Contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor.

The extracellular portion of the macrophage mannose receptor is composed of several cysteine-rich domains, including a fibronectin type II repeat and eight segments related in sequence to Ca(2+)-dependent carbohydrate-recognition domains (CRDs) of animal lectins. Expression of portions of the receptor in vitro, in fibroblasts and in bacteria, has been used to determine which of the extracellular domains are involved in binding and endocytosis of ligand. The NH2-terminal cysteine-rich domain and the fibronectin type II repeat are not necessary for endocytosis of mannose-terminated glycoproteins. CRDs 1-3 have at most very weak affinity for carbohydrate, so the carbohydrate binding activity of the receptor resides in CRDs 4-8. CRD 4 shows the highest affinity binding and has multispecificity for a variety of monosaccharides. However, CRD 4 alone cannot account for the binding of the receptor to glycoproteins. At least 3 CRDs (4, 5, and 7) are required for high affinity binding and endocytosis of multivalent glycoconjugates. In this respect, the mannose receptor is like other carbohydrate-binding proteins, in which several CRDs, each with weak affinity for single sugars, are clustered to achieve high affinity binding to oligosaccharides. In the mannose receptor, these multiple weak interactions are achieved through several active CRDs in a single polypeptide chain rather than by oligomerization of polypeptides each containing a single CRD.     

Purification of the human alveolar macrophage mannose receptor

We report here the first isolation of a mannose receptor from human lung, and identify the alveolar macrophage as the cell of origin. The receptor was purified from detergent-solubilized lung tissue by absorption to mannose- and fucose-Sepharose, and elution with EDTA. The eluted protein had a molecular weight of 175 kD. Maximum binding of 125I-mannan-2 to the isolated receptor occurred at pH 7.5. Binding was inhibited by 40 micrograms/ml mannan (75%); 200 mM mannose (89%); and 200 mM fucose (93%). Galactose (200 mM) had no effect. Polyclonal antibodies raised against the purified receptor reacted with the purified 175 kD protein and a 175 kD protein from detergent extracts of human alveolar macrophages by immunoblot analysis. The antibody immunoprecipitated a 175 kD protein from solubilized 125I-labeled human alveolar macrophage membranes. These studies indicate that the 175 kD protein purified from human lung is the cell surface alveolar macrophage mannose receptor.     

An extended conformation of the macrophage mannose receptor

The macrophage mannose receptor mediates phagocytosis of pathogenic microorganisms and endocytosis of potentially harmful soluble glycoproteins by recognition of their defining carbohydrate structures. The mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of 8-10 domains related to C-type carbohydrate recognition domains (CRDs), a fibronectin type II repeat and an N-terminal cysteine-rich domain. Hydrodynamic analysis and proteolysis experiments performed on fragments of the extracellular region of the receptor have been used to investigate its conformation. Size and shape parameters derived from sedimentation and diffusion coefficients indicate that the receptor is a monomeric, elongated and asymmetric molecule. Proteolysis experiments indicate the presence of close contacts between several pairs of domains and exposed linker regions separating CRDs 3 and 6 from their neighboring domains. Hydrodynamic coefficients predicted for modeled receptor conformations are consistent with an extended conformation with close contacts between three pairs of CRDs. The N-terminal cysteine-rich domain and the fibronectin type II repeat appear to increase the rigidity of the molecule. The rigid, extended conformation of the receptor places domains with different functions at distinct positions with respect to the membrane.     

The mannose receptor ligands and the macrophage glycome

A unique glycan-binding protein expressed in macrophages and some types of other immune cells is the mannose receptor (MR, CD206). It is an endocytic, transmembrane protein with multiple glycan-binding domains and different specificities in binding glycans. The mannose receptor is important as it has major roles in diverse biological processes, including regulation of circulating levels of reproductive hormones, homeostasis, innate immunity, and infections. These different functions involve the recognition of a wide range of glycans, and their nature is currently under intense study. But the mannose receptor is just one of many glycan-binding proteins expressed in macrophages, leading to an interest in the potential relationship between the macrophage glycome and how it may regulate cognate glycan-binding protein activities. This review focuses primarily on the mannose receptor and its carbohydrate ligands, as well as macrophages and their glycomes.     

Modulation of macrophage mannose receptor affects the uptake of virulent and avirulent Leishmania donovani promastigotes.

The effect of oxidants and the anti-inflammatory steroid dexamethasone on the attachment and internalization of virulent and avirulent Leishmania donovani promastigotes by the macrophage mannosyl fucosyl receptor was examined. Oxidants and dexamethasone are known to down- and upregulate the expression of the mannose receptor. Macrophages, when treated with 500 microM H2O2 at 37 C for 30 min, stimulate about 45% inhibition in uptake of an avirulent strain (UR6), and 30 and 25% inhibition for virulent strains AG-83 and GE-I, respectively. Treatment of macrophages with dexamethasone for 20 hr resulted in a stimulation in uptake of the parasite. When UR6 was used, a 3-fold increase in uptake was observed compared with the controls. Parasite uptake was also inhibited by the H2O2-generating system, glucose/glucose oxidase; inhibition was blocked by catalase. Treatment of macrophages either with H2O2 or dexamethasone did not affect the binding of the advanced glycosylation end product-bovine serum albumin (AGE-BSA), the ligand for AGE receptor of macrophages. Similarly, indirect evidence also shows that both types 1 and 3 complement receptors (CR1, CR3) are not affected by these treatments, indicating that, besides the mannosyl fucosyl receptor, other receptors are minimally altered in the identified condition. These results suggest that the up- and downregulation of the mannose receptor of macrophages may play a role in affecting L. donovani infection.     

Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor

The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)-dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed.     

Glycosylation influences the lectin activities of the macrophage mannose receptor

The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man5-GlcNAc2 glycans (Man5 -MR) or non-sialylated complex glycans (SA0-MR) did not bind mannosylated glycans, but could recognize SO4-3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA0-MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands.     

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.     

Organization of the gene encoding the human macrophage mannose receptor (MRC1).

The gene for the human macrophage mannose receptor (MRC1) has been characterized by isolation of clones covering the entire coding region. Sequence analysis reveals that the gene is divided into 30 exons. The first three exons encode the signal sequence, the NH2-terminal cysteine-rich domain, and the fibronectin type II repeat, while the final exon encodes the transmembrane anchor and the cytoplasmic tail. The intervening 26 exons encode the eight carbohydrate-recognition domains and intervening spacer elements. However, no simple correlation between intron boundaries and functional carbohydrate-recognition domains is apparent. The pattern of intron positions as well as comparison of the sequences of the carbohydrate-recognition domains suggests that the duplication of these domains was an evolutionarily ancient event.     

Involvement of mannose receptor in glycopeptidolipid-mediated inhibition of phagosome-lysosome fusion

We previously reported that glycopeptidolipid (GPL) isolated from Mycobacterium avium serovar 4 inhibited phagosome-lysosome (P-L) fusion when macrophages phagocytosed heat-killed Staphylococcus aureus (SA). In the present study we analyzed the underlying inhibitory mechanism of GPL coated on SA. Elimination of oligosaccharide from GPL abrogated its inhibitory activity. GPL did not inhibit P-L fusion of opsonized SA phagocytosed via complement receptors. The inhibitory activity of GPL was competitively reduced by the presence of alpha-methyl-D-mannoside and anti-mannose receptor antibody, suggesting that inhibition of P-L fusion by GPL is mediated through mannose receptor. Recruitment of early endosome antigen 1 and Ca2+/calmodulin kinase II in human macrophage-like THP-1 cells were significantly suppressed by GPL, indicating that GPL inhibits steps for leading to the P-L fusion.     

肺泡巨噬细胞表面甘露糖受体在免疫抑制大鼠肺隐球菌感染中的实验研究

目的探讨免疫抑制大鼠肺泡巨噬细胞(AM)表面表达的甘露糖受体在AM吞噬隐球菌过程中的作用。方法建立免疫抑制(FK506)大鼠模型,流式细胞法检测AM表面甘露糖受体mRNA的表达量,观察免疫缺陷对AM表面甘露糖受体表达的影响。将实验(FK506)组与对照(正常)组大鼠的AM分别与荧光标记的新生隐球菌共培养,观察AM对隐球菌的吞噬情况。结果成功建立免疫抑制大鼠模型,免疫抑制大鼠表达的甘露糖受体与正常大鼠无统计学差别,对隐球菌的吞噬亦无差别。结论免疫抑制状态不是影响甘露糖受体表达的主要因素。     

Structural insights into the mechanism of pH-dependent ligand binding and release by the cation-dependent mannose 6-phosphate receptor

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a key component of the lysosomal enzyme targeting system that binds newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and transports them to endosomal compartments. The interaction between the MPRs and its ligands is pH-dependent; the homodimeric CD-MPR binds lysosomal enzymes optimally in the pH environment of the trans Golgi network (pH approximately 6.5) and releases its cargo in acidic endosomal compartments (相似文献   

Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells.

Increased levels of agalactosyl IgG (G0 IgG) are found in several autoimmune diseases, including rheumatoid arthritis, in which they are correlated with severity of the disease. To investigate whether structural alteration of IgG may lead to aberrant processing and presentation of IgG peptides as autoantigens, we have studied uptake of G0 IgG by human dendritic cells and macrophages cultured from PBMC. We found that enzymatic removal of terminal galactose residues, which exposes N-acetylglucosamine residues, increases uptake of soluble IgG mediated by mannose receptor on macrophages and dendritic cells. Efficient uptake appears to require recycling of the receptor, can be blocked by saccharides or Abs reactive with mannose receptor, and is dependent upon the state of maturation of the dendritic cells. No differences between IgG isotypes in ability to be internalized by APC were identified, suggesting that uptake would not be limited to a particular subset of Abs. These results suggest a novel pathway by which Abs or Ag-Ab complexes can be taken into dendritic cells and macrophages, and potentially generate epitopes recognized by T cells. These findings may have particular relevance for autoimmune disorders characterized by high levels of G0 IgG.     

Structural model for the mannose receptor family uncovered by electron microscopy of Endo180 and the mannose receptor

The mannose receptor family comprises four members in mammals, Endo180 (CD280), DEC-205 (CD205), phospholipase A(2) receptor (PLA(2)R) and the mannose receptor (MR, CD206), whose extracellular portion contains a similar domain arrangement: an N-terminal cysteine-rich domain (CysR) followed by a single fibronectin type II domain (FNII) and 8-10 C-type lectin-like domains (CTLDs). These proteins mediate diverse functions ranging from extracellular matrix turnover through collagen uptake to homeostasis and immunity based on sugar recognition. Endo180 and the MR are multivalent transmembrane receptors capable of interacting with multiple ligands; in both receptors FNII recognizes collagens, and a single CTLD retains lectin activity (CTLD2 in Endo180 and CTLD4 in MR). It is expected that the overall conformation of these multivalent molecules would deeply influence their function as the availability of their binding sites could be altered under different conditions. However, conflicting reports have been published on the three-dimensional arrangement of these receptors. Here, we have used single particle electron microscopy to elucidate the three-dimensional organization of the MR and Endo180. Strikingly, we have found that both receptors display distinct three-dimensional structures, which are, however, conceptually very similar: a bent and compact conformation built upon interactions of the CysR domain and the lone functional CTLD. Biochemical and electron microscopy experiments indicate that, under a low pH mimicking the endosomal environment, both MR and Endo180 experience large conformational changes. We propose a structural model for the mannose receptor family where at least two conformations exist that may serve to regulate differences in ligand selectivity.     

CD47, a ligand for the macrophage fusion receptor, participates in macrophage multinucleation

The macrophage fusion receptor (MFR), also called P84/BIT/SIRPalpha/SHPS-1, is a transmembrane glycoprotein that belongs to the superfamily of immunoglobulins. Previously, we showed that MFR expression is highly induced at the onset of fusion in macrophages, and that MFR appears to play a role in macrophage-macrophage adhesion/fusion leading to multinucleation. The recent finding that IAP/CD47 acts as a ligand for MFR led us to hypothesize that it interacts with CD47 at the onset of cell-cell fusion. CD47 is a transmembrane glycoprotein, which, like MFR, belongs to the superfamily of immunoglobulins. We show that macrophages express the hemopoietic form of CD47, the expression of which is induced at the onset of fusion, but to a lower level than MFR. A glutathione S-transferase CD47 fusion protein engineered to contain the extracellular domain of CD47, binds macrophages, associates with MFR, and prevents multinucleation. CD47 and MFR associate via their amino-terminal immunoglobulin variable domain. Of the nine monoclonal antibodies raised against the extracellular domain of CD47, three block fusion, as well as MFR-CD47 interaction, whereas the others have no effect. Together, these data suggest that CD47 is involved in macrophage multinucleation by virtue of interacting with MFR during adhesion/fusion.     

Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed (125)I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.     

Differential inhibition of macrophage proliferation by anti-transferrin receptor antibody ER-MP21: correlation to macrophage differentiation stage

Monoclonal antibodies (mAbs) directed against the transferrin receptor are known to inhibit proliferation of cells due to iron deprivation. Some cell types, however, escape from growth inhibition by a mechanism which is unclear at present. This mechanism is the subject of the present study. We investigated the differential growth inhibition caused by anti-transferrin receptor mAb ER-MP21 in connection with the differentiation of murine macrophages (M phi). Therefore, we applied two models of M phi differentiation, namely, culture of bone marrow cells in the presence of M-CSF and a panel of M phi cell lines ordered in a linear differentiation sequence. In both models we observed that proliferation of M phi precursors was strongly inhibited by ER-MP21. In contrast, proliferation of more mature stages of M phi differentiation was hardly affected. Remarkably, iron uptake by M phi precursor and mature M phi cell lines was inhibited by ER-MP21 to the same extent. However, mature M phi cell lines showed an iron uptake two- to threefold higher than that of M phi precursor cell lines. These observations strongly suggest that mature M phi escape from ER-MP21-mediated growth inhibition, because these cells take up more iron than is actually needed for proliferation. Furthermore, we found that enhanced iron uptake by mature M phi is not necessarily accompanied by a higher cell surface expression of transferrin receptors, thus suggesting an increased recycling of transferrin receptors in mature M phi.     

Binding of tissue-type plasminogen activator by the mannose receptor

Previous studies have shown that tissue-type plasminogen activator (t-PA) in blood is cleared by the liver partially through a mannose-specific uptake system. The present study was undertaken to investigate, in a purified system, whether t-PA is recognized by the mannose receptor which is expressed on macrophages and liver sinusoidal cells. The mannose receptor was isolated and purified from bovine alveolar macrophages and migrated as a single protein band at Mr 175,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Ligand blotting revealed that this protein specifically bound t-PA. The t-PA-receptor interaction was further characterized in a binding assay, which showed saturable binding with an apparent dissociation constant of 1 nM. t-PA binding required calcium ions and was negligible in the presence of EDTA or at acid pH. Mannose-albumin was an effective inhibitor, whereas galactose-albumin did not have a significant effect. From a series of monosaccharides tested, D-mannose and L-fucose were the most potent inhibitors, N-acetyl-D-glucosamine was a moderate inhibitor, whereas D-galactose and N-acetyl-D-galactosamine were ineffective. t-PA, deglycosylated by endoglycosidase H, did not interact with the receptor. It is concluded that the mannose receptor specifically binds t-PA, probably through its high mannose-type oligosaccharide.     

Oxidant-mediated mitochondrial injury in eosinophil apoptosis: enhancement by glucocorticoids and inhibition by granulocyte-macrophage colony-stimulating factor

The mainstay of asthma therapy, glucocorticosteroids (GCs) have among their therapeutic effects the inhibition of inflammatory cytokine production and induction of eosinophil apoptosis. In the absence of prosurvival cytokines (e.g., GM-CSF), eosinophils appear to be short-lived, undergoing apoptosis over 96 h in vitro. In a dose-dependent manner, GC further enhances apoptosis, while prosurvival cytokines inhibit apoptosis and antagonize the effect of GC. The mechanisms of eosinophil apoptosis, its enhancement by GC, and antagonism of GC by GM-CSF are not well-understood. As demonstrated in this study, baseline apoptosis of eosinophils resulted from oxidant-mediated mitochondrial injury that was significantly enhanced by GC. Mitochondrial injury was detected by early and progressive loss of mitochondrial membrane potential and the antioxidant protein, Mn superoxide dismutase (SOD). Also observed was the activation/translocation of the proapoptotic protein, Bax, to mitochondria. Underscoring the role of oxidants was the inhibition of mitochondrial changes and apoptosis with culture in hypoxia, or pretreatment with a flavoprotein inhibitor or a SOD mimic. GCs demonstrated early (40 min) and late (16 h) activation of proapoptotic c-Jun NH2-terminal kinase (JNK) and decreased the antiapoptotic protein X-linked inhibitor of apoptosis, a recently demonstrated inhibitor of JNK activation. Similarly, inhibition of JNK prevented GC-enhanced mitochondrial injury and apoptosis. Importantly, GM-CSF prevented GC-induced loss of X-linked inhibitor of apoptosis protein, late activation of JNK, and mitochondrial injury even in the face of unchanged oxidant production, loss of MnSOD, and early JNK activation. These data demonstrate that oxidant-induced mitochondrial injury is pivotal in eosinophil apoptosis, and is enhanced by GC-induced prolonged JNK activation that is in turn inhibited by GM-CSF.     

Dermal microvascular endothelial cells express the 180-kDa macrophage mannose receptor in situ and in vitro

Expression of the 180-kDa mannose receptor (MR) is mainly found on cells of the macrophage lineage. MR mediates the uptake of micro-organisms and host-derived glycoproteins. We demonstrate that endothelium of the human skin in situ and dermal microvascular endothelial cells (DMEC) in vitro expressed MR at both the protein and mRNA levels. In contrast, HUVEC were consistently negative for MR expression. DMEC internalized dextran as well as Escherichia coli by the way of MR into acidic phagosomes, only a few of which fused with CD63- and lysosomal-associated membrane glycoprotein-2-positive lysosomes. This contrasts with the situation in monocyte-derived dendritic cells, where almost all of the MR-Ag complexes reached CD63- and lysosomal-associated membrane glycoprotein-2-positive compartments, indicating differences in the phagolysosomal fusion rate between DMEC and dendritic cells. In conclusion, DMEC express functional MR, a finding that corroborates a role of skin endothelium in Ag capture/clearing.