Structure and biological properties of scavenger receptor MARCO

Sammanfattning: Macrophages are monocyte-derived cells that play an important role in the innate immune response against invading pathogens. These cells express several host defense receptors that can be divided into two classes; those dependent on opsonizing components for recognition of pathogens, and those that can recognize pathogens directly, pattern recognition receptors (PRRs). Class A scavenger receptors are a family of PRRs composed of three members: Scavenger Receptor A (SRA), MAcrophage Receptor with COllagenous structure (MARCO), and a recently identified protein Scavenger Receptor with C-type Lectin (SRCL). MARCO is a trimeric membrane protein containing an Nterminal intracellular domain, a transmembrane domain, and an extracellular portion composed of a short spacer domain, a triple- helical collagenous domain, and a C-terminal cysteine-rich domain (SRCR). In unstimulated mice, MARCO expression is restricted to the marginal zone macrophages in spleen, macrophages in medullary cord in lymph nodes, and peritoneal macrophages. Its expression can be induced in cultured macrophages or in macrophages of various tissues after administering LPS, BCG, Listeria monocytogenes or zymosan. These findings suggest a role in host defense. Furthermore, cells transfected with MARCO cDNA avidly bind both gram-negative and gram-positive bacteria, but not yeast. Preliminary data shows that the bacteria-binding region is in the SRCR domain. Interestingly, MARCO was found to be the major receptor on alveolar macrophages for binding of unopsonized environmental particles such as TiO2 and Fe203 To examine the regulatory mechanisms of MARCO, and its potential role in disease, the structures of human and murine MARCO genes were determined. Both genes have 17 exons, of which exons 4-15 encode the collagenous domain. Two major transcription initiation sites were identified, one starting at position +1, 27 bp downstream of a TATA box, and another at position -64 downstream of an AT-rich region. Several potential binding sites for transcription factors involved in host defense were identified in the promoter region. The human and mouse genes were localized to syntenic regions on chromosomes 2 and 1, respectively. We could also show that MARCO most likely has a direct effect on the phenotype of activated macrophages, since the expression of MARCO in different cell lines induces dramatic cell shape changes. Typically these changes include formation of large lamellipodia-like structures and long dendritic processes. The morphological changes are accompanied by disassembly of actin stress fibers and often also by complete loss of focal adhesions. The MARCO- induced changes are dependent on cell adhesion and are inhibited when the cells are plated on fibronectin-coated surfaces. Similarly, a dominant-negative mutant of Rac- I partially inhibits the morphogenic effects of MARCO in CHO cells. Our data indicate that the proximal segment of the SRCR domain is important for the morphoregulatory activity. We used a large number of human and mouse MARCO variants to show that the predominant bacteria- binding region of MARCO resides in the SRCR domain. This result demonstrates the first function for a SRCR domain of the scavenger receptor A family. In further analysis, we found that an arginine-rich segment in the cysteine-rich domain is responsible for the high-affinity binding. More precisely, the motif arginine-X-arginine was found to be of importance in this regard. This was demonstrated by comparing the bacteria-binding activity of two truncated constructs of MARCO. One form, which contains an argininedoublet in its SRCR domain, binds bacteria avidly, while the other, containing one single arginine residue, exhibits insignificant binding. To obtain a tool for ligand-binding studies, we produced the extracellular part of MARCO as a recombinant protein, sMARCO. Several studies indicated that sMARCO has a triple-helical structure. When imaged in electron microscope after rotary shadowing, the protein appeared as 82.7 nm long rod-like molecules with small globes at both ends. Furthermore, the molecules were often found to be associated with each other. Binding studies with sMARCO, as well as with MARCO-expressing cells, indicated that MARCO binds gram-negative strains expressing either the smooth- or rough- form of I VS. Additional studies revealed that LPS is a ligand of MARCO. Finally, we also produced the SRCR domain as a recombinant protein, and compared the bacteria-binding properties of this protein and sMARCO. These studies indicated that the SRCR domain has to be in a trimeric form in order to bind bacteria efficiently.