The key unsolved problems in the glycosciences are the temporal and spatial expression of glycans, as well as the identification of glycans, the proteins to which they are attached, and their biological partners (glycan-binding proteins or GBPs) that recognize them. To define glycan expression new techniques are needed that are applicable to flow cytometry, immunohistochemistry, and small numbers of cells and small amounts of glycoconjugates. Here we propose to develop a suite of anti-glycan reagents whose glycan-binding specificities are well defined. Such reagents and novel approaches to create them will provide novel information about the temporal and spatial expression of glycans in cells and tissues, and provide accessible reagents for `non-glyco' experts. We will exploit new technologies to generate `smart anti-glycan reagents' (SAGRs) whose anti-glycan specificities are defined. These will be created from both murine single-chain variable fragments (scfv) and lamprey-derived antibodies (variable lymphocyte receptors or VLRs).
(Aim 1) We will assemble a panel of 100 glycans with different glycan determinants, which include well-defined glycans generated by chemo/enzymatic methods, purified and available at milligram scale. These glycans will be derivatized to contain a primary amine for covalent coupling to solid surfaces for screening.
(Aim 2 a) We will screen scfv libraries for recognition of specific glycan determinants and characterize the scfv for their specificity of glycan recognition using glycan microarray technologies.
(Aim 2 b) We will immunize lampreys with cells expressing known and unknown glycan determinants, and select and screen for specificity as above.
(Aim 3) We will use this suite of novel SAGRs to explore glycan expression in many different primary cells, immortalized cell lines, and human tissues. These highly specific and targeted reagents will allow researchers to begin mapping the Human Glycome Atlas, which will compile information about glycan expression.
The repertoire of glycan determinants recognized by glycan-binding proteins is large but the temporal and spatial expression of such determinants is relatively unknown in human and animal cells. Here we propose to expand on two novel technologies to generate 100 `smart anti-glycan reagents' (SAGRs) using antibodies based on single-chain variable fragments (scfv) and VLRs derived from lampreys. SAGRs will be identified that recognize specific glycan determinants. SAGRs in kit form will help to define the human glycome atlas.
|Collins, Bernard C; Nakahara, Hiro; Acharya, Sharmistha et al. (2017) Crystal structure of an anti-idiotype variable lymphocyte receptor. Acta Crystallogr F Struct Biol Commun 73:682-687|
|Collins, Bernard C; Gunn, Robin J; McKitrick, Tanya R et al. (2017) Structural Insights into VLR Fine Specificity for Blood Group Carbohydrates. Structure 25:1667-1678.e4|