High-mannose Oligosaccharides Research Articles

Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli.

We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.

Biosynthesis, Intracellular Targeting, and Degradation of the EAAC1 Glutamate/Aspartate Transporter in C6 Glioma Cells

Rat C6 glioma cells were used as a model system to study the biosynthesis, intracellular targeting, and degradation of the EAAC1 transporter, a sodium-dependent glutamate/aspartate transport protein that encodes System X(-)A,G activity. At steady state, nearly 70% of the EAAC1 transporter was located at the cell surface. The newly synthesized EAAC1 protein was co-translationally N-glycosylated with high mannose oligosaccharide chains that were processed into complex-type sugar chains as the protein matured. The final maturation steps for EAAC1 protein coincided with its plasma membrane arrival, which was first detected at about 45 min after the initial synthesis. The newly synthesized EAAC1 protein was protected from degradation during the maturation and targeting process, as well as during the first 5 h after plasma membrane arrival. After this initial lag period, both the newly synthesized transporter and the total cellular EAAC1 pool were degraded by first order kinetics with a half-life of 6 h. These results represent the first analysis of the synthesis and degradation of the EAAC1 amino acid transporter.

Multisite and Multivalent Binding between Cyanovirin-N and Branched Oligomannosides: Calorimetric and NMR Characterization

Binding of the protein cyanovirin-N to oligomannose-8 and oligomannose-9 of gp120 is crucially involved in its potent virucidal activity against the human immunodeficiency virus (HIV). The interaction between cyanovirin-N and these oligosaccharides has not been thoroughly characterized due to aggregation of the oligosaccharide-protein complexes. Here, cyanovirin-N's interaction with a nonamannoside, a structural analog of oligomannose-9, has been studied by nuclear magnetic resonance and isothermal titration calorimetry. The nonamannoside interacts with cyanovirin-N in a multivalent fashion, resulting in tight complexes with an average 1:1 stoichiometry. Like the nonamannoside, an α1→2-linked trimannoside substructure interacts with cyanovirin-N at two distinct protein subsites. The chitobiose and internal core trimannoside substructures of oligomannose-9 are not recognized by cyanovirin-N, and binding of the core hexamannoside occurs at only one of the sites on the protein. This is the first detailed analysis of a biologically relevant interaction between cyanovirin-N and high-mannose oligosaccharides of HIV-1 gp120.

H2, the Minor Subunit of the Human Asialoglycoprotein Receptor, Trafficks Intracellularly and Forms Homo-oligomers, but Does Not Bind Asialo-orosomucoid

The functional human hepatic asialoglycoprotein receptor (ASGP-R) is a hetero-oligomer composed of two subunits, designated H1 and H2, which are highly homologous. Despite their extensive homology, the major H1 subunit is stably expressed by itself, whereas in the absence of H1 most of the H2 subunits are degraded in the ER. In this study, we were able to investigate the capability of the minor ASGP-R subunit, H2, to function independently of H1, because it was apparently stabilized by fusing its NH(2) terminus with an epitope tag. We could thus create stable cell lines in hepatoma-derived SK-Hep-1 cells that expressed the H2 subunit alone. H2 was expressed on the cell surface and was internalized, predominantly through the clathrin-coated pit pathway. Since the internal pool of H2 was also able to traffick to the cell surface, we conclude that H2 recycles between the surface and intracellular compartments, similar to the constitutive recycling of hetero-oligomeric ASGP-R complexes. However, the rate of H2 recycling and internalization was approximately 25-33% that of H1. Similar to H1, the H2 polypeptides were also able to self-associate to form homo-oligomers, including trimers and tetramers. However, unlike H1, which can bind the ligand asialo-orosomucoid (ASOR) when overexpressed in COS-7 cells, H2 failed to bind or endocytose ASOR. In summary, the H2 subunit of the human ASGP-R contains functional, although weak, signal(s) for endocytosis and recycling and has the ability to oligomerize. H2 homo-oligomers, however, do not create binding sites for desialylated glycoproteins, such as ASOR, that contain tri- and tetra-antennary N-linked oligosaccharides. Nonetheless, these results raise the intriguing possibility that naturally occurring H2 homo-oligomers may exist in human hepatocytes and have an as yet undiscovered function.

E3 ubiquitin ligase that recognizes sugar chains.

N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control. Here we report that N-glycan serves as a signal for degradation by the Skp1-Cullin1-Fbx2-Roc1 (SCF(Fbx2)) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin beta 1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2 Delta F, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway. Our results indicate that SCF(Fbx2) ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.

Orientation of Bound Ligands in Mannose-binding Proteins: IMPLICATIONS FOR MULTIVALENT LIGAND RECOGNITION

Mannose-binding proteins (MBPs) are C-type animal lectins that recognize high mannose oligosaccharides on pathogenic cell surfaces. MBPs bind to their carbohydrate ligands by forming a series of Ca(2+) coordination and hydrogen bonds with two hydroxyl groups equivalent to the 3- and 4-OH of mannose. In this work, the determinants of the orientation of sugars bound to rat serum and liver MBPs (MBP-A and MBP-C) have been systematically investigated. The crystal structures of MBP-A soaked with monosaccharides and disaccharides and also the structure of the MBP-A trimer cross-linked by a high mannose asparaginyl oligosaccharide reveal that monosaccharides or alpha1-6-linked mannose bind to MBP-A in one orientation, whereas alpha1-2- or alpha1-3-linked mannose binds in an orientation rotated 180 degrees around a local symmetry axis relating the 3- and 4-OH groups. In contrast, a similar set of ligands all bind to MBP-C in a single orientation. The mutation of MBP-A His(189) to its MBP-C equivalent, valine, causes Man alpha 1-3Man to bind in a mixture of orientations. These data combined with modeling indicate that the residue at this position influences the orientation of bound ligands in MBP. We propose that the control of binding orientation can influence the recognition of multivalent ligands. A lateral association of trimers in the cross-linked crystals may reflect interactions within higher oligomers of MBP-A that are stabilized by multivalent ligands.

Potent Inhibition of HIV-1 Fusion by Cyanovirin-N Requires Only a Single High Affinity Carbohydrate Binding Site: Characterization of Low Affinity Carbohydrate Binding Site Knockout Mutants

Cyanovirin-N (CVN) is a novel cyanobacterial protein that potently inhibits viral entry by human immunodeficiency viruses (HIV) via high affinity carbohydrate-mediated binding to the surface envelope glycoprotein gp120. Bearing C 2 pseudo-symmetry, CVN contains two carbohydrate binding sites of differing affinities located at opposite ends of the protein. CVN selectively binds with nanomolar affinity the mammalian high mannose oligosaccharides oligomannose-8 D1D3 and oligomannose-9, which also govern binding to gp120. At nanomolar concentrations CVN binds these oligosaccharides only through its high affinity site, while at micromolar to millimolar concentrations the oligosaccharides are bound through both sites leading to divalent protein–carbohydrate interactions. Similarly, two modes of binding to gp120 can be envisioned where CVN either binds gp120 solely through the high affinity site, or binds divalently using both carbohydrate binding sites. To determine the role of the low affinity site in binding to gp120, we sought to design a variant of CVN that lacks the low affinity carbohydrate binding site but retains a fully functional high affinity site. Thus, we constructed a series of CVN mutants possessing cumulative mutations in the low affinity site only, and characterized by NMR the overall structure and carbohydrate binding ability of each of these mutants. We demonstrate that carbohydrate binding by the low affinity site is completely absent in two mutants bearing three or four mutations (namely, m3-CVN=Lys3Asn, Glu23Ile, Asn93Ala; and m4-CVN=Lys3Asn, Thr7Ala, Glu23Ile, Asn93Ala), while the high affinity site binds the high affinity ligand Manα(1-2)Manα with a K d value equal to that measured for CVN. Using an HIV-1 cell fusion assay, we show that all of the mutants inhibit HIV-1 fusion with nearly identical IC 50 values as wild-type CVN. We interpret these results as indicating that the low affinity carbohydrate binding site of CVN is not necessary for high affinity binding to gp120, and HIV-1 fusion can therefore be blocked by monovalent protein–carbohydrate interactions.

Interaction of Diocleinae lectins with glycoproteins based in surface plasmon resonance.

Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was followed in real time using surface plasmon resonance technology. The lectins which share many biochemical and structural features could be clearly differentiated in terms of their specificity for complex glycoconjugates. The most prominent interaction of the lectins with PHA-E comparing with soybean agglutinin, both glycoproteins exhibiting high mannose oligosaccharides, suggests that the whole structure of the glycoproteins themselves, may interfere in affinity. These findings also support the hypothesis that minor amino acid replacements in the primary sequence of the lectins might be responsible for their divergence in fine specificity and biological activities. This is the first report using surface plasmon resonance technology that evidences differences of Diocleinae lectins in respect their fine glycan-specificity.

A Linear Synthesis of Branched High-Mannose Oligosaccharides from the HIV-1 Viral Surface Envelope Glycoprotein gp120

Described is a linear solution-phase synthesis of the HIV-1 viral surface envelope glycoprotein gp120 high-mannose nonasaccharide pentyl glycoside. Envisioning the automated solid-phase assembly of complex carbohydrates, the synthesis of the nonasaccharide and the related tri- and hexamannosides demonstrates the facile assembly of highly branched structures in a stepwise fashion incorporating monosaccharide building blocks. A differentially protected core trisaccharide was prepared and further elongated in two high-yielding tri-mannosylations to furnish the triantennary structure. The tri-, hexa-, and nonamannoside n-pentyl glycosides obtained via the described synthesis are currently being used for detailed study of the carbohydrate protein interactions responsible for binding of the anti-HIV protein cyanovirin-N to the glycoprotein gp120. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

Interaction of C1q and Mannan-Binding Lectin with Viruses

As soluble recognition molecules of innate immunity, C1q and MBL are able to bind directly to various viruses, including retroviruses and influenza viruses. Interaction of C1q with retroviruses and certain infected cells was shown to involve the globular region of C1q and viral envelope glycoproteins, such as p15E of MuLV, gp41 and gp120 of HIV-1, gp21 of HTLV-1. C1q binding was found to trigger antibody-independent activation of the classical pathway of complement, but did not lead to virus destruction and had even an adverse effect on infection in humans, because of subversion of the complement system by the virus. Binding of MBL or of the pulmonary collectin SP-D to influenza A virus was shown to involve the carbohydrate recognition domain of the molecule and high-mannose oligosaccharides of the viral proteins haemagglutinin and neuraminidase. These interactions lead to virus inactivation, are independent of complement activation and are influenced by the oligomerization state of the collectin.

Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR.

Dendritic cell specific intracellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a C-type lectin present on the surface of dendritic cells, mediates the initial interaction of dendritic cells with T cells by binding to ICAM-3. DC-SIGN and DC-SIGNR, a related receptor found on the endothelium of liver sinusoids, placental capillaries, and lymph nodes, bind to oligosaccharides that are present on the envelope of human immunodeficiency virus (HIV), an interaction that strongly promotes viral infection of T cells. Crystal structures of carbohydrate-recognition domains of DC-SIGN and of DC-SIGNR bound to oligosaccharide, in combination with binding studies, reveal that these receptors selectively recognize endogenous high-mannose oligosaccharides and may represent a new avenue for developing HIV prophylactics.

Structural analysis and antibody response to the extracellular glutathione S-transferases from Onchocerca volvulus.

Onchocerca volvulus is a human pathogenic filarial parasite which, like other parasitic nematodes, is capable of surviving in an immunologically competent host by employing a variety of immune evasion strategies and defense mechanisms including the detoxification and repair mechanisms of the glutathione S-transferases (GSTs). In this study we analyzed the glycosylation pattern and the immunological properties of extracellular O. volvulus GST1a and -1b (OvGST1a and -1b). The enzymes differ in only 10 amino acids, and both are glycoproteins that have cleavable signal peptides and unusual N-terminal extensions. These characteristics have not been described for other GSTs so far. Mass spectrometry analyses indicate that both enzymes carry high-mannose type oligosaccharides on at least four glycosylation sites. Glycosylation sites 1 to 3 of OvGST1a (OvGST1b sites 2 to 4) are occupied by truncated N-glycans (Man(2)GlcNAc2 to Man(5)GlcNAc(2)), and N glycosylation site 4 of OvGST1a (OvGST1b site 5) carries Man(5)GlcNAc2 to Man(9)GlcNAc(2). To analyze the capacity of these secretory GSTs to stimulate host immune responses, we studied the antibody responses of onchocerciasis patients against the native affinity-purified OvGST1a and -1b. By enzyme-linked immunosorbent assay we showed that OvGST1a and -1b are immunodominant antigens, with less than 7% nonresponder patients. A direct comparison of the antibody responses to the glycosylated and deglycosylated forms demonstrates the high immunogenicity of the N-glycans. Analyses of the antibody responses to the unusual N-terminal extension show an enhanced recognition of this portion by patients as opposed to recognition of the recombinant protein without extension.

A Conserved Flagellar Pocket Exposed High Mannose Moiety Is Used by African Trypanosomes as a Host Cytokine Binding Molecule

Trypanosomes use antigenic variation of their variant-specific surface glycoprotein (VSG) coat as defense against the host immune system. However, in order to sustain their growth, they need to expose conserved epitopes, allowing host macromolecule binding and receptor-mediated endocytosis. Here we show that Trypanosoma brucei uses the conserved chitobiose-oligomannose (GlcNAc(2)-Man(5-9)) moieties of its VSG as a binding ligand for tumor necrosis factor (TNF), a host cytokine with lectin-like properties. As endocytosis in trypanosomes is restricted to the flagellar pocket, we show that soluble flagellar pocket extracts, and in particular soluble VSG, inhibit the binding of (125)I-TNF to trypanosomes. The interaction between TNF and VSG is confirmed by affinity chromatography, biosensor, and dot-blot affinity measurements, and soluble VSG inhibition of TNF-mediated trypanolysis. In all approaches, removal of N-linked carbohydrates abrogates the TNF-VSG interaction. In addition, synthetic high mannose oligosaccharides can block TNF-VSG interactions, and a VSG glycopeptide carrying the GlcNAc(2)-Man(5-9) moiety is shown to inhibit TNF-mediated trypanosome killing in mixed parasite/macrophage cell cultures. Together, these results support the observation that TNF plays a role in growth control of trypanosomes and, moreover, suggest that, by the use of conserved VSG carbohydrates as lectin-binding epitopes, trypanosomes can limit the necessity to express large numbers of invariant surface exposed receptors.

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-α- D-mannosidase for N-glycan engineering in Pichia pastoris

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-α- D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of α-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man 5GlcNAc 2 was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.

Pharmacokinetic studies of Gln117 tissue-type plasminogen activator in rats.

Gln117 t-PA is a mutant type of tissue-type plasminogen activator (mt-PA), which is generated by the removal of a high mannose oligosaccharide resulting from the mutation of amino acid #117, from asparagine (Asn) to glutamine (Gln). The plasma concentration, distribution, metabolism and excretion of Gln117 t-PA were investigated after a single intravenous administration of 125I-Gln117 t-PA or Gln117 t-PA, comparing with wild-type t-PA (WT t-PA). The plasma concentration of Gln117 t-PA decreased more slowly than that of WT t-PA, plasma clearance (CL(p)), and that of Gln117 t-PA in rats was approximately 2.6 times lower than that of WT t-PA. The highest radioactivity was found in the liver at 5 min after intravenous administration of [125I]Gln117 t-PA to rats, but the radioactivity in the liver was lower than that after intravenous administration of [125I]WT t-PA in our previous paper. Within 288 h after intravenous administration of [125I]Gln117 t-PA to rats, 88.5 and 5.5% of administered radioactivity were excreted into urine and feces, respectively. In a gel-filtration chomatographic (GFC) analysis of plasma, Gln117 t-PA formed complexes with plasma proteins, similarly to WT t-PA. The hepatic clearance (CL(hep)) of both t-PAs was evaluated by comparing the plasma concentration after a constant intravenous infusion with that after a constant intraportal venous infusion. The CL(hep) of Gln117 t-PA was 6.5 times lower than that of WT t-PA. These results indicate that the low uptake of Gln117 t-PA to the liver reduces CL(p) compared with WT t-PA.

Suppression of Integrin Expression and Tumorigenicity by Sulfation of Lactosylceramide in 3LL Lewis Lung Carcinoma Cells

To investigate the cellular functions of sulfated glycosphingolipids, we introduced the cerebroside sulfotransferase (CST) gene into J5 cells, a subclone of 3LL Lewis lung carcinoma cells. The J5 cells lack acidic glycosphingolipids but accumulate their common biosynthetic precursor, lactosylceramide. We established the stable CST transfectants, J5/CST-1 and J5/CST-2 clones, highly expressing sulfated lactosylceramide (SM3). Both clones exhibited more spherical morphology in comparison to mock transfectant, and their adhesiveness to fibronectin and laminin was significantly lower. The loss of cell-substratum interactions in these SM3-expressing cells could be attributed to decreased expression of integrins (alpha(5), alpha(6), and beta(1)) on the cell surface and their whole cellular levels. However, the levels of H-2K(b) and H-2D(b) antigens remained unchanged. Reverse transcriptase-polymerase chain reaction and Northern blot analyses for these integrins exhibited significant decrease of beta(1) gene expression in J5/CST-1 and 2, but there was no change in the levels of alpha(5) and alpha(6) transcripts. Deglycosylation by endoglycosidase H treatment clearly demonstrated that the precursor form of beta(1) integrin, possessing high mannose oligosaccharide chains, was preferentially decreased in the CST transfectants. These results demonstrate that endogenous SM3 negatively regulates beta(1) integrin expression at the transcriptional level, and the decrease of alpha integrin proteins in the CST transfectants was due to the post-transcriptional modification. We suggest the putative importance of the intracellular pre-beta(1) integrin pool for normal integrin maturation and subsequent function. Although the rates of cell proliferation in vitro for mock and CST transfectants were similar, tumorigenicity of J5/CST-1 and -2 cells inoculated into syngeneic C57/BL6 mice was greatly decreased or even absent. This was probably due to global loss of the efficient cell-matrix interactions, which are essential for the development of malignant tumors in vivo. Thus, we showed the evidence that cellular SM3 negatively regulates the cell-substratum interaction, resulting in the loss of tumorigenicity.

Biological activities of human mannose-binding lectin bound to two different ligand sugar structures, Lewis A and Lewis B antigens and high-mannose type oligosaccharides

The biological activities of mannose-binding lectin (MBL) which binds to different ligands on mammalian cells were examined using two types of Colo205 cells, a human colon adenocarcinoma cell line: one naturally expressing Lewis A and Lewis B antigens as ligands for MBL (NT-Colo205), and the other modified to express high-mannose type oligosaccharides by treatment with benzyl-2-acetamide-2-deoxy-α-galactopyranoside and 1-deoxymannojirimycin (Bz+dMM-Colo205). Although the final lysis was not observed, the deposition of C4 and C3 was observed on both types of Colo205 cells after treatment with MBL and complements as a result of complement activation by MBL. MBL bound to Bz+dMM-Colo205 could also activate human peripheral blood leukocytes and induce superoxide production; however, MBL bound to NT-Colo205 could not. This may be explained by the lower affinity of MBL to Lewis A and Lewis B antigens than to high-mannose type oligosaccharides under physiological conditions, since MBL bound to NT-Colo205 was more easily released from the cell surface than that bound to Bz+dMM-Colo205 at 37°C. These findings suggest that the difference in the affinity of MBL to its ligands could influence the expression of some biological activities of MBL.

Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner.

Herein we report that the novel HIV-inactivating protein cyanovirin-N (CV-N) targets specific, N-linked high-mannose oligosaccharides found on the viral envelope of HIV-1. First, we released the oligosaccharides by PnGase-treatment of HIV-gp120 (containing high-mannose, hybrid-type and complex-type oligosaccharides) or HSV-1 gC (containing only complex-type). Then, in an affinity chromatographic system, we found that CV-N bound to the free oligosaccharides from gp120 but not from gC-1, suggesting that high-mannose oligosaccharides constitute a target structure for CV-N. This was supported by the affinity of CV-N for high-mannose glycans released from gp120 by endo-H as well as high-mannose glycans released from castanospermine-treated HSV-1 gC. Furthermore, free Man-8 or Man-9 oligosaccharides partially inhibited the binding of CV-N to gp120, although neither oligosaccharides smaller than Man-7 nor monosaccharides interfered with CV-N/gp120 interaction, thereby establishing the oligosaccharide-specific affinity of CV-N to high-mannose glycans. This affinity for high-mannose oligosaccharides may explain the broad antiviral activity of CV-N against human and primate immunodeficiency retroviruses as well as certain other viruses that carry these oligosaccharides.

Retention of Glucose on Oligosaccharide Chains Linked to the LH/hCG Receptor Prevents Cell Surface Expression

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [125I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.