Carbohydrate-protein interactions play a critical role in a wide range of biological
processes such as inflammation and metastasis, and carbohydrate-binding proteins
such as lectins and antibodies can be used as diagnostic and therapeutic agents.
Traditional methods for studying carbohydrate recognition are slow and/or require
large amounts of material. As a result, it has been
difficult to determine if a particular protein is a carbohydrate-binding protein,
to identify carbohydrate ligands to modulate biological processes, and to develop
carbohydrate-binding antibodies as research and diagnostic tools. Over the last
few years, our group and others have been developing carbohydrate microarray technology
for the high-throughput analysis of carbohydrate-macromolecule interactions. Carbohydrate
microarrays, or glycan arrays, are analogous to DNA and protein arrays but contain
many different carbohydrate structures spotted on a microscope slide in a spatially-defined
arrangement. The miniaturized format permits analysis of many potential interactions
while using only tiny amounts of precious materials. Since most carbohydrate-protein
interactions require multivalent binding to achieve a high functional affinity,
presentation on the array surface is a key design feature for carbohydrate microarrays.
Other groups print monovalent glycans and then utilize the array surface as a multivalent
scaffold. To construct our arrays, we attach carbohydrates to a carrier protein
such as bovine serum albumin (BSA) and then print these neoglycoconjugates, along
with natural glycoproteins, on epoxide-coated glass microscope slides using a robotic
microarrayer. With this approach, the carrier protein serves both as a linker for
immobilization and as a multivalent scaffold. The advantage of this approach is
that the neoglycoproteins can be used as multivalent inhibitors, reagents, and immunogens
without having to identify an alternative multivalent scaffold that mimics the array
surface. In addition, this approach provides unique strategies for varying carbohydrate
presentation. As of February 2009, our array has 190 array components.
One of the major challenges for development of a glycan array is obtaining a diverse
set of carbohydrates for the array. Our group relies heavily on synthetic organic
chemistry to acquire homogeneous, structurally-defined oligosaccharides for the
array. In support of this, we also develop better synthetic methods for obtaining
many oligosaccharides.
Application of Carbohydrate Microarray Technology to Cancer Research
Cancer cells undergo dramatic changes in carbohydrate expression during the onset
and progression of disease. Many of these alterations are found on the cell surface
or on secreted glycoproteins making them appealing molecular targets for the development
of diagnostics and therapeutic agents. As a result, there are major efforts to develop
carbohydrate binding monoclonal antibodies and/or lectins as research tools and
diagnostic reagents. In addition, many cancer vaccines currently in clinical trials
generate immune responses to glycans, glycoproteins, and or glycolipids. We are
applying carbohydrate microarray technology in several areas:
- Evaluation of antibody and lectin specificity
We have used the array to evaluate the specificity of over 60 different antibodies
and lectins. These reagents have been used for many years to detect expression of
carbohydrate antigens on cells, glycoproteins, and biopsy samples. We have found
that many of the reagents thought to be specific for a single carbohydrate antigen
can cross-react with other carbohydrate structures, suggesting that much of the
information in the literature on carbohydrate expression is inaccurate.
- Profiling serum antibodies
We are using the array to profile the repertoire of carbohydrate-binding antibodies
in serum. Serum antibody profiles can be used to identify new biomarkers, discover
new carbohydrate antigens, and evaluate responses to cancer vaccines.
Collaborations and Carbohydrate Microarray Screening
We are frequently asked to screen lectins, antibodies, and other entities on our
array. Although we are not a screening core facility, we are happy to help out in
many cases. Please contact Jeff Gildersleeve for more details.