title: Characterization of recombinant soluble macrophage scavenger receptor MARCO.
abstract: 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.
authors.affiliation: Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-171 77 Stockholm, Sweden.
authors.forename: Marko
authors.initials: M
authors.lastname: Sankala
authors.forename: Annika
authors.initials: A
authors.lastname: Brännström
authors.forename: Therese
authors.initials: T
authors.lastname: Schulthess
authors.forename: Ulrich
authors.initials: U
authors.lastname: Bergmann
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Morgunova
authors.forename: Jürgen
authors.initials: J
authors.lastname: Engel
authors.forename: Karl
authors.initials: K
authors.lastname: Tryggvason
authors.forename: Timo
authors.initials: T
authors.lastname: Pikkarainen
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D002460:Cell Line; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008070:Lipopolysaccharides / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology
chemical_list: D008024:Ligands; D008070:Lipopolysaccharides; C490460:MARCO protein, human; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase