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Our group is working at the interface of chemistry and biology. We are interested in developing and applying new chemical methods for tackling problems of biological and/or biomedical significance. Our current projects are focused on carbohydrate chemistry and glycobiology with relevance to HIV and cancer.
1. Chemo-enzymatic synthesis of glycopeptides and glycoproteins
Glycosylation is one of the most common posttranslational modifications in eukaryotes. The oligosaccharide components of glycoproteins affect protein's structure and a wide range of biological functions. Yet we are only beginning to understand the specific roles of oligosaccharides and the structure-function relationships of glycoproteins.
A major challenge comes from the micro-heterogeneity of this class of biomlecules. Glycoproteins are typically produced as a mixture of glycoforms that differ only in the pendant oligosaccharide chains, from which homogeneous glycforms are difficult to isolate. To overcome this problem, various chemical and chemoenzymatic synthetic methods have been developed in the past decade. But each method has its own limitations, and the construction of large, homogeneous glycopeptides and glycoproteins is still a challenging task. We seek to systematically explore the tranglycosylation potential of endo- -N-acetylglucosaminidases (ENGases), a special class of endoglycosidases, for glycopeptide and glycoprotein synthesis.
In contrast to typical glycosyltransferases that extend sugar chains by adding monosaccharides one by one, some ENGases has the unique ability to transfer a large intact N-glycan to a GlcNAc-containing peptide to form a new glycopeptide in a single step. But the intrinsic problems associated with ENGase-catalyzed synthesis is the low transglycosylation yield (5-20%) and the limitation to use only natural N-glycans as the donor substrates. We have recently moved one step forward to enhance the efficiency of the chemoenzymatic method by exploring synthetic oligosaccharide oxazolines corresponding to the core structure of N-glycans as the donor substrates.
We have demonstrated that the use of the transition state mimics as donor substrates of ENGases resulted in a highly efficient synthesis of large N-glycopeptides in a regio- and stereo-specific manner. The new chemoenzymatic method not only expended the substrate availability, but also led to a substantial enhancement of transglycosylation yield (from 10% to over 80%). We have also found that the enzymatic transglycosylation can tolerate certain modifications on the substrates, thus allowing the synthesis of specifically modified glycopeptides. We are now extending the chemoenzymatic method to the construction of homogeneous natural and modified glycoproteins.
With the availability of the synthetic homogeneous glycopeptides and glycoproteins, we are particularly interested in probing the effects of glycosylation on the conformations, protease stability, and immunogenicity of the peptide and protein domains. Among other targets, the new synthetic approach is currently applied for the synthesis of HIV- and cancer-associated glycopeptides for vaccine development.
2. Design and synthesis of carbohydrate-based HIV vaccines
We are exploring conserved HIV-1 oligosaccharides and glycopeptides as novel antigenic structures for immunogen design. Neutralizing antibodies are an important component of protection that provides sterilizing immunity against HIV-1 infection. However, design of immunogens capable of eliciting broadly reactive antibodies against HIV-1 has turned out to be a difficult task.
The rationales behind a carbohydrate-based HIV-1 vaccine are:
- HIV-1 is heavily glycosylated and some N-glycans are highly conserved;
- the carbohydrates are well exposed to the immune system;
- structural analysis have implicated the existence of unusual carbohydrate antigenic structures on the envelope glycoproteins; and
- the identification of a novel oligomannose cluster on gp120 as the epitope of the broadly neutralizing antibody 2G12 implicates the feasibility of targeting HIV-1 carbohydrates as a vaccine approach.
Using the 2G12 epitope as a template, we have designed and synthesized a series of carbohydrate epitope mimics and evaluated their antigenicity toward the neutralizing antibody 2G12. Theses studies provide important information for designing better immunogens in the hope of eliciting 2G12-like neutralizing antibodies. On the other hand, we are particularly interested in exploring conserved HIV-1 glycopeptides as the basis for vaccine design.
We believe that highly conserved HIV-1 glycopeptides, which combine the viral peptide and oligosaccharide portions as an integrated moiety, represent a new type of antigenic structure that is conceptually different from the peptide or carbohydrate antigen alone.
Focusing on the V3 domain glycopeptides that are the "principal neutralizing determinant" of HIV-1, we are currently investigating how the conserved carbohydrates affect the V3 domain's conformations and immunological properties, and whether the glycopeptides as an integrated immunogen can raise neutralizing antibodies that recognize both the HIV-1 carbohydrate and peptide antigenic structures.
3. Molecular targeting
Targeted drug delivery is crucial for the in vivo efficiency of therapeutic agents. We are interested in developing novel bi-functional molecules as "mediators" for precise, site-specific targeting in vivo. Glycobiology concepts including specific ligand-receptor recognition are employed in this pursuit.
Our current interest is to explore novel glycoconjugates for targeting HIV DNA vaccine to dendritic cells and for directing intrinsic human antibodies to HIV virus.
We are also interested in using the concept for enhancing cancer immunotherapy.
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