Involvement of serum mannan binding proteins and mannose receptors in uptake of mannosylated liposomes by macrophages

The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.     

Uptake of recombinant iduronate-2-sulfatase into neuronal and glial cells in vitro

The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man-liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man-liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man-liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.     

Serum mannan binding protein inhibits mannosylated liposome-mediated transfection to macrophages

The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man–liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man–liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-d-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man–liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.     

Mannose receptor dependent uptake of a ricin A chain--antibody conjugate by rat liver non-parenchymal cells

Mannose receptor mediated uptake by the reticuloendothelial system has been suggested as an explanation for the rapid removal of ricin A chain antibody conjugates from the circulation after their administration. We have measured, in the rat, hepatic uptake of a ricin A chain antibody conjugate in vivo and its susceptibility to inhibition by a mannosylated protein and have measured uptake of the conjugate in vitro by rat parenchymal and non-parenchymal liver cells. The results indicate that rapid hepatic uptake of conjugate does occur in vivo; cultured non-parenchymal cells accumulate the conjugate to a much greater degree than cultured parenchymal cells and that mannose receptors appear to be involved in the process.     

Pharmacokinetics and disposition characteristics of recombinant decorin after intravenous injection into mice

The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent.     

The increase in mannose receptor recycling favors arginase induction and Trypanosoma cruzi survival in macrophages

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular parasite growth.     

The adjuvant activity of lactoferrin in the generation of DTH to ovalbumin can be inhibited by bovine serum albumin bearing alpha-D-mannopyranosyl residues

Lactoferrin (LF) is an iron-binding glycoprotein present in the cytoplasmic granules of neutrophils and in external secretions of mammals. Although the biological role of human and bovine lactoferrin has been extensively studied, there is still uncertainty as to the nature and function of lactoferrin receptors. We recently determined that methyl-alpha-D-mannopyranoside given intraperitoneally (i.p.) could suppress the adjuvant activity of LF in the generation of delayed-type hypersensitivity (DTH) to ovalbumin (OVA). We concluded that the lactoferrin effects in DTH are mediated by carbohydrate-recognizing receptors like the mannose receptor (MR). This study indicates that subcutaneous (s.c.) administration of very small doses of the Man-bovine serum albumin (Man-BSA) complex, together with a sensitizing dose of the antigen, gives the same effects as i.p. administration of methyl-alpha-D-mannopyranoside. The latter is also a blocker of MR, although of a much lower affinity to the receptor than Man-BSA. The blocking of the adjuvant effect of LF by the Man-BSA complex (when given together with the sensitising dose of antigen) suggests that the function of antigen-presenting cells in the skin (presumably immature dendritic cells expressing MR) is inhibited. The results of our study indicate that a receptor with an affinity for mannose is essential for the mediation of adjuvant lactoferrin function in the generation of DTH.     

Biomimetic liposomes and planar supported bilayers for the assessment of glycodendrimeric porphyrins interaction with an immobilized lectin

Photodynamic therapy is a potentially efficient treatment for various solid tumours, among which retinoblastoma. Its efficacy depends on the preferential accumulation of photosensitizers in the malignant tissues and their accessibility to light. The specificity of drugs for retinoblastoma cells can be improved by targeting a mannose receptor overexpressed at their surface. With the aim of assessing the recognition of newly synthesized glycodendrimeric porphyrins by such receptors, we have built and characterized an original synthetic biomimetic membrane having similar lipidic composition to that of the retinal cell membranes and bearing Concanavalin A, as a model of the mannose receptor. The interaction of the porphyrin derivatives with liposomes and supported planar bilayers has been studied by dynamic light scattering and quartz crystal microbalance with dissipation monitoring (QCM-D). Only mannosylated porphyrins interacted significantly with the membrane model. The methodology used proved to be efficient for the selection of potentially active compounds.     

Enhancement of macromolecular ligand binding by rabbit alveolar macrophages by mannose oligosaccharides and related compounds

When rabbit alveolar macrophages were incubated with 10 mM D-mannose, binding of macromolecular ligands containing D-mannose, such as bovine serum albumin modified with mannose (Man-BSA), was enhanced more than 100%, but was inhibited at higher concentrations [C.A. Hoppe and Y. C. Lee (1982) J. Biol. Chem. 257, 12831-12834]. This phenomenon was further investigated with ovalbumin-derived glycopeptide, Asn(GlcNA2,Man5), and with a wide variety of synthetic mannose oligosaccharides. The extent of enhancement is related to the fine structure of the oligosaccharide groups, but the results are complicated by concurrent inhibition exerted by these compounds. It appears that the more inhibitory a compound is, the less capable it is of exerting the enhancement effect. Thus, small mannose derivatives such as glycosides, including clustered mannosides based on tris(hydroxymethyl)aminomethane [Y. C. Lee (1978) Carbohydr. Res. 67, 509-514], and most of the biantennary mannose oligosaccharides were found to be effective in enhancing the binding of radiolabeled Man-BSA. Triantennary oligosaccharides, on the other hand, showed only a slight enhancement effect and a much stronger inhibitory effect. The effects of ligand size, valency, as well as the fine structure on enhancement are discussed.     

Extremely rapid endocytosis mediated by the mannose receptor of sinusoidal endothelial rat liver cells.

Isolated sinusoidal endothelial rat liver cells (EC) in suspension bound and internalized ovalbumin, a mannose-terminated glycoprotein, in a saturable manner. The binding and uptake were Ca2+-dependent and were effectively inhibited by alpha-methyl mannoside and yeast mannan, but not by galactose or asialoglycoproteins. This corresponds to the binding specificity described for the mannose receptor of macrophages and non-parenchymal liver cells. Binding studies indicated a surface pool of 20,000-25,000 mannose receptors per cell, with a dissociation constant of 6 x 10(-8) M. Uptake and degradation of ovalbumin by isolated EC were inhibited by weak bases and ionophores which inhibit acidification of endocytic vesicles and dissociation of receptor-ligand complexes. Cycloheximide had no effect on uptake or degradation. Degradation, but not uptake, was inhibited by leupeptin. We conclude that ovalbumin dissociates from the mannose receptors in the endosomal compartment and the receptors are recycled to the cell surface, while the ovalbumin is directed to the lysosomes for degradation. A fraction of the internalized ovalbumin was recycled intact to the cell surface and escaped degradation (retroendocytosis). The rate of internalization of ovalbumin by isolated EC was very fast, with a Ke (endocytotic rate constant) of 4.12 min-1, which corresponds to a half-life of 10 s for the surface pool of receptor-ligand complexes. To our knowledge, this is the highest Ke reported for a receptor-mediated endocytosis system.     

The effect of a mannose binding protein on macrophage interactions with Candida albicans.

A soluble mannose binding protein (MBP), obtained from rabbit serum, was found to inhibit phagocytosis of Candida albicans by bone marrow derived, cultured murine macrophages. During in vitro incubation of yeast with lymphocyte-free macrophage populations uptake of the yeast was significantly reduced at MBP concentrations of 5 micrograms/ml. A similar reduction in yeast phagocytosis was produced by dextrose, d-fucose, l-fucose, d-mannose and alpha-methyl-d-mannoside but required saccharide concentrations of 25-50 mg/ml. Inhibition of phagocytosis of the yeast also resulted from pretreatment of either the macrophages or the yeasts with MBP followed by washing. As expected, the addition of mannan to the assay medium blocked the inhibitory effect of MBP for uptake of C. albicans. These findings suggest that both cell bound and soluble mannose receptors may be important modulators of macrophage-Candida interactions.     

Uptake by macrophages of a biotinylated oligo-alpha-deoxythymidylate by using mannosylated streptavidin.

Streptavidin substituted with mannose residues increased by 20-fold the intracellular concentration of a biotinylated dodecakis(alpha-deoxythymidylate) in macrophages by comparison with the uptake of free oligodeoxynucleotide. Streptavidin, the bacterial homologue of the very basic avidin, which does not contain any carbohydrate moieties and is a neutral protein, was substituted with 12 mannose residues in order to be recognized and internalized by mannose-specific lectins on the surface of macrophages. A 3'-biotinylated and 5'-fluoresceinylated dodecakis (alpha-deoxythymidylate) was synthesized and bound onto mannosylated streptavidin. The conjugate was isolated, and by using flow cytometry, it was shown that the uptake of fluoresceinylated oligodeoxynucleotides bound to mannosylated streptavidin by macrophages is 20-fold higher than that of free oligodeoxynucleotides and that the uptake was competively inhibited by mannosylated serum albumin. Glycosylated streptavidin conjugates recognizing specific membrane lectins on different cells provide the possibility to target biotinylated antisense oligodeoxynucleotides and to increase the biological effect of these chemotherapeutic agents.     

Cryptococcus neoformans glycoantigens are captured by multiple lectin receptors and presented by dendritic cells

Cell-mediated immune responses to glycoantigens have been largely uncharacterized. Protective T cell responses to the pathogenic yeast Cryptococcus neoformans are dependent on heavily mannosylated Ags termed mannoproteins. In the work presented, the innate immune response to mannoprotein was determined. Purified murine splenic dendritic cells (DC), B cells, and macrophages were used to stimulate mannoprotein-specific T cells. Only DC were capable of any measurable stimulation. Depletion of DC resulted in the abrogation of the T cell response. Human and murine DC rapidly captured fluorescent-labeled mannoprotein by a mannose receptor-mediated process. Using transfected cell lines, the type II C-type lectin receptor DC-specific ICAM-3-grabbing nonintegrin (CD209) was determined to have affinity for mannoprotein. Taken together with prior work demonstrating that mannoprotein was captured by the macrophage mannose receptor (CD206), these data suggest that multiple mannose receptors on DC recognize mannoprotein. Pulsing experiments demonstrated that DC captured sufficient mannoprotein over 2 h to account for 50% of total stimulation. Capture appeared dependent on mannose receptors, as competitive mannosylated inhibitors and calcium chelators each interfered with T cell stimulation. By confocal microscopy, intracellular mannoprotein trafficked to an endo-lysosomal compartment in DC, and at later time points extended into tubules in a similar fashion to the degradation marker DQ-OVA. Mannoprotein colocalized intracellularly with CD206 and CD209. These data suggest that DC provide the crucial link between innate and adaptive immune responses to C. neoformans via a process that is dependent upon the efficient uptake of mannoprotein by mannose receptors.     

Isolation and characterization of a mannose-specific endocytosis receptor from human placenta

A receptor which recognizes glycoproteins bearing terminal mannose residues has been isolated from human placental membranes. Washed membranes were solubilized with buffer containing 1% Triton X-100 and applied to a mannose-Sepharose affinity column. The column was eluted with buffer containing 200 mM mannose and 1% cholate. The major protein eluted exhibited a molecular weight of 175 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein binds 125I-labeled mannosylated bovine serum albumin in a saturable fashion with a dissociation constant of 4 nM. Ligand binding is pH-dependent with maximal binding above pH 6.5. This binding can be inhibited with EDTA, mannose, fucose, mannan, beta-glucuronidase, and bovine serum albumin conjugated to fucose. Polyclonal antibodies generated against the mannose binding protein immunoprecipitate a single 175-kDa protein species from both surface-iodinated and biosynthetically labeled human monocyte-derived macrophages.     

Endocytosis of α1-acid glycoprotein variants and of neoglycoproteins containing mannose derivatives by a mouse hybridoma cell line (2C11–12). Comparison with mouse peritoneal macrophages

Macrophages from various origins are known to express membrane lectins that mediate the endocytosis of mannose-bearing glycoconjugates. Most macrophage tumor cell-lines lack such receptors. In this paper we show by flow cytometry analysis that a newly generated macrophage hybridoma (2C_11–12), which displays several macrophage characteristics, also expresses mannose membrane lectins, resulting in the internalization of fluoresceinylated neoglycoproteins into acidic compartments.Thioglycolate elicited mouse peritoneal macrophages and the 2C_11–12 hybridomas were compared by flow cytometry with regard to the binding and endocytosis of ₁-acid glycoprotein (AGP) variants separated by affinity chromatography on immobilized concanavalin A. AGP C eluted specifically with methyl -mannopyranoside, which contains two bi-antennary oligosaccharides, was endocytosed as mannosylated serum albumin (Man-BSA). In both types of macrophages, the fluoresceinylated ligands were internalized in acidic compartments as demonstrated by the fluorescence intensity increase upon monensin post-incubation. However the behaviour of the internalized ligands was found to be quite different. AGP C and Man-BSA were rapidly degraded by thioglycolate elicited peritoneal macrophages and excreted in the medium as small peptide fragments; conversely they remained a longer time in the 2C_11–12 hybridoma.     

Highly ordered supra-molecular organization of the mycobacterial lipoarabinomannans in solution. Evidence of a relationship between supra-molecular organization and biological activity

The complex mycobacterial mannosylated lipoarabinomannans (ManLAMs) are currently considered to be the major virulence factors of the pathogenic Mycobacterium tuberculosis. The recognition and the interaction of ManLAMs with immune system receptors have been shown to promote M.tuberculosis phagocytosis but also to down-regulate the bactericidal immune response of the host in favor of the survival of the pathogenic bacilli. To date these original biological activities were mainly associated to the presence of mannose residues capping the non-reducing ends of the ramified polysaccharide moiety of these complex lipoglycans. However, we demonstrated recently that the molecular recognition of ManLAM terminal mannose units by human pulmonary surfactant protein A (hSP-A) carbohydrate recognition domains depends on the presence of the lipid moiety of the ManLAMs as proposed by Sidobre et al. in 2002. Thus, we investigated the putative role of the ManLAM aglycon moiety. The data presented here, indicate that the hydrophobic aglycon part of ManLAM is associated to a characteristic concentration-dependent supra-molecular organization of these complex molecules. Furthermore, we observed that the deacylated ManLAMs or the lipid-free mannosylated arabinomannans, which do not exhibit characteristic ManLAM activities, do not display this supra-molecular organization. These observations strongly suggest that the ManLAMs immunomodulatory activities might be associated to their particular organization. Finally, the determination of the critical micellar concentration of ManLAMs obviously supports the notion that this supra-molecular organization may be responsible for the specific biological activities of these complex molecules.     

Cooperative stimulation of dendritic cells by Cryptococcus neoformans mannoproteins and CpG oligodeoxynucleotides

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4(+) T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.     

Synthesis of lupane-type saponins bearing mannosyl and 3,6-branched trimannosyl residues and their evaluation as anticancer agents

The saponins modified with mono- or trimannosyl residues can provide a convenient means of delivering drugs to certain human cells via interactions with mannose receptors. In the study reported therein, we developed a convenient approach for the synthesis of 3-O-mannoside and branched trimannoside derivatives of the saponin lupeol and of C-28 acyl esters of 3-O-acetyl-betulinic acid bearing the same mannosyl entities. Lupeol and 3-O-acetyl-betulinic acid were mannosylated with tetra-O-benzoyl- or tetra-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidates. De-esterification followed by regioselective dimannosylation of the unprotected monosaccharide derivatives with 2equiv of tetra-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate selectively yielded O-3,O-6-linked trimannosides. The cytotoxic activity of selected lupane-type saponins (derivatives of lupeol, betulinic acid, and betulin) toward normal human fibroblasts and various cancer cell lines was also compared.     

Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system

Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG(2b)) carrying mostly O-glycans and, as a control, the α1-acid glycoprotein (AGP/mIgG(2b)) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. Pichia pastoris-produced PSGL-1/mIgG(2b) was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by α-mannosidases including an α1,2- but not an α1,6-mannosidase. Electrospray ionization ion-trap mass spectrometry confirmed the presence of O-glycans containing up to nine hexoses with the penta- and hexasaccharides being the predominant ones. α1,2- and α1,3-linked, but not α1,6-linked, mannose residues were detected by (1)H-nuclear magnetic resonance spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG(2b) to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG(2b) expressed in P. pastoris carried O-glycans mainly comprised of α-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo.     

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.