Macrophage Lectin Research Articles

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses

Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple ‘glycogenes’ that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses.

Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple 'glycogenes' that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.

Carrageenan from red algae: an application in the development of inhalable tuberculosis therapy targeting the macrophages.

Macrophages have unique surface receptors that might recognize preferentially several moieties present on the surface of infecting organisms, including in the bacterial cell wall. Benefiting from a similar composition regarding the referred moieties, polysaccharides might be good candidates to compose the matrix of drug carriers aimed at macrophage targeting, as they can use the same recognition pathways of the infecting organisms. Carrageenan (CRG), a polysaccharide extracted from red edible seaweed, is an interesting possibility for the approach of directly targeting alveolar macrophages, as its composition is reported to be recognized by several macrophage lectin receptors. Inhalable starch/CRG microparticles were successfully produced, effectively associating isoniazid (96%) and rifabutin (74%) simultaneously. Furthermore, the produced microparticles presented adequate aerodynamic properties for pulmonary delivery with potential to reach the respiratory zone, with a mass median aerodynamic diameter (MMAD) between 3.3 and 3.9μm. It was further demonstrated that the antitubercular activity of the drugs remained unchanged after encapsulation. The formulation evidenced no cytotoxic effects on lung epithelial cells (A549), although mild toxicity was observed on macrophage-differentiated THP-1 cells for the drug-loaded formulation. Starch/CRG microparticles also exhibited a propensity to be captured by macrophages in a dose-dependent manner, as well as an ability to activate the target cells. This work provides indications on the potential of the starch/CRG carriers to interact with macrophages, thus providing a platform for drug delivery in the context of macrophage intracellular diseases. Additionally, if tuberculosis is focused, these microparticles can be used as inhalable drug carriers. Graphical abstractz.

Carbohydrate-based nanocarriers and their application to target macrophages and deliver antimicrobial agents.

Many deadly infections are produced by microorganisms capable of sustained survival in macrophages. This reduces exposure to chemadrotherapy, prevents immune detection, and is akin to criminals hiding in police stations. Therefore, the use of glyco-nanoparticles (GNPs) as carriers of therapeutic agents is a burgeoning field. Such an approach can enhance the penetration of drugs into macrophages with specific carbohydrate targeting molecules on the nanocarrier to interact with macrophage lectins. Carbohydrates are natural biological molecules and the key constituents in a large variety of biological events such as cellular communication, infection, inflammation, enzyme trafficking, cellular migration, cancer metastasis and immune functions. The prominent characteristics of carbohydrates including biodegradability, biocompatibility, hydrophilicity and the highly specific interaction of targeting cell-surface receptors support their potential application to drug delivery systems (DDS). This review presents the 21st century development of carbohydrate-based nanocarriers for drug targeting of therapeutic agents for diseases localized in macrophages. The significance of natural carbohydrate-derived nanoparticles (GNPs) as anti-microbial drug carriers is highlighted in several areas of treatment including tuberculosis, salmonellosis, leishmaniasis, candidiasis, and HIV/AIDS.

Bone Marrow Macrophage Galectin-3 Regulates Platelet Production through Recognition of O-Glycans on Megakaryocytes

Bone marrow (BM) macrophages maintain both survival and retention of hematopoietic stem cells and regulate erythropoiesis. The role of macrophage lectins and glycans in thrombopoiesis remains unclear. We report a novel role for bone marrow macrophage galectin-3 in maintaining platelet counts, by phagocytosing megakaryocytes (MKs) expressing the Thomsen-Friedenreich (TF) antigen, which is often exposed under pathological conditions, such as cancer and malignancies.The TF antigen is a disaccharide presented in cryptic form on O-glycans and covered by a sialic acid moiety. The sialyltransferase ST3Gal1 transfers sialic acid onto the TF antigen. To investigate the role of O-glycans in thrombopoiesis, we generated mice with increased TF antigen in MKs by generating St3gal1loxP/PF4+ mice specifically lacking ST3Gal1 in the MK lineage. As expected, St3gal1loxP/PF4+ circulating platelets and BM MKs had increased TF antigen expression, compared to controls, as evidenced by peanut agglutinin (PNA) binding. Other blood cell lineages had no increase in TF antigen expression. St3gal1loxP/PF4+ mice developed mild thrombocytopenia, but surprisingly had virtually normal platelet clearance. BM MK colony forming units and in vitro proplatelet production were normal in St3gal1loxP/PF4+ mice, suggesting that extrinsic factors in the St3gal1loxP/PF4+BM environment affected platelet production. St3gal1loxP/PF4+ BM smears revealed increased hemophagocytosis, indicative of an increase in phagocytic macrophages. In vivo macrophage ablation by injection of clodronate-encapsulated liposomes significantly reduced the numbers of activated macrophages, thereby normalizing blood platelet counts and size. Flow cytometric phenotypic analysis of BM-derived macrophages showed an increased population of activated macrophages in St3gal1loxP/PF4+ mice, compared to controls, specifically macrophages with increased galectin-3 expression, a ligand for the TF antigen. Immunofluorescence staining of BM sections using a specific antibody towards the TF antigen showed that MK progenitors and pro-platelet-like structures expressed TF antigen in control BMs, which is significantly increased in St3gal1loxP/PF4+ mice and co-localized with galectin-3 expressing macrophages, supporting the notion that MK O-glycans and macrophage galectin-3 play a role in thrombopoiesis under steady state and pathological conditions. Consistent with this notion, galectin-3 deficient mice have slightly, but significantly increased blood platelet counts. We conclude that galactin-3 plays a minor role in normal thrombopoiesis. Activation of galectin-3 expressing macrophages by the MK TF antigen leads to MK phagocytosis, inhibition of platelet formation and thrombocytopenia. DisclosuresNo relevant conflicts of interest to declare.

Galectin-3 Regulates Desmoglein-2 and Intestinal Epithelial Intercellular Adhesion

The desmosomal cadherins, desmogleins, and desmocollins mediate strong intercellular adhesion. Human intestinal epithelial cells express the desmoglein-2 isoform. A proteomic screen for Dsg2-associated proteins in intestinal epithelial cells identified a lectin referred to as galectin-3 (Gal3). Gal3 bound to N-linked β-galactosides in Dsg2 extracellular domain and co-sedimented with caveolin-1 in lipid rafts. Down-regulation of Gal3 protein or incubation with lactose, a galactose-containing disaccharide that competitively inhibits galectin binding to Dsg2, decreased intercellular adhesion in intestinal epithelial cells. In the absence of functional Gal3, Dsg2 protein was internalized from the plasma membrane and degraded in the proteasome. These results report a novel role of Gal3 in stabilizing a desmosomal cadherin and intercellular adhesion in intestinal epithelial cells.

Mechanism for Recognition of an Unusual Mycobacterial Glycolipid by the Macrophage Receptor Mincle

Binding of the macrophage lectin mincle to trehalose dimycolate, a key glycolipid virulence factor on the surface of Mycobacterium tuberculosis and Mycobacterium bovis, initiates responses that can lead both to toxicity and to protection of these pathogens from destruction. Crystallographic structural analysis, site-directed mutagenesis, and binding studies with glycolipid mimics have been used to define an extended binding site in the C-type carbohydrate recognition domain (CRD) of bovine mincle that encompasses both the headgroup and a portion of the attached acyl chains. One glucose residue of the trehalose Glcα1-1Glcα headgroup is liganded to a Ca(2+) in a manner common to many C-type CRDs, whereas the second glucose residue is accommodated in a novel secondary binding site. The additional contacts in the secondary site lead to a 36-fold higher affinity for trehalose compared with glucose. An adjacent hydrophobic groove, not seen in other C-type CRDs, provides a docking site for one of the acyl chains attached to the trehalose, which can be targeted with small molecule analogs of trehalose dimycolate that bind with 52-fold higher affinity than trehalose. The data demonstrate how mincle bridges between the surfaces of the macrophage and the mycobacterium and suggest the possibility of disrupting this interaction. In addition, the results may provide a basis for design of adjuvants that mimic the ability of mycobacteria to stimulate a response to immunization that can be employed in vaccine development.

Porcine Sialoadhesin: A Newly Identified Xenogeneic Innate Immune Receptor

Extracorporeal porcine liver perfusion is being developed as a bridge to liver allotransplantation for patients with fulminant hepatic failure. This strategy is limited by porcine Kupffer cell destruction of human erythrocytes, mediated by lectin binding of a sialic acid motif in the absence of antibody and complement. Sialoadhesin, a macrophage restricted lectin that binds sialic acid, was originally described as a sheep erythrocyte binding receptor. Given similarities between sialoadhesin and the unidentified macrophage lectin in our model, we hypothesized porcine sialoadhesin contributed to recognition of human erythrocytes. Two additional types of macrophages were identified to bind human erythrocytes-spleen and alveolar. Expression of sialoadhesin was confirmed by immunofluorescence in porcine tissues and by flow cytometry on primary macrophages. A stable transgenic cell line expressing porcine sialoadhesin (pSn CHO) bound human erythrocytes, while a sialoadhesin mutant cell line did not. Porcine macrophage and pSn CHO recognition of human erythrocytes was inhibited approximately 90% by an antiporcine sialoadhesin monoclonal antibody and by human erythrocyte glycoproteins. Furthermore, this binding was substantially reduced by sialidase treatment of erythrocytes. These data support the hypothesis that porcine sialoadhesin is a xenogeneic receptor that mediates porcine macrophage binding of human erythrocytes in a sialic acid-dependent manner.

Regulation of Prostate Cancer Progression by Galectin-3

Galectin-3, a beta-galactoside-binding protein, has been implicated in a variety of biological functions including cell proliferation, apoptosis, angiogenesis, tumor progression, and metastasis. The present study was undertaken to understand the role of galectin-3 in the progression of prostate cancer. Immunohistochemical analysis of galectin-3 expression revealed that galectin-3 was cleaved during the progression of prostate cancer. Galectin-3 knockdown by small interfering RNA (siRNA) was associated with reduced cell migration, invasion, cell proliferation, anchorage-independent colony formation, and tumor growth in the prostates of nude mice. Galectin-3 knockdown in human prostate cancer PC3 cells led to cell-cycle arrest at G(1) phase, up-regulation of nuclear p21, and hypophosphorylation of the retinoblastoma tumor suppressor protein (pRb), with no effect on cyclin D1, cyclin E, cyclin-dependent kinases (CDK2 and CDK4), and p27 protein expression levels. The data obtained here implicate galectin-3 in prostate cancer progression and suggest that galectin-3 may serve as both a diagnostic marker and therapeutic target for future disease treatments.

Galectin-3 Gene Inactivation Reduces Atherosclerotic Lesions and Adventitial Inflammation in ApoE-Deficient Mice

This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n = 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoE-deficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36- to 44-week group of ApoE(-/-)/GaL3(-/-) mice had a significantly lower number of atherosclerotic lesions (P < 0.004) and fewer atheromatous plaques (P < 0.008) when compared with ApoE(-/-)/GaL3+/+ mice of the same age. ApoE(-/-)/GaL3(-/-) mice had a lower number of perivascular inflammatory infiltrates and mast cells than those found in ApoE(-/-)/GaL3+/+ mice. The reduced number of perivascular mast cells may have resulted in a low level of interleukin-4 that contributed to the reduction in the morphological parameters of atherogenesis correlated with the lack of GaL3 expression. The effect of GaL3 deficiency on atherogenesis decrease could be related to its function as a multifunctional protein implicated in macrophage chemotaxis, angiogenesis, lipid loading, and inflammation.

Sialoside-binding macrophage lectins in phagocytosis of apoptotic bodies

Elimination of apoptotic bodies is one of the important functions of macrophages. The aim of this work was to study the role of macrophage lectins in this process. Macrophage lectins were probed with neoglycoconjugates Glyc-PAA-fluo where carbohydrate is linked to fluorescein-labeled polyacrylamide (MW 30 kD). It was shown that neoglycoconjugates containing a Neu5Acalpha2-3Gal fragment bound to macrophages isolated from blood of healthy donors. Besides, carbohydrate chains containing the same fragment were revealed on apoptotic bodies. Phagocytosis of apoptotic bodies by macrophages was inhibited with sialooligosaccharide ligands of siglec-5 and MAbs to siglec-5. Thus, siglec-5 expressed on macrophages could participate in phagocytosis of apoptotic bodies. In addition, the role of siglecs in engulfment of apoptotic bodies by tumor-associated macrophages was studied. The phagocytic potency of macrophages isolated from blood of breast cancer patients was lower than engulfment ability of macrophages obtained from healthy donors and depended on tumor degree. Staining of macrophages obtained from blood of tumor patients with sialylated Glyc-PAA-fluo probes was more intense than that of macrophages from healthy donors; phagocytosis of apoptotic bodies by tumor-associated macrophages was inhibited by carbohydrates that are known to be ligands for siglecs.

Receptor-mediated stimulatory effect of oligochitosan in macrophages

Oligochitosan, which has greater than 3 but less than 10 saccharide ( N-acetylglucosamine or glucosamine) residues, is obtained by either chemical or enzymatic hydrolysis of chitosan. In this work, we demonstrated that oligochitosan had an in vitro stimulatory effect on the release of tumor necrosis factor-α and interleukin-1 β in macrophages. Moreover, we observed that oligochitosan could be uptaken by macrophages through confocal laser microscopy. Scatchard analysis of internalization of 2-aminoacridone-oligochitosan in macrophages indicated its internalization was mediated by a specific receptor on macrophage membrane with a K d of 2.1 × 10 −5 M. Competition studies showed that mannose could inhibit oligochitosan internalization, while lipopolysaccharide and β-glucan could not do it. Inhibition of mannose–BSA, fucose–BSA, and N-acetylglucosamine–BSA on oligochitosan internalization further suggests that oligochitosan internalization is mediated by a macrophage lectin receptor like with mannose specificity.

Dectin-1 is a major beta-glucan receptor on macrophages.

Zymosan is a β-glucan– and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a β-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the β-glucan receptor mediating this activity. To address the role of the recently described β-glucan receptor, Dectin-1, we generated a novel anti–Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the β-glucan–dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte β-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of β-glucans for therapeutic drug design.

The Macrophage C-type Lectin Specific for Galactose/N-Acetylgalactosamine Is an Endocytic Receptor Expressed on Monocyte-derived Immature Dendritic Cells

Lectins on antigen presenting cells are potentially involved in the antigen uptake and the cellular recognition and trafficking. Serial analysis of gene expression in monocyte-derived dendritic cells (DCs), monocytes, and macrophages revealed that 7 of the 19 C-type lectin mRNA were present in immature DCs. Two of these, the macrophage mannose receptor and the macrophage lectin specific for galactose/N-acetylgalactosamine (MGL), were found only in immature DCs, as confirmed by reverse transcriptase-PCR and flow cytometric analysis. By subcloning and sequencing the amplified mRNA, we obtained nucleotide sequences encoding seven different human MGL (hMGL) subtypes, which were apparently derived from alternatively spliced mRNA. In addition, the hMGL gene locus on human chromosome 17p13 contains one gene. A single nucleotide polymorphism was identified at a position in exon 3 that corresponds to the cytoplasmic region proximal to the transmembrane domain. Of all the splicing variants, the hMGL variant 6C was expressed at the highest levels on immature DCs from all donors tested. Immature DCs could incorporate alpha-GalNAc-modified soluble acrylamide polymers, and this was significantly inhibited by pretreatment of the cells with an anti-hMGL monoclonal antibody that blocks the lectin-carbohydrate interaction. We propose that hMGL is a marker of imDCs and that it functions as an endocytic receptor for glycosylated antigens.

Human macrophage lectin specific for galactose/N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages.

We studied the expression of a human macrophage lectin specific for galactose/N-acetylgalactosamine (hMGL) during macrophage differentiation. The expression of hMGL during the in vitro differentiation induced by human serum was examined by immunostaining and Western blotting with a specific mAb, MLD-1, as well as with RT-PCR analysis. hMGL was detected on cells at an intermediate stage of differentiation. These cells were round, slightly larger in size (12.7 +/- 0.2 microm) than monocytes (9.8 +/- 0.1 microm) and expressed the macrophage marker CD14, but lacked the dendritic cell marker CD1a. The highest levels of expression occurred after 2-4 days of culture. At this time point, MLD-1 prominently stained 20-40% of the cells. Monocytes cultured for 16 h or fully differentiated monocyte-derived macrophages were negative or weak for hMGL expression. Similar transient expression was also observed during granulocyte macrophage colony stimulating factor- or macrophage colony stimulating factor-dependent macrophage differentiation. The lectin was characterized as a functional endocytic receptor for glycosylated macromolecules, since the uptake of carbohydrate polymers was partially inhibited by the addition of MLD-1. The distribution of hMGL(+) cells in normal human skin was found by immunostaining to be mainly in the upper dermis distant from vascular structures. More than 90% of the hMGL(+) cells were double stained with anti-CD68 mAb and constituted approximately 20% of the CD68(+) cells. We suggest that the dermal hMGL(+) cells are a subset of differentiated cells derived from monocytes and that hMGL is a unique marker for cells at an intermediate stage of macrophage differentiation.

Immature human dendritic cells express asialoglycoprotein receptor isoforms for efficient receptor-mediated endocytosis.

In a search for genes expressed by dendritic cells (DC), we have cloned cDNAs encoding different forms of an asialoglycoprotein receptor (ASGPR). The DC-ASGPR represents long and short isoforms of human macrophage lectin, a Ca(2+)-dependent type II transmembrane lectin displaying considerable homology with the H1 and H2 subunits of the hepatic ASGPR. Immunoprecipitation from DC using an anti-DC-ASGPR mAb yielded a major 40-kDa protein with an isoelectric point of 8.2. DC-ASGPR mRNA was observed predominantly in immune tissues. Both isoforms were detected in DC and granulocytes, but not in T, B, or NK cells, or monocytes. DC-ASGPR species were restricted to the CD14-derived DC obtained from CD34(+) progenitors, while absent from the CD1a-derived subset. Accordingly, both monocyte-derived DC and tonsillar interstitial-type DC expressed DC-ASGPR protein, while Langerhans-type cells did not. Furthermore, DC-ASGPR is a feature of immaturity, as expression was lost upon CD40 activation. In agreement with the presence of tyrosine-based and dileucine motifs in the intracytoplasmic domain, mAb against DC-ASGPR was rapidly internalized by DC at 37 degrees C. Finally, intracellular DC-ASGPR was localized to early endosomes, suggesting that the receptor recycles to the cell surface following internalization of ligand. Our findings identify DC-ASGPR/human macrophage lectin as a feature of immature DC, and as another lectin important for the specialized Ag-capture function of DC.

Adsorbed IgG: a potent adhesive substrate for human macrophages.

Previous reports from our laboratory have demonstrated qualitatively that preabsorbed IgG can enhance long-term macrophage adhesion in vitro. This investigation further characterizes and quantifies the biological effect of adsorbed human IgG on human macrophages and probes the potential mechanisms. Ten-day human monocyte/macrophage cultures on Plastek M (PM), a normally poor cellular substrate for macrophages, confirmed the ability of preabsorbed IgG to dramatically enhance long-term macrophage adhesion. An adsorption solution concentration of 200 microg/mL of IgG was necessary to provide a consistent, optimal cellular response. (125)I adsorption studies indicated Langmuir-style IgG adsorption at low concentrations; however, no adsorption maximum was observed. Additional adsorption analysis revealed that the IgG fragments Fab, F(ab')(2), and Fc adsorb at levels only 20-40% that of the whole molecule. Despite the lower adsorption levels, both preabsorbed Fab and F(ab')(2) were shown to be as effective as whole molecule IgG at enhancing long-term macrophage adhesion. Surprisingly, the preabsorbed Fc fragment demonstrated no IgG-like activity, thereby eliminating the possibility of an Fc receptor-based mechanism. Other possible mechanisms, such as macrophage lectins, novel macrophage Fab receptors, and complement activation by adsorbed IgG and IgG fragments, are discussed.

Epitope mapping of monoclonal antibodies specific for a macrophage lectin: a calcium-dependent epitope is in the carbohydrate recognition domain

Mouse macrophage galactose/ N-acetylgalactosamine-specific calcium-type lectin (mMGL) has a calcium-dependent conformational epitope which is a ligand-induced binding site. A monoclonal antibody (mAb) specific for this epitope (LOM-11) stabilize lectin activity. We performed mapping for this conformational epitope using trypsin fragments that contain a carbohydrate recognition domain (CRD) and chimeric recombinant proteins between mMGL and a human counterpart of this molecule. Binding site for the mAb LOM-11 was mapped within the C-terminal 59 amino acids of CRD. Binding sites for all four mAbs that block carbohydrate ligand binding were also mapped in the C-terminal half of CRD. These results indicated that the calcium-dependent site potentially involved in protein-protein interaction, regulatory or for coordinated binding, is mapped within CRD in addition to the independent carbohydrate binding site, and that both of the distinct sites may have spatial proximity.

APCs Express DCIR, a Novel C-Type Lectin Surface Receptor Containing an Immunoreceptor Tyrosine-Based Inhibitory Motif

We have identified a novel member of the calcium-dependent (C-type) lectin family. This molecule, designated DCIR (for dendritic cell (DC) immunoreceptor), is a type II membrane glycoprotein of 237 aa with a single carbohydrate recognition domain (CRD), closest in homology to those of the macrophage lectin and hepatic asialoglycoprotein receptors. The intracellular domain of DCIR contains a consensus immunoreceptor tyrosine-based inhibitory motif. A mouse cDNA, encoding a homologous protein has been identified. Northern blot analysis showed DCIR mRNA to be predominantly transcribed in hematopoietic tissues. The gene encoding human DCIR was localized to chromosome 12p13, in a region close to the NK gene complex. Unlike members of this complex, DCIR displays a typical lectin CRD rather than an NK cell type extracellular domain, and was expressed on DC, monocytes, macrophages, B lymphocytes, and granulocytes, but not detected on NK and T cells. DCIR was strongly expressed by DC derived from blood monocytes cultured with GM-CSF and IL-4. DCIR was mostly expressed by monocyte-related rather than Langerhans cell related DC obtained from CD34+ progenitor cells. Finally, DCIR expression was down-regulated by signals inducing DC maturation such as CD40 ligand, LPS, or TNF-alpha. Thus, DCIR is differentially expressed on DC depending on their origin and stage of maturation/activation. DCIR represents a novel surface molecule expressed by Ag presenting cells, and of potential importance in regulation of DC function.

Interaction of Human Macrophage C-type Lectin withO-Linked N-Acetylgalactosamine Residues on Mucin Glycopeptides

A fluorescein-labeled synthetic peptide, PTTTPITTTTK, was converted into O-glycosylated glycopeptides with various numbers of attached N-acetyl-D-galactosamines (GalNAcs) by in vitro glycosylation with UDP-GalNAc and a microsomal fraction of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAcTGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G6. A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density. Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble gly-copeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML. Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylated O-linked glycopeptides should be due to the trimer formation of rHML.