Glycomic analyses and glycan array formats for characterizing glycan-binding proteins

CARB 14

Richard Cummings, rdcummi@emory.edu1, Baoyun Xia2, Ziad S. Kawar3, Anne Leppänen4, Tongzhong Ju3, Richard Alvarez, richard-alvarez@ouhsc.edu5, and Goverdhan Sachdev2. (1) Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road – Suite 4001, Atlanta, GA 30322, (2) Department of Medicinal Chemistry, University of Oklahoma, College of Pharmacy, 1110N Stonewall Avenue, Oklahoma city, OK 73117, (3) Department of biochemistry and molecular biology, The Oklahoma Center for Medical Glycobiology, (4) Department of Bio and Environmental Sciences, Division of Biochemistry, University of Helsinki, (5) Biochemistry and Molecular Biology, BRC 411B, University of Oklahoma, 975 NE 10th Street, Oklahoma City, OK 73104
The new field of functional glycomics encompasses information about both glycan structure and recognition by glycan-binding proteins (GBPs), which is currently being explored through glycan array technology. To aid in analyzing the specificity of GBPs in a variety of formats, we have developed new semi-synthetic methods of generating glycans and glycopeptides. One of the most important new formats used for these compounds is high-throughput covalent glycan arrays printed on glass slides. These arrays are also useful for studying interactions of cells, protozoan, viruses, and bacteria to specific glycan determinants. However, while a tremendous variety of naturally-occurring free glycans can be generated directly from biological sources, glycan array construction from free glycans is limited by the complexity of efficiently generating derivatives with primary amines for conjugation. To solve this problem we developed a new method to derivatize glycans by reductive amination with diaminopyridine, which generates glycan conjugates that are fluorescent and contain a primary arylamine for further conjugation (GDAPs). A wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans, and chitin-derived glycans, are readily converted to GDAPs regardless of the reducing sugar moiety. These GDAP derivatives along with synthetic glycopeptides have been covalently conjugated in high yield to NHS-activated glass slides, maleimide-activated protein, carboxylated-microspheres, and to N-hydroxysuccinimide-biotin. An advantage of using GDAPs is that they can be easily visualized by fluorescent imaging techniques and directly quantified. All of the immobilized GDAPs and glycopeptides studied are well-recognized by appropriate GBPs. Importantly, we have also used immobilized GDAPs to purify novel carbohydrate-binding proteins. Thus, GDAP derivatives and synthetic glycopeptides provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for generating novel glycan arrays from naturally-occurring glycans and provide new approaches for exploring GBP functions in biology and pathogenesis.
 

Carbohydrate Recognition Mechanisms and Applications
2:00 PM-4:30 PM, Monday, 27 March 2006 Georgia World Congress Center -- B409, Oral

Division of Carbohydrate Chemistry

The 231st ACS National Meeting, Atlanta, GA, March 26-30, 2006