Antitumor activity of a lectibody targeting cancer-associated high-mannose glycans.

Aberrant protein glycosylation is a hallmark of cancer, but few drugs targeting cancer glycobiomarkers are currently available. Here, we showed that a lectibody consisting of the high-mannose glycan-binding lectin Avaren and human immunoglobulin G1 (IgG1) Fc (AvFc) selectively recognizes a range of cell lines derived from lung, breast, colon, and blood cancers at nanomolar concentrations. Binding of AvFc to the non-small cell lung cancer (NSCLC) cell lines A549 and H460 was characterized in detail. Co-immunoprecipitation proteomics analysis revealed that epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) are among the lectibody's common targets in these cells. AvFc blocked the activation of EGFR and IGF1R by their respective ligands in A549 cells and inhibited the migration of A549 and H460 cells upon stimulation with EGF and IGF1. Furthermore, AvFc induced potent Fc-mediated cytotoxic effects and significantly restricted A549 and H460 tumor growth in severe combined immunodeficiency (SCID) mice. Immunohistochemistry analysis of primary lung tissues from NSCLC patients demonstrated that AvFc preferentially binds to tumors over adjacent non-tumor tissues. Our findings provide evidence that increased abundance of high-mannose glycans in the glycocalyx of cancer cells can be a druggable target, and AvFc may provide a new tool to probe and target this tumor-associated glycobiomarker.

Cyanovirin-N Binds Viral Envelope Proteins at the Low-Affinity Carbohydrate Binding Site without Direct Virus Neutralization Ability.

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV-N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV-N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV-N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV-N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.

Systematic Interaction Analysis of Anti-Human Immunodeficiency Virus Type-1 Neutralizing Antibodies with High Mannose Glycans Using Fragment Molecular Orbital and Molecular Dynamics Methods.

A series of broadly neutralizing antibodies called PGT have been shown to be bound directly to human immunodeficiency virus type-1 via high mannose glycans on glycoprotein gp120. Despite the sequence similarities of amino acids of the antibodies, their affinities to the glycan differ. Glycan-antibody interactions among these antibodies are systematically compared with quantum chemical fragment molecular orbital calculations and molecular dynamics simulations. The differences among structural stability of the glycan in the active site of the complexes and total interaction energies as well as binding free energies between the glycan and antibodies agree well with the experimentally shown affinities of the glycan to the antibodies. The terminal saccharide, Man D3, is structurally stable and responsible for the glycan-antibody binding through electrostatic and dispersion interactions. The structural stability of nonterminal saccharides such as Man 4 or Man C plays substantial roles in the interaction via direct hydrogen bonds. © 2019 Wiley Periodicals, Inc.

Altered glycosylation associated with dedifferentiation of hepatocellular carcinoma: a lectin microarray-based study.

BACKGROUND: Altered glycosylation associated with hepatocellular carcinoma (HCC) is well documented. However, few reports have investigated the association between dedifferentiation and glycosylation. Therefore, the aim of this study was to analyze glycosylation associated with dedifferentiation of HCC within the same nodule and to investigate glycosyltransferase related to the glycosylation. METHODS: We analyzed resected HCC specimens (n = 50) using lectin microarray to comprehensively and sensitively analyze glycan profiles, and identify changes to glycosylation between well- and moderately-differentiated components within the same nodule. Moreover, we performed immunohistochemical staining of mannosyl(α-1,3-)-glycoprotein ß-1,2-N-acetylglucosaminyltransferase (MGAT1), which is an essential glycosyltransferase that converts high-mannose glycans to complex- or hybrid-type N-glycans. RESULTS: Four lectins from Narcissus pseudonarcissus agglutinin (NPA), Concanavalin A, Galanthus nivalis agglutinin, and Calystegia sepium agglutinin were significantly elevated in moderately-differentiated components of HCC compared with well-differentiated components, and all lectins showed binding specificity to high-mannose glycans. Therefore, these structures were represented to a greater extent in moderately-differentiated components than in well-differentiated ones. Immunohistochemical staining revealed significantly increased NPA expression and decreased MGAT1 expression in moderately-differentiated components. Low MGAT1 expression in moderately-differentiated components of tumors was associated with intrahepatic metastasis and had tendency for poor prognosis. CONCLUSION: Dedifferentiation of well-differentiated HCC is associated with an increase in high-mannose glycans. MGAT1 may play a role in the dedifferentiation of HCC.

Structural basis for receptor recognition and pore formation of a zebrafish aerolysin-like protein.

Various aerolysin-like pore-forming proteins have been identified from bacteria to vertebrates. However, the mechanism of receptor recognition and/or pore formation of the eukaryotic members remains unknown. Here, we present the first crystal and electron microscopy structures of a vertebrate aerolysin-like protein from Danio rerio, termed Dln1, before and after pore formation. Each subunit of Dln1 dimer comprises a ß-prism lectin module followed by an aerolysin module. Specific binding of the lectin module toward high-mannose glycans triggers drastic conformational changes of the aerolysin module in a pH-dependent manner, ultimately resulting in the formation of a membrane-bound octameric pore. Structural analyses combined with computational simulations and biochemical assays suggest a pore-forming process with an activation mechanism distinct from the previously characterized bacterial members. Moreover, Dln1 and its homologs are ubiquitously distributed in bony fishes and lamprey, suggesting a novel fish-specific defense molecule.

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin.

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.

Lectibodies as antivirals.

Growing concerns regarding the emergence of highly transmissible viral diseases highlight the urgent need to expand the repertoire of antiviral therapeutics. For this reason, new strategies for neutralizing and inhibiting these viruses are necessary. A promising approach involves targeting the glycans present on the surfaces of enveloped viruses. Lectins, known for their ability to recognize specific carbohydrate molecules, offer the potential for glycan-targeted antiviral strategies. Indeed, numerous studies have reported the antiviral effects of various lectins of both endogenous and exogenous origins. However, many lectins in their natural forms, are not suitable for use as antiviral therapeutics due to toxicity, other unfavorable pharmacological effects, and/or unreliable manufacturing sources. Therefore, improvements are crucial for employing lectins as effective antiviral therapeutics. A novel approach to enhance lectins' suitability as pharmaceuticals could be the generation of recombinant lectin-Fc fusion proteins, termed "lectibodies." In this review, we discuss the scientific rationale behind lectin-based antiviral strategies and explore how lectibodies could facilitate the development of new antiviral therapeutics. We will also share our perspective on the potential of these molecules to transcend their potential use as antiviral agents.

Loss of α-1,2-mannosidase MAN1C1 promotes tumorigenesis of intrahepatic cholangiocarcinoma through enhancing CD133-FIP200 interaction.

CD133 is widely used as a marker to isolate tumor-initiating cells in many types of cancers. The structure of N-glycan on CD133 is altered during the differentiation of tumor-initiating cells. However, the relationship between CD133 N-glycosylation and stem cell characteristics remains elusive. Here, we found that the level of α-1,2-mannosylated CD133 was associated with the level of stemness genes in intrahepatic cholangiocarcinoma (iCCA) tissues. α-1,2-mannosylated CD133+ cells possessed the characteristics of tumor-initiating cells. The loss of the Golgi α-mannosidase I coding gene MAN1C1 resulted in the formation of α-1,2-mannosylated CD133 in iCCA-initiating cells. Mechanistically, α-1,2-mannosylation promoted the cytoplasmic distribution of CD133 and enhanced the interaction between CD133 and the autophagy gene FIP200, subsequently promoting the tumorigenesis of α-1,2-mannosylated CD133+ cells. Analysis of iCCA samples showed that the level of cytoplasmic CD133 was associated with poor iCCA prognosis. Collectively, α-1,2-mannosylated CD133 is a functional marker of iCCA-initiating cells.

Legume Lectins with Different Specificities as Potential Glycan Probes for Pathogenic Enveloped Viruses.

Pathogenic enveloped viruses are covered with a glycan shield that provides a dual function: the glycan structures contribute to virus protection as well as host cell recognition. The three classical types of N-glycans, in particular complex glycans, high-mannose glycans, and hybrid glycans, together with some O-glycans, participate in the glycan shield of the Ebola virus, influenza virus, human cytomegalovirus, herpes virus, human immunodeficiency virus, Lassa virus, and MERS-CoV, SARS-CoV, and SARS-CoV-2, which are responsible for respiratory syndromes. The glycans are linked to glycoproteins that occur as metastable prefusion glycoproteins on the surface of infectious virions such as gp120 of HIV, hemagglutinin of influenza, or spike proteins of beta-coronaviruses. Plant lectins with different carbohydrate-binding specificities and, especially, mannose-specific lectins from the Vicieae tribe, such as pea lectin and lentil lectin, can be used as glycan probes for targeting the glycan shield because of their specific interaction with the α1,6-fucosylated core Man3GlcNAc2, which predominantly occurs in complex and hybrid glycans. Other plant lectins with Neu5Ac specificity or GalNAc/T/Tn specificity can also serve as potential glycan probes for the often sialylated complex glycans and truncated O-glycans, respectively, which are abundantly distributed in the glycan shield of enveloped viruses. The biomedical and therapeutical potential of plant lectins as antiviral drugs is discussed.

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model.

BACKGROUND & AIMS: Infection with hepatitis C virus (HCV) remains a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, there is no approved therapeutic targeting this potentially druggable biomarker. METHODS: The anti-HCV activity of a fusion protein consisting of Avaren lectin and the fragment crystallizable (Fc) region of a human immunoglobulin G1 antibody, Avaren-Fc (AvFc) was evaluated through the use of in vitro neutralization assays as well as an in vivo challenge in a chimeric human liver (PXB) mouse model. Drug toxicity was assessed by histopathology, serum alanine aminotransferase, and mouse body weights. RESULTS: AvFc was capable of neutralizing cell culture-derived HCV in a genotype-independent manner, with 50% inhibitory concentration values in the low nanomolar range. Systemic administration of AvFc in a histidine-based buffer was well tolerated; after 11 doses every other day at 25 mg/kg there were no significant changes in body or liver weights or in blood human albumin or serum alanine aminotransferase activity. Gross necropsy and liver pathology confirmed the lack of toxicity. This regimen successfully prevented genotype 1a HCV infection in all animals, although an AvFc mutant lacking HMG binding activity failed. CONCLUSIONS: These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection, and AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

Effects of microvirin monomers and oligomers on hepatitis C virus.

Microvirin (MVN) is a carbohydrate-binding protein which shows high specificity for high-mannose type N-glycan structures. In the present study, we tried to identify whether MVN could bind to high-mannose containing hepatitis C virus (HCV) envelope glycoproteins, which are heavily decorated high-mannose glycans. In addition, recombinantly expressed MVN oligomers in di-, tri- and tetrameric form were evaluated for their viral inhibition. MVN oligomers bound more efficiently to HCV virions, and displayed in comparison with the MVN monomer a higher neutralization potency against HCV infection. The antiviral effect was furthermore affected by the peptide linker sequence connecting the MVN monomers. The results indicate that MVN oligomers such as trimers and tetramers may be used as future neutralization agents against HCV infections.

Influence of aglycone structures on N-glycan processing reactions in the endoplasmic reticulum.

Glycoprotein N-linked oligosaccharides in the endoplasmic reticulum function as tags to regulate glycoprotein folding, sorting, secretion and degradation. Since the N-glycan structure of a glycoprotein should reflect the folding state, N-glycan processing may be affected by the aglycone state. In this study, we examined the influence of aglycone structures on N-glycan processing using synthetic substrates. We prepared (Glc1)Man9GlcNAc2 linked to hydrophobic BODIPY-dye with a systematic series of different linker lengths. With these compounds, glucose transfer, glucose trimming and mannose trimming reactions of an endoplasmic reticulum fraction were examined. The results showed that substrates with shorter linkers between the N-glycan and hydrophobic patch had higher activities for both the glucose transfer and the mannose trimming reactions. In contrast, the glucose trimming reaction showed lower activity when substrates had shorter linkers. Thus, the reactivity for N-linked oligosaccharide processing of glycoproteins in the endoplasmic reticulum might be tunable by the aglycone structure, e.g., protein portion of glycoproteins.

Two carbohydrate recognizing domains from Cycas revoluta leaf lectin show the distinct sugar-binding specificity-A unique mannooligosaccharide recognition by N-terminal domain.

Cycas revoluta leaf lectin (CRLL) of mannose-recognizing jacalin-related lectin (mJRL) has two tandem repeated carbohydrate recognition domains, and shows the characteristic sugar-binding specificity toward high mannose-glycans, compared with other mJRLs. We expressed the N-terminal domain and C-terminal domain (CRLL-N and CRLL-C) separately, to determine the fine sugar-binding specificity of each domain, using frontal affinity chromatography, glycan array and equilibrium dialysis. The specificity of CRLL toward high mannose was basically derived from CRLL-N, whereas CRLL-C had affinity for α1-6 extended mono-antennary complex-type glycans. Notably, the affinity of CRLL-N was most potent to one of three Man 8 glycans and Man 9 glycan, whereas the affinity of CRLL-C decreased with the increase in the number of extended α1-2 linked mannose residue. The recognition of the Man 8 glycans by CRLL-N has not been found for other mannose recognizing lectins. Glycan array reflected these specificities of the two domains. Furthermore, it was revealed by equilibrium dialysis method that the each domain had two sugar-binding sites, similar with Banlec, banana mannose-binding Jacalin-related lectin.

Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars.

Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn(2+) in the basal medium to 32% with 16 µM Mn(2+) supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn(2+) concentration. By varying Mn(2+) and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn(2+) concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn(2+) on high mannose glycan content depends on glucose availability. At each Mn(2+) supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non-limiting concentration.