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

Abstract

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.MARCO, a close relative of scavenger receptor A (see Ref. 1), is a trimeric type II transmembrane protein with an N-terminal intracellular domain, a transmembrane domain, and an extracellular portion composed of a short spacer domain, a long triple-helical collagenous domain, and a C-terminal scavenger receptor cysteine-rich (SRCR) 3 domain (2). MARCO has a very restricted expression pattern in adult mice living under pathogen-free conditions. It is expressed at significant levels only in the marginal zone macrophages of the spleen, in macrophages of the medullary cord of lymph nodes, and in the peritoneal macrophages (2). 4 In bacterial infections MARCO expression is up-regulated in macrophages of most tissues (3-6). Cells transfected with a plasmid encoding MARCO avidly bind both Gram-negative and -positive bacteria, but not yeast (2, 3). MARCO also has other than microbial ligand...

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

“…In accordance with the finding that soluble MARCO binds the polyanionic macromolecule poly I with a high affinity in the BIAcore system (19), expression of the full-length protein conferred CHO cells also with the ability to adhere to this polyanion immobilized on glass coverslips. Cell adhesion was in fact somewhat more effective (20 -25% more cells adhered) to poly I than to gelatin (Fig.…”

“…Regarding this assay, we found in previous studies avid binding to cells expressing wild-type mouse MARCO, whereas binding was undetectable to cells expressing a MARCO form lacking the SRCR domain (19). The double mutant 431/433, which exhibited severe defects in adhesion to gelatin, was found to have a slightly lower ability than wild-type MARCO to bind AcLDL (Fig.…”

“…The binding of AcLDL was tested as described previously (19). For these assays, transfectants were seeded on coverslips 24 h post-transfection.…”

“…This form (M496 -518) was tested, because we found previously that replacement of the C-terminal segment of the mouse MARCO SRCR domain with that of SCARA5 greatly decreased AcLDL binding (19). This new chimera exhibited slightly higher binding than the one with an intact human SRCR domain (Fig.…”

Deterioration of atherosclerosis in mice lacking angiotensin II type 1A receptor in bone marrow-derived cells

“…Colec12 has been reported to react with oxidized LDL 38 and MARCO was recently shown to have profound effects on binding of acetylated LDL. 39 Little is known about the role of Ang II on phagocytosis by macrophages. Keidar et al 11 reported that although systemic Ang II administration in mice resulted in an increase in modified LDL uptake by macrophages in vivo, no change of phagocytic activity was observed in peritoneal macrophage cell culture in vitro following addition of Ang II or ARB.…”