CD163 Binding to Haptoglobin-Hemoglobin Complexes Involves a Dual-point Electrostatic Receptor-Ligand Pairing

Formation of the haptoglobin (Hp)-hemoglobin (Hb) complex in human plasma leads to a high affinity recognition by the endocytic macrophage receptor CD163. A fast segregation of Hp-Hb from CD163 occurs at endosomal conditions (pH <6.5). The ligand binding site of CD163 has previously been shown to involve the scavenger receptor cysteine-rich (SRCR) domain 3. This domain and the adjacent SRCR domain 2 of CD163 contain a consensus motif for a calcium-coordinated acidic amino acid triad cluster as originally identified in the SRCR domain of the scavenger receptor MARCO. Here we show that site-directed mutagenesis in each of these acidic triads of SRCR domains 2 and 3 abrogates the high affinity binding of recombinant CD163 to Hp-Hb. In the ligand, Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding. These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors.     

Characterization of recombinant soluble macrophage scavenger receptor MARCO

MARCO is a type II transmembrane protein of the class A scavenger receptor family. It has a short N-terminal cytoplasmic domain, a transmembrane domain, and a large extracellular part composed of a 75-residue long spacer domain, a 270-residue collagenous domain, and a 99-residue long scavenger receptor cysteine-rich (SRCR) domain. Previous studies have indicated a role for this receptor in anti-microbial host defense functions. In this work we have produced the extracellular part of MARCO as a recombinant protein, and analyzed its binding properties. The production of this protein, soluble MARCO (sMARCO), has made it possible for the first time to study MARCO and its binding properties in a cell-free system. Using circular dichroism analyses, a protease-sensitive assay, and rotary shadowing electron microscopy, sMARCO was shown to have a triple-helical collagenous structure. Rotary shadowing also demonstrated that the molecules often associate with each other via the globes. sMARCO was found to bind avidly both heat-killed and living bacteria. Lipopolysaccharide, an important component of the outer membrane of Gram-negative bacteria, was shown to be a ligand of MARCO. Studies with different bacterial strains indicated that the O-side chain of lipopolysaccharide is not needed for the bacterial recognition. Finally, the C-terminal SRCR domain was also produced as a recombinant protein, and its bacteria-binding capability was studied. Although the transfection experiments with transmembrane MARCO variants have indicated a crucial role for this domain in bacterial binding, the monomeric domain exhibited low, barely detectable bacteria-binding activity. Thus, it is possible that cooperation between the SRCR domain and the collagenous domain is needed for high-affinity bacterial binding, or that the SRCR domain has to be in a trimeric form to effectively bind to bacteria.     

Arginine residues in domain V have a central role for bacteria-binding activity of macrophage scavenger receptor MARCO

MARCO is a bacteria-binding macrophage-specific scavenger receptor that plays a role in innate immune response. MARCO has short intracellular and transmembrane domains, as well as a large extracellular domain composed of a spacer domain, a long collagenous domain, and a C-terminal scavenger receptor cysteine-rich domain (SRCR), domain V. As yet, no specific function has been assigned to the SRCR domain of scavenger receptors. In the present study, we generated several human and mouse MARCO variants with deletions or single amino acid substitutions and localized the primary bacteria-binding region to domain V. Furthermore, analysis of the MARCO variants containing only portions of domain V demonstrated a crucial role for an arginine-rich segment for this function. More precisely, the motif RXR was identified as an essential element for high-affinity bacterial binding. The results indicate that the binding properties of MARCO differ from those of the other class A scavenger receptors, SR-A and SRCL, whose ligand-binding function has been localized to the collagenous domain.     

A phage display screen and binding studies with acetylated low density lipoprotein provide evidence for the importance of the scavenger receptor cysteine-rich (SRCR) domain in the ligand-binding function of MARCO

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.     

Crystal structure of a scavenger receptor cysteine-rich domain sheds light on an ancient superfamily

Scavenger receptor cysteine-rich (SRCR) domains are found widely in cell surface molecules and in some secreted proteins, where they are thought to mediate ligand binding. We have determined the crystal structure at 2.0 A resolution of the SRCR domain of Mac-2 binding protein (M2BP), a tumor-associated antigen and matrix protein. The structure reveals a curved six-stranded beta-sheet cradling an alpha-helix. Structure-based sequence alignment demonstrates that the M2BP SRCR domain is a valid template for the entire SRCR protein superfamily. This allows an interpretation of previous mutagenesis data on ligand binding to the lymphocyte receptor CD6.     

Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains

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.     

Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin.hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin.hemoglobin complexes.     

Identification of human immunodeficiency virus type 1 (HIV-1) gp120-binding sites on scavenger receptor cysteine rich 1 (SRCR1) domain of gp340

Background

gp340, a member of scavenger receptor cysteine rich family encoded by Deleted in Malignant Brain Tumors 1 (DMBT1), is an important component in innate immune defense. The first scavenger receptor cysteine rich domain (SRCR1) of gp340 has been shown to inhibit HIV-1 infection through binding to the N-terminal flank of the V3 loop of HIV-1 gp120.

Results

Through homology modeling and docking analysis of SRCR1 to a gp120-CD4-X5 antibody complex, we identified three loop regions containing polar or acidic residues that directly interacted with gp120. To confirm the docking prediction, a series of over-lapping peptides covering the SRCR1 sequence were synthesized and analyzed by gp120-peptide binding assay. Five peptides coincide with three loop regions showed the relative high binding index. An alanine substitution scan revealed that Asp34, Asp35, Asn96 and Glu101 in two peptides with the highest binding index are the critical residues in SRCR1 interaction with gp120.

Conclusion

We pinpointed the vital gp120-binding regions in SRCR1 and narrowed down the amino acids which play critical roles in contacting with gp120.     

Three-Dimensional Analysis of CD6 Site-Directed Mutagenesis and Monoclonal Antibody Binding Studies Using the X-ray Structure of Mac-2 Binding Protein and a Molecular Model of the CD6 Ligand Binding Domain

The extracellular region of CD6 consists of three scavenger receptor cysteine-rich (SRCR) domains and binds activated leukocyte cell adhesion molecule (ALCAM), a member of the immunoglobulin superfamily (IgSF). Residues important for the CD6-ALCAM interaction have previously been identified by mutagenesis. A total of 22 CD6 residues were classified according to their importance for anti-CD6 monoclonal antibody (mAb) and/or ALCAM binding. The three-dimensional structure of the SRCR domain of Mac-2 binding protein has recently been determined, providing a structural prototype for the SRCR protein superfamily. This has made a thorough three-dimensional analysis of CD6 mutagenesis and mAb binding experiments possible. Mutation of buried residues compromised both mAb and ALCAM binding, consistent with the presence of structural perturbations. However, several residues whose mutation affected both mAb and ALCAM binding or, alternatively, only ligand binding were found to map to the surface in the same region of the domain. This suggests that the CD6 ligand binding site and epitopes of tested mAbs overlap and provides an explanation for the finding that these mAbs effectively block ALCAM binding. An approximate molecular model of CD6 was used to delineate the ALCAM binding site.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089490050263Abbreviations ALCAM activated leukocyte cell adhesion molecule - CD6D3 third (membrane-proxi-mal) extracellular domain of CD6 - IgSF immunoglobulin superfamily - mAb monoclonal antibody - M2BP Mac-2 binding protein - SRCR scavenger receptor cysteine-rich domain - SRCRSF scavenger receptor cysteine-rich protein superfamily     

Recognition of lipoproteins by scavenger receptor class A members

Scavenger receptor class A (SR-A) proteins are type II transmembrane glycoproteins that form homotrimers on the cell surface. This family has five known members (SCARA1 to 5, or SR-A1 to A5) that recognize a variety of ligands and are involved in multiple biological pathways. Previous reports have shown that some SR-A family members can bind modified low-density lipoproteins (LDLs); however, the mechanisms of the interactions between the SR-A members and these lipoproteins are not fully understood. Here, we systematically characterize the recognition of SR-A receptors with lipoproteins and report that SCARA1 (SR-A1, CD204), MARCO (SCARA2), and SCARA5 recognize acetylated or oxidized LDL and very-low-density lipoprotein in a Ca²⁺-dependent manner through their C-terminal scavenger receptor cysteine-rich (SRCR) domains. These interactions occur specifically between the SRCR domains and the modified apolipoprotein B component of the lipoproteins, suggesting that they might share a similar mechanism for lipoprotein recognition. Meanwhile, SCARA4, a SR-A member with a carbohydrate recognition domain instead of the SRCR domain at the C terminus, shows low affinity for modified LDL and very-low-density lipoprotein but binds in a Ca²⁺-independent manner. SCARA3, which does not have a globular domain at the C terminus, was found to have no detectable binding with these lipoproteins. Taken together, these results provide mechanistic insights into the interactions between SR-A family members and lipoproteins that may help us understand the roles of SR-A receptors in lipid transport and related diseases such as atherosclerosis.     

Crystal structure of the third extracellular domain of CD5 reveals the fold of a group B scavenger cysteine-rich receptor domain

Scavenger receptor cysteine-rich (SRCR) domains are ancient protein modules widely found among cell surface and secreted proteins of the innate and adaptive immune system, where they mediate ligand binding. We have solved the crystal structure at 2.2 A of resolution of the SRCR CD5 domain III, a human lymphocyte receptor involved in the modulation of antigen specific receptor-mediated T cell activation and differentiation signals. The first structure of a member of a group B SRCR domain reveals the fold of this ancient protein module into a central core formed by two antiparallel beta-sheets and one alpha-helix, illustrating the conserved core at the protein level of genes coding for group A and B members of the SRCR superfamily. The novel SRCR group B structure permits the interpretation of site-directed mutagenesis data on the binding of activated leukocyte cell adhesion molecule (ALCAM/CD166) binding to CD6, a closely related lymphocyte receptor homologue to CD5.     

Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily

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.     

Cysteine-rich domain of scavenger receptor AI modulates the efficacy of surface targeting and mediates oligomeric Aβ internalization

Background

Insufficient clearance of soluble oligomeric amyloid-β peptide (oAβ) in the central nervous system leads to the synaptic and memory deficits in Alzheimer''s disease (AD). Previously we have identified scavenger receptor class A (SR-A) of microglia mediates oligomeric amyloid-β peptide (oAβ) internalization by siRNA approach. SR-A is a member of cysteine-rich domain (SRCR) superfamily which contains proteins actively modulating the innate immunity and host defense, however the functions of the SRCR domain remain unclear. Whether the SRCR domain of SR-AI modulates the receptor surface targeting and ligand internalization was investigated by expressing truncated SR-A variants in COS-7 cells. Surface targeting of SR-A variants was examined by live immunostaining and surface biotinylation assays. Transfected COS-7 cells were incubated with fluorescent oAβ and acetylated LDL (AcLDL) to assess their ligand-internalization capabilities.

Result

Genetic ablation of SR-A attenuated the internalization of oAβ and AcLDL by microglia. Half of oAβ-containing endocytic vesicles was SR-A positive in both microglia and macrophages. Clathrin and dynamin in SR-AI-mediated oAβ internalization were involved. The SRCR domain of SR-AI is encoded by exons 10 and 11. SR-A variants with truncated exon 11 were intracellularly retained, whereas SR-A variants with further truncations into exon 10 were surface-targeted. The fusion of exon 11 to the surface-targeted SR-A variant lacking the SRCR domain resulted in the intracellular retention and the co-immunoprecipitation of Bip chaperon of the endoplasmic reticulum. Surface-targeted variants were N-glycosylated, whereas intracellularly-retained variants retained in high-mannose states. In addition to the collagenous domain, the SRCR domain is a functional binding domain for oAβ and AcLDL. Our data suggest that inefficient folding of SR-AI variants with truncated SRCR domain was recognized by the endoplasmic reticulum associated degradation which leads to the immature N- glycosylation and intracellular retention.

Conclusion

The novel functions of the SRCR domain on regulating the efficacy of receptor trafficking and ligand binding may lead to possible approaches on modulating the innate immunity in Alzheimer’s disease and atherosclerosis.     

The crystal structure and mutational binding analysis of the extracellular domain of the platelet-activating receptor CLEC-2

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.     

Structure of an LDLR-RAP complex reveals a general mode for ligand recognition by lipoprotein receptors

Proteins of the low-density lipoprotein receptor (LDLR) family are remarkable in their ability to bind an extremely diverse range of protein and lipoprotein ligands, yet the basis for ligand recognition is poorly understood. Here, we report the 1.26 A X-ray structure of a complex between a two-module region of the ligand binding domain of the LDLR and the third domain of RAP, an escort protein for LDLR family members. The RAP domain forms a three-helix bundle with two docking sites, one for each LDLR module. The mode of recognition at each site is virtually identical: three conserved, calcium-coordinating acidic residues from each LDLR module encircle a lysine side chain protruding from the second helix of RAP. This metal-dependent mode of electrostatic recognition, together with avidity effects resulting from the use of multiple sites, represents a general binding strategy likely to apply in the binding of other basic ligands to LDLR family proteins.     

Structural characterization of the third scavenger receptor cysteine‐rich domain of murine neurotrypsin

Neurotrypsin (NT) is a multi‐domain serine protease of the nervous system with only one known substrate: the large proteoglycan Agrin. NT has seen to be involved in the maintenance/turnover of neuromuscular junctions and in processes of synaptic plasticity in the central nervous system. Roles which have been tied to its enzymatic activity, localized in the C‐terminal serine‐protease (SP) domain. However the purpose of NT's remaining 3–4 scavenger receptor cysteine‐rich (SRCR) domains is still unclear. We have determined the crystal structure of the third SRCR domain of murine NT (mmNT‐SRCR3), immediately preceding the SP domain and performed a comparative structural analysis using homologous SRCR structures. Our data and the elevated degree of structural conservation with homologous domains highlight possible functional roles for NT SRCRs. Computational and experimental analyses suggest the identification of a putative binding region for Ca²⁺ ions, known to regulate NT enzymatic activity. Furthermore, sequence and structure comparisons allow to single out regions of interest that, in future studies, might be implicated in Agrin recognition/binding or in interactions with as of yet undiscovered NT partners.     

MARCO is the major binding receptor for unopsonized particles and bacteria on human alveolar macrophages

Alveolar macrophages (AMs) avidly bind and ingest inhaled environmental particles and bacteria. To identify the particle binding receptor(s) on human AMs, we used functional screening of anti-human AM hybridomas and isolated a mAb, PLK-1, which inhibits AM binding of unopsonized particles (e.g., TiO2, latex beads; 63 +/- 5 and 67 +/- 4% inhibition, respectively, measured by flow cytometry; n = 11) and unopsonized bacteria ( approximately 84 and 41% inhibition of Escherichia coli and Staphylococcus aureus binding by mAb PLK-1, respectively). The PLK-1 Ag was identified as the human class A scavenger receptor (SR) MARCO (macrophage receptor with collagenous structure) by observing specific immunolabeling of COS cells transfected with human MARCO (but not SR-AI/II) cDNA and by immunoprecipitation by PLK-1 of a protein of appropriate molecular mass (approximately 70 kDa) from both normal human bronchoalveolar lavage cells (>90% AMs) and human MARCO-transfected COS cells. PLK-1 also specifically inhibited particle binding by COS cells, only after transfection with human MARCO cDNA. Immunostaining showed specific labeling of AMs within human lung tissue, bronchoalveolar lavage samples, as well as macrophages in other sites (e.g., lymph node and liver). Using COS transfectants with different truncated forms of MARCO, allowed epitope mapping for the PLK-1 Ab to MARCO domain V between amino acid residues 420 and 431. A panel of Abs to various SRs identified expression on AMs, but failed to inhibit TiO2 or S. aureus binding. The data support a dominant role for MARCO in the human AM defense against inhaled particles and pathogens.