Site-Specific Multi-Functionalization of the Carrier Protein CRM197 by Disulfide Rebridging for Conjugate Vaccine Development

Conjugation of an antigen to a carrier protein is widely used for vaccine development. To develop the next generation of conjugate vaccines, we describe here a method for the controlled multi-functionalization of the widely employed carrier protein CRM197 with a carbohydrate-based antigen and an immune potentiator. The approach is based on the selective reduction of one of the disulfides of CRM197 followed by disulfide rebridging employing an appropriately functionalized dibromopyridazinedione. Efficient protein modification required that the reduction and functionalization with a dibromopyridazinedione was performed as a one-step procedure with control over the reaction temperature. Furthermore, ligations were most successful when dibromopyridazinediones were employed having a functional entity such as a TLR7/8 agonist and a cyclooctyne for further modification. Site-specific conjugation avoids modification of T-epitopes of the carrier protein and covalent attachment of an immune potentiator will ensure that cytokines are produced where the vaccine interacts with relevant immune cells resulting in efficient immune potentiation

Conjugation of a Toll-Like Receptor Agonist to Glycans of an HIV Native-Like Envelope Trimer Preserves Neutralization Epitopes

Small molecule adjuvants are attractive for enhancing broad protection and durability of immune responses elicited by subunit vaccines. Covalent attachment of an adjuvant to an immunogen is particularly attractive because it simultaneously delivers both entities to antigen presenting cells resulting in more efficient immune activation. There is, however, a lack of methods to conjugate small molecule immune potentiators to viral glycoprotein immunogens without compromising epitope integrity. We describe herein a one-step enzymatic conjugation approach for the covalent attachment of small molecule adjuvants to N-linked glycans of viral glycoproteins. It involves the attachment of an immune potentiator to CMP-Neu5AcN 3 by Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition followed by sialyltransferase-mediated transfer to N-glycans of a viral glycoprotein. The method was employed to modify a native-like HIV envelope trimer with a Toll-like receptor 7/8 agonist. The modification did not compromise Env-trimer recognition by several broadly neutralization antibodies. Electron microscopy confirmed structural integrity of the modified immunogen

Selective Exoenzymatic Labeling of Lipooligosaccharides of Neisseria gonorrhoeae with α2,6-Sialoside Analogues

The interactions between bacteria and their host often rely on recognition processes that involve host or bacterial glycans. Glycoengineering techniques make it possible to modify and study the glycans on the host's eukaryotic cells, but only a few are available for the study of bacterial glycans. Here, we have adapted selective exoenzymatic labeling (SEEL), a chemical reporter strategy, to label the lipooligosaccharides of the bacterial pathogen Neisseria gonorrhoeae, using the recombinant glycosyltransferase ST6Gal1, and three synthetic CMP-sialic acid derivatives. We show that SEEL treatment does not affect cell viability and can introduce an α2,6-linked sialic acid with a reporter group on the lipooligosaccharides by Western blot, flow cytometry and fluorescent microscopy. This new bacterial glycoengineering technique allows for the precise modification, here with α2,6-sialoside derivatives, and direct detection of specific surface glycans on live bacteria, which will aid in further unravelling the precise biological functions of bacterial glycans

Detection of Bacterial α-l-Fucosidases with an Ortho-Quinone Methide-Based Probe and Mapping of the Probe-Protein Adducts.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria

Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder

Sialylglycopeptide (SGP) is a complex bi-antennary N-glycan bearing a short peptide fragment that can be isolated from the yolk of hen eggs. This natural product has gained popularity as a starting material for the semi-synthesis of N-glycans. We have found that current isolation methods provide a glycopeptide contaminated with several related structures, one being a glycopeptide having a hexose directly attached to peptide backbone, most like through the hydroxyl containing side chain of the threonine moiety. Furthermore, current methods employ fresh egg yolks that need to be lyophilized and involve several tedious purification steps. Herein, we report a convenient method for the isolation of gram quantities of homogeneous SGP from commercially available egg yolk powder using solid/liquid extraction and HILIC-HPLC purification

Improved isolation and characterization procedure of sialylglycopeptide from egg yolk powder

Sialylglycopeptide (SGP) is a complex bi-antennary N-glycan bearing a short peptide fragment that can be isolated from the yolk of hen eggs. This natural product has gained popularity as a starting material for the semi-synthesis of N-glycans. We have found that current isolation methods provide a glycopeptide contaminated with several related structures, one being a glycopeptide having a hexose directly attached to peptide backbone, most like through the hydroxyl containing side chain of the threonine moiety. Furthermore, current methods employ fresh egg yolks that need to be lyophilized and involve several tedious purification steps. Herein, we report a convenient method for the isolation of gram quantities of homogeneous SGP from commercially available egg yolk powder using solid/liquid extraction and HILIC-HPLC purification

Conjugation of a Toll-Like Receptor Agonist to Glycans of an HIV Native-Like Envelope Trimer Preserves Neutralization Epitopes

Small molecule adjuvants are attractive for enhancing broad protection and durability of immune responses elicited by subunit vaccines. Covalent attachment of an adjuvant to an immunogen is particularly attractive because it simultaneously delivers both entities to antigen presenting cells resulting in more efficient immune activation. There is, however, a lack of methods to conjugate small molecule immune potentiators to viral glycoprotein immunogens without compromising epitope integrity. We describe herein a one-step enzymatic conjugation approach for the covalent attachment of small molecule adjuvants to N-linked glycans of viral glycoproteins. It involves the attachment of an immune potentiator to CMP-Neu5AcN3 by Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition followed by sialyltransferase-mediated transfer to N-glycans of a viral glycoprotein. The method was employed to modify a native-like HIV envelope trimer with a Toll-like receptor 7/8 agonist. The modification did not compromise Env-trimer recognition by several broadly neutralization antibodies. Electron microscopy confirmed structural integrity of the modified immunogen

Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch-selective manner to give asymmetrical structures commonly found in nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in ten or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart, allowing each arm to be elaborated into a unique appendage

Streamlining the chemoenzymatic synthesis of complex N-glycans by a stop and go strategy

Contemporary chemoenzymatic approaches can provide highly complex multi-antennary N-linked glycans. These procedures are, however, very demanding and typically involve as many as 100 chemical steps to prepare advanced intermediates that can be diversified by glycosyltransferases in a branch-selective manner to give asymmetrical structures commonly found in nature. Only highly specialized laboratories can perform such syntheses, which greatly hampers progress in glycoscience. Here we describe a biomimetic approach in which a readily available bi-antennary glycopeptide can be converted in ten or fewer chemical and enzymatic steps into multi-antennary N-glycans that at each arm can be uniquely extended by glycosyltransferases to give access to highly complex asymmetrically branched N-glycans. A key feature of our approach is the installation of additional branching points using recombinant MGAT4 and MGAT5 in combination with unnatural sugar donors. At an appropriate point in the enzymatic synthesis, the unnatural monosaccharides can be converted into their natural counterpart, allowing each arm to be elaborated into a unique appendage

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown. Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems