The macrophage receptor MARCO

MARCO (macrophage receptor with collagenous structure) belongs to the class A scavenger receptor molecules. The structure and function of the molecule is described. Although it is expressed on subsets of macrophages, it can be upregulated on other macrophages after bacterial infection. The strategic position of MARCO-expressing cells in lymphoid organs suggests an important role for this bacteria-binding molecule in removal of pathogens.     

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.     

Expression of macrophage MARCO receptor induces formation of dendritic plasma membrane processes

MARCO is a novel macrophage-specific receptor structurally related to macrophage class A scavenger receptors. It is constitutively expressed in macrophages of the marginal zone of the spleen and in lymph nodes and is up-regulated in other tissues during systemic bacterial infections. In this study, we show that ectopic expression of MARCO in cell lines such as Chinese hamster ovary, HeLa, NIH3T3, and 293 induces dramatic cell shape changes. Typically these changes include formation of large lamellipodia-like structures and of 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, but not completely abolished, when the cells are plated on fibronectin-coated surfaces. Similarly, a dominant-negative mutant of the Rho family GTPase Rac1 partially inhibited the morphogenic effects of MARCO in Chinese hamster ovary cells, whereas a dominant-negative form of a related protein, Cdc42, did not. Expression studies with a variety of truncated MARCO forms indicated that the proximal segment of the cysteine-rich domain V is important for the morphoregulatory activity. The results indicate that expression of MARCO has a direct effect in generating the phenotype of activated macrophages necessary for the trapping and removal of pathogens and other foreign substances.     

Oligonucleotide aggregates bind to the macrophage scavenger receptor.

We have prepared a model receptor containing a Lys cluster (320-340) in the collagen-like domain of the bovine macrophage scavenger receptor, and have shown that it has a similar binding specificity to the native scavenger receptor. The native scavenger receptor is reported to bind the quadruplex structure of nucleotides. In this study, we analyzed the model receptor binding of nucleotides with various structures, random, parallel or antiparallel quadruplex and aggregate forms. This was carried out by direct binding assays using labeled oligonucleotides or surface plasmon resonance, and by an inhibition assay using Chinese hamster ovary (CHO) cells expressing the scavenger receptor. The results showed that the nucleotides forming the quadruplex structure did not exhibit any binding. Only the aggregate forms of the nucleotide could bind to the model receptor. They also inhibited the degradation of acetylated low density lipoprotein by CHO cells expressing the native scavenger receptor, whereas nucleotides that did not bind to the model receptor had no effect on cellular acetylated low density lipoprotein degradation. Our results suggest that the quadruplex structure is not essential but may be required for the formation of the nucleotide aggregates, which can interact with the scavenger receptor.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Stimulation of macrophages by mucins through a macrophage scavenger receptor.

We found that phorbol ester-primed THP-1 cells (a human monocyte cell line), which express a scavenger receptor, were stimulated by mucins through the macrophage scavenger receptor, resulting in enhanced secretion of IL-1beta. The activity was abolished by treatment of the mucins with sialidase, indicating that sialic acid is involved in binding. (125)I-Labeled ovine submaxillary mucin could bind to COS 7 cells transfected with cDNA encoding the scavenger receptor. Binding was inhibited by mucins, fucoidan, and polyinosinic acid but not by polycytidylic acid, this being consistent with the characteristics of the scavenger receptor. When phorbol ester-primed THP-1 cells were cocultured with colon cancer cells producing mucins, IL-1beta secreted from the THP-1 cells increased significantly. Adhesion between colon cancer cells and a scavenger receptor transfectant was observed, and binding was inhibited partly by mucins and ligands for the scavenger receptor.     

Molecular flypaper and atherosclerosis: structure of the macrophage scavenger receptor.

Macrophage scavenger receptors have been implicated both in the deposition of lipoprotein cholesterol in artery walls during the formation of atherosclerotic plaques and in host defense against pathogenic infections. The receptor's unusual ability to bind tightly to a very wide variety of ligands and its novel mosaic structure comprising alpha-helical coiled-coil, collagenous and cysteine-rich domains are described.     

N-glycosylation profile of recombinant human soluble Fcgamma receptor III

N-glycans of human Fcgamma receptor III (FcgammaR III) are believed to be involved in the interaction with complement receptor type 3 (CR3) (Sehgal et al. [1993] J. Immunol., 150, 4571-4580). Recombinant human soluble FcgammaRIII (rhsFcgammaRIII), which is produced in baby hamster kidney (BHK) cells, has been shown to interact with CR3 in a manner similar to native FcgammaRIII. We elucidated the N-glycosylation profiles of rhsFcgammaRIII by the 3D high-performance liquid chromatography mapping technique. It was revealed that the N-glycans of rhsFcgammaRIII are much more divergent (consisting of 20 neutral, 7 monosialyl, 4 disialyl, 5 trisialyl, and 1 tetrasialyl oligosaccharides) than those previously determined for BHK-expressed mouse sFcgammaRII, notwithstanding close structural similarity of polypeptide chains between the two sFcgammaRs. Particularly, high-mannose type oligosaccharides are specifically expressed on rhsFcgammaRIII.     

Characterization of recombinant soluble carcinoembryonic antigen cell adhesion molecule 1

Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a type 1 transmembrane, homotypic cell adhesion protein expressed on epithelial and hematopoietic cells. CEACAM1 has four major isoforms with three or four immunoglobulin (Ig)-like ectodomains and either long or short cytoplasmic domains. In a 3D model of breast epithelial cell morphogenesis, CEACAM1 plays an essential role in lumen formation [J. Cell Sci. 112 (1999) 4193]. Two soluble ectodomain isoforms of CEACAM1 expressed in myeloma cells were immunologically active and highly glycosylated. The molecular weights of the 3-ecto- and 4-ectodomain isoforms were 90 and 110kDa, respectively, and monomers by sedimentation equilibrium centrifugation. Both isoforms were prolate ellipsoids with axial ratios of 6 for the 3-ecto- and 8 for 4-ectodomain isoforms, respectively, by size exclusion chromatography and analytical ultracentrifugation. Both isoforms caused a significant reduction in lumen formation when tested in the 3D model culture system.     

Identification and characterization of naturally occurring variants of the macrophage scavenger receptor (SR-A)

The scavenger receptor (SR) family comprises a group of cell surface proteins functionally defined by their ability to bind chemically modified lipoproteins. In macrophages, the class A Type I and Type II SRs (SR-AI/II) are thought to play a key role in adherence to and phagocytosis of infectious agents. Immunoprecipitation studies show that the rat anti-SR-AI/II monoclonal antibody 2F8 detects the mature, trimeric form of the receptor expressed in peritoneal macrophages from A/J, but not from C57Bl/6J (B6) mice. Subsequent sequencing of cDNA and genomic clones indicates that SR-AI and AII of A/J and B6 mice differ in sequence at nine positions, two in the cytoplasmic domain and seven in the extracellular spacer and α-helical coiled coil domains. These sequence polymorphisms are non-conservative and produce distinct receptor molecules that differ by four charged residues and alter recognition of the receptor by the monoclonal 2F8 antibody. The B6 SR-AI/II haplotype appears unique, since most inbred strains analyzed show the A/J-type haplotype. Interestingly, several of the B6 polymorphic variant residues are conserved in human and bovine receptors, suggesting a recent divergence of the A/J haplotype. Initial studies in CHO-derived cells expressing individual receptor isoforms indicate that the A/J and B6 receptors are stable and can mature into oligomers expressed in the membrane fractions of these cells. In these transfectants, no major functional differences were detected between receptors of the two haplotypes with respect to internalization and degradation of ¹²⁵I-labeled acetylated LDL. However, since SR-AI/II recognizes a large number of structurally unrelated anionic molecules, the possibility that different haplotypes may affect either binding and release of other ligands, or receptor recycling, cannot be excluded. Received: 11 January 2000 / Accepted: 30 March 2000     

Cholesterol-induced macrophage apoptosis requires ER stress pathways and engagement of the type A scavenger receptor

Macrophage death in advanced atherosclerosis promotes necrosis and plaque destabilization. A likely cause of macrophage death is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) and C/EBP homologous protein (CHOP)-induced apoptosis. Here we show that p38 MAPK signaling is necessary for CHOP induction and apoptosis. Additionally, two other signaling pathways must cooperate with p38-CHOP to effect apoptosis. One involves the type A scavenger receptor (SRA). As evidence, FC loading by non-SRA mechanisms activates p38 and CHOP, but not apoptosis unless the SRA is engaged. The other pathway involves c-Jun NH2-terminal kinase (JNK)2, which is activated by cholesterol trafficking to the ER, but is independent of CHOP. Thus, FC-induced apoptosis requires cholesterol trafficking to the ER, which triggers p38-CHOP and JNK2, and engagement of the SRA. These findings have important implications for understanding how the UPR, MAPKs, and the SRA might conspire to cause macrophage death, lesional necrosis, and plaque destabilization in advanced atherosclerotic lesions.     

Disparate regulation and function of the class A scavenger receptors SR-AI/II and MARCO

The macrophage class A scavenger receptors, macrophage receptor with a collagenous structure (MARCO) and type I/II class A scavenger receptor (SR-AI/II), share structural features and roles in host defense, but little is known about their regulation and signaling properties. Ligation of MARCO on mouse thioglycollate-elicited peritoneal macrophages (PEMs) with immobilized mAb costimulated IL-12 production, in contrast to previously reported inhibition by SR-AI/II. PEMs from MARCO-deficient mice exhibited 2.7 times lower IL-12 production in responses to stimulation with LPS and IFN-gamma and lack of significant IL-12 production on stimulation with LPS alone. Conversely, SR-AI/II-deficient PEMs produced 2.4 and 1.8 times more IL-12 than wild-type PEMs in response to LPS or LPS and IFN-gamma, respectively. Corresponding differences in regulation of SR-A and MARCO expression were also observed. Th1 adjuvants (LPS, a CpG motif-containing oligodeoxynucleotide (CpG-ODN), IL-12, and GM-CSF) increased, whereas Th2-polarizing factors (IL-4, M-CSF, and non-CpG ODN) decreased expression of MARCO on J774 macrophage-like cells. Expression of SR-A was regulated in the opposite manner to MARCO or not affected. Whereas MARCO was involved in opsonin-independent phagocytosis in CpG-ODN-pretreated but not in IL-4-pretreated J774 cells, anti-SR-A Abs inhibited particle uptake in untreated and IL-4-pretreated but not in CpG-ODN-pretreated cells. SR-A and MARCO are regulated differently and mediate distinct negative and positive effects on IL-12 production in macrophages. These differences may contribute to sustained Th1 or Th2 polarization of ongoing immune responses.     

Role of the buried glutamate in the alpha-helical coiled coil domain of the macrophage scavenger receptor

The macrophage scavenger receptor exhibits a pH-dependent conformational change around the carboxy-terminal half of the alpha-helical coiled coil domain, which has a representative amino acid sequence of a (defgabc)n heptad. We previously demonstrated that a peptide corresponding to this region formed a random coil structure at pH 7 and an alpha-helical coiled coil structure at pH 5 [Suzuki, K., Doi, T., Imanishi, T., Kodama, T., and Tanaka, T. (1997) Biochemistry 36, 15140-15146]. To determine the amino acid responsible for the conformational change, we prepared several peptides in which the acidic amino acids were replaced with neutral amino acids. Analyses of their structures by circular dichroism and sedimentation equilibrium gave the result that the presence of Glu242 at the d position was sufficient to induce the pH-dependent conformational change of the alpha-helical coiled coil domain. Furthermore, we substituted a Glu residue for the Ile residue at the d or a position of a de novo designed peptide (IEKKIEA)4, which forms a highly stable triple-stranded coiled coil. These peptides exhibited a pH-dependent conformational change similar to that of the scavenger receptor. Therefore, we conclude that a buried Glu residue in the hydrophobic core of a triple-stranded coiled coil has the potential to induce the pH-dependent conformational change. This finding makes it possible to elucidate the functions of natural proteins and to create a de novo protein designed to undergo a pH-dependent conformational change.