 (19). SRCRP2 was found to inhibit the binding of r0843 in an ELISA (Table 3).…”

“…There are few studies suggesting that both carbohydrates and the protein core of the gp340 can be involved in these interactions. For example, a VEVLXXXXW peptide derived from the SRCR domain of gp340 is shown to bind different types of bacteria (19), whereas sialic acid residues may mediate binding to, for example, influenza virus (20). However, the molecular basis of the ability of gp340 or peptides derived thereof to bind a large ligand repertoire is not understood.…”

Crystal Structure of the Cysteine-rich Domain of Scavenger Receptor MARCO Reveals the Presence of a Basic and an Acidic Cluster That Both Contribute to Ligand Recognition

“…The lymphocyte cell surface receptor CD6 interacts with activated leukocyte cell adhesion molecule (ALCAM/CD166) via the SRCR3 domain (15); in gp-340/DMBT1 (deleted in malignant tumors 1), the peptide QGRVEVLYRGSWGTVC, present in eight of its 14 SRCR domains, binds and agglutinates Streptococcus mutans and various other bacterial strains (16,17), and the first of the three SRCR domains present in the SP␣, a soluble human glycoprotein expressed by several types of macrophages, binds to Escherichia coli and Staphylococcus aureus (18). In the case of MARCO, it has been shown that the binding site for bacteria, lipopolysaccharide, acetylated LDL (AcLDL), and a hydrophobic peptide isolated with a phage display screen resides in the SRCR domain (5, 19 -21).…”

The macrophage soluble receptor AIM/Api6/CD5L displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression

“…They do not possess enzymatic activity, although some SRCR domains have been involved in protein-protein interactions, the best studied examples being those of CD6 with CD166/ALCAM, 6 and of CD163 with the haptoglobin-haemoglobin complex. 7 In recent years, a number of studies also support the recognition of PAMPs by some SRCR-SF members such as MARCO, 8 DMBT1/SAG/gp340, 9 Spa, 10 CD6, 11 CD5, 12 S5D-SRCRB, 13 SCARA5 14 and CD163. 15 The apoptosis inhibitor of macrophages (AIMs), also known as Api6 (for apoptosis inhibitor 6) or CD5L (for CD5-like), is a mouse soluble member of the SRCR-SF which is secreted by mature tissue macrophages.…”