Mohammed N. Amin

Structural basis for diverse N-glycan recognition by HIV-1–neutralizing V1–V2–directed antibody PG16

HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

Synthetic glycopeptides reveal the glycan specificity of HIV-neutralizing antibodies.

A new class of glycan-reactive HIV-neutralizing antibodies, including PG9 and PG16, has been recently discovered that appear to recognize novel glycopeptide epitopes on HIV-1 gp120. However, further characterization and reconstitution of the precise neutralizing epitopes are complicated by the heterogeneity of glycosylation. We report here the design, synthesis, and antigenic evaluation of novel cyclic V1V2 glycopeptides carrying defined N-linked glycans at the conserved glycosylation sites (N160 and N156/N173) derived from gp120 of two HIV-1 isolates. Antibody binding studies confirmed the necessity of a Man5GlcNAc2 glycan at N160 for recognition by PG9 and PG16, and further revealed a critical role of a sialylated N-glycan at the secondary site (N156/N173) in the context of glycopeptides for antibody binding. In addition to defining the glycan specificities of PG9 and PG16, the identified synthetic glycopeptides provide a valuable template for HIV-1 vaccine design.

Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

Glycoproteins are an important class of biomolecules involved in a number of biological recognition processes. However, natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ in the structures of the pendent glycans, which are difficult to separate in pure glycoforms. As a result, synthetic homogeneous glycopeptides and glycoproteins have become indispensable probes for detailed structural and functional studies. A number of elegant chemical and biological strategies have been developed for synthetic construction of tailor-made, full-size glycoproteins to address specific biological problems. In this review, we highlight recent advances in chemical and chemoenzymatic synthesis of homogeneous glycoproteins. Selected examples are given to demonstrate the applications of tailor-made, glycan-defined glycoproteins for deciphering glycosylation functions.

Convergent Synthesis of Homogeneous Glc1Man9GlcNAc2-Protein and Derivatives as Ligands of Molecular Chaperones in Protein Quality Control

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.

A two-step enzymatic glycosylation of polypeptides with complex N-glycans

A chemoenyzmatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymatic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our experiments demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymatic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc-Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc-Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addition, the Glc-Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc-Asn linked glycopeptide. The chemoenzymatic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery.

The Galectin CvGal1 from the Eastern Oyster (Crassostrea virginica) Binds to Blood Group A Oligosaccharides on the Hemocyte Surface

Background: The carbohydrate specificity of the oyster galectin CvGal1 for endogenous and exogenous glycans was unresolved. Results: CvGal1 recognizes blood group A tetrasaccharides on oyster hemocytes, which are absent on the surface of the P. marinus parasite. Conclusion: Oyster hemocytes and P. marinus display structurally distinct ligands for CvGal1. Significance: Galectins may function as pattern recognition receptors by binding microbial glycans structurally different from endogenous ligands. The galectin CvGal1 from the eastern oyster (Crassostrea virginica), which possesses four tandemly arrayed carbohydrate recognition domains, was previously shown to display stronger binding to galactosamine and N-acetylgalactosamine relative to d-galactose. CvGal1 expressed by phagocytic cells is “hijacked” by the parasite Perkinsus marinus to enter the host, where it proliferates and causes systemic infection and death. In this study, a detailed glycan array analysis revealed that CvGal1 preferentially recognizes type 2 blood group A oligosaccharides. Homology modeling of the protein and its oligosaccharide ligands supported this preference over type 1 blood group A and B oligosaccharides. The CvGal ligand models were further validated by binding, inhibition, and competitive binding studies of CvGal1 and ABH-specific monoclonal antibodies with intact and deglycosylated glycoproteins, hemocyte extracts, and intact hemocytes and by surface plasmon resonance analysis. A parallel glycomic study carried out on oyster hemocytes (Kurz, S., Jin, C., Hykollari, A., Gregorich, D., Giomarelli, B., Vasta, G. R., Wilson, I. B. H., and Paschinger, K. (2013) J. Biol. Chem. 288,) determined the structures of oligosaccharides recognized by CvGal1. Proteomic analysis of the hemocyte glycoproteins identified β-integrin and dominin as CvGal1 “self”-ligands. Despite strong CvGal1 binding to P. marinus trophozoites, no binding of ABH blood group antibodies was observed. Thus, parasite glycans structurally distinct from the blood group A oligosaccharides on the hemocyte surface may function as potentially effective ligands for CvGal1. We hypothesize that carbohydrate-based mimicry resulting from the host/parasite co-evolution facilitates CvGal1-mediated cross-linking to β-integrin, located on the hemocyte surface, leading to cell activation, phagocytosis, and host infection.

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins.

Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptorsubstrate specificity toward α1,6-fucosyl-GlcNAc-Asn or α1,6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi- and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.

Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding.

The continued threat of worldwide influenza pandemics, together with the yearly emergence of antigenically drifted influenza A virus (IAV) strains, underscore the urgent need to elucidate not only the mechanisms of influenza virulence, but also those mechanisms that predispose influenza patients to increased susceptibility to subsequent infection with Streptococcus pneumoniae. Glycans displayed on the surface of epithelia that are exposed to the external environment play important roles in microbial recognition, adhesion, and invasion. It is well established that the IAV hemagglutinin and pneumococcal adhesins enable their attachment to the host epithelia. Reciprocally, the recognition of microbial glycans by host carbohydrate-binding proteins (lectins) can initiate innate immune responses, but their relevance in influenza or pneumococcal infections is poorly understood. Galectins are evolutionarily conserved lectins characterized by affinity for β-galactosides and a unique sequence motif, with critical regulatory roles in development and immune homeostasis. In this study, we examined the possibility that galectins expressed in the airway epithelial cells might play a significant role in viral or pneumococcal adhesion to airway epithelial cells. Our results in a mouse model for influenza and pneumococcal infection revealed that the murine lung expresses a diverse galectin repertoire, from which selected galectins, including galectin 1 (Gal1) and galectin 3 (Gal3), are released to the bronchoalveolar space. Further, the results showed that influenza and subsequent S. pneumoniae infections significantly alter the glycosylation patterns of the airway epithelial surface and modulate galectin expression. In vitro studies on the human airway epithelial cell line A549 were consistent with the observations made in the mouse model, and further revealed that both Gal1 and Gal3 bind strongly to IAV and S. pneumoniae, and that exposure of the cells to viral neuraminidase or influenza infection increased galectin-mediated S. pneumoniae adhesion to the cell surface. Our results suggest that upon influenza infection, pneumococcal adhesion to the airway epithelial surface is enhanced by an interplay among the host galectins and viral and pneumococcal neuraminidases. The observed enhancement of pneumococcal adhesion may be a contributing factor to the observed hypersusceptibility to pneumonia of influenza patients.

Chemoenzymatic Synthesis and Receptor Binding of Mannose-6-Phosphate (M6P)-Containing Glycoprotein Ligands Reveal Unusual Structural Requirements for M6P Receptor Recognition

Mannose-6-phosphate (M6P)-terminated oligosaccharides are important signals for M6P-receptor-mediated targeting of newly synthesized hydrolases from Golgi to lysosomes, but the precise structural requirement for the M6P ligand-receptor recognition has not been fully understood due to the difficulties in obtaining homogeneous M6P-containing glycoproteins. We describe here a chemoenzymatic synthesis of homogeneous phosphoglycoproteins carrying natural M6P-containing N-glycans. The method includes the chemical synthesis of glycan oxazolines with varied number and location of the M6P moieties and their transfer to the GlcNAc-protein by an endoglycosynthase to provide homogeneous M6P-containing glycoproteins. Simultaneous attachment of two M6P-oligosaccahrides to a cyclic polypeptide was also accomplished to yield bivalent M6P-glycopeptides. Surface plasmon resonance binding studies reveal that a single M6P moiety located at the low α-1,3-branch of the oligomannose context is sufficient for a high-affinity binding to receptor CI-MPR, while the presence of a M6P moiety at the α-1,6-branch is dispensable. In addition, a binding study with the bivalent cyclic and linear polypeptides reveals that a close proximity of two M6P-oligosaccharide ligands is critical to achieve high affinity for the CI-MPR receptor. Taken together, the present study indicates that the location and valency of the M6P moieties and the right oligosaccharide context are all critical for high-affinity binding with the major M6P receptor. The chemoenzymatic method described here provides a new avenue for glycosylation remodeling of recombinant enzymes to enhance the uptake and delivery of enzymes to lysosomes in enzyme replacement therapy for the treatment of lysosomal storage diseases.

Galectin CvGal2 from the Eastern Oyster (Crassostrea virginica) Displays Unique Specificity for ABH Blood Group Oligosaccharides and Differentially Recognizes Sympatric Perkinsus Species.

Galectins are highly conserved lectins that are key to multiple biological functions, including pathogen recognition and regulation of immune responses. We previously reported that CvGal1, a galectin expressed in phagocytic cells (hemocytes) of the eastern oyster (Crassostrea virginica), is hijacked by the parasite Perkinsus marinus to enter the host, where it causes systemic infection and death. Screening of an oyster hemocyte cDNA library revealed a novel galectin, which we designated CvGal2, with four tandemly arrayed carbohydrate recognition domains (CRDs). Phylogentic analysis of the CvGal2 CRDs suggests close relationships with homologous CRDs from CvGal1. Glycan array analysis, however, revealed that, unlike CvGal1 which preferentially binds to the blood group A tetrasaccharide, CvGal2 recognizes both blood group A and B tetrasaccharides and related structures, suggesting that CvGal2 has broader binding specificity. Furthermore, SPR analysis demonstrated significant differences in the binding kinetics of CvGal1 and CvGal2, and structural modeling revealed substantial differences in their interactions with the oligosaccharide ligands. CvGal2 is homogeneously distributed in the hemocyte cytoplasm, is released to the extracellular space, and binds to the hemocyte surface. CvGal2 binds to P. marinus trophozoites in a dose-dependent and β-galactoside-specific manner. Strikingly, negligible binding of CvGal2 was observed for Perkinsus chesapeaki, a sympatric parasite species mostly prevalent in the clams Mya arenaria and Macoma balthica. The differential recognition of Perkinsus species by the oyster galectins is consistent with their relative prevalence in oyster and clam species and supports their role in facilitating parasite entry and infectivity in a host-preferential manner.