N glycan specific antibodies for advancing glycobiology research

Project Summary Carbohydrate chains known as glycans are fundamental to a wide spectrum of biological processes including anticoagulation cell growth and development cell cell communication immune recognition response and microbial pathogenesis Glycans also feature prominently in human disease For example glycans are commonly expressed at atypical levels on tumor cells and have been shown to regulate tumor proliferation invasion hematogenous metastasis and angiogenesis A better understanding of the role that glycans play in both health and disease could be achieved with a comprehensive set of affinity reagents that specifically recognize defined glycoforms Unfortunately the number of commercially available glycan binding proteins such as antibodies or lectins is greatly eclipsed by the number of biologically important glycan epitopes glycotopes and there are no commercially available antibodies specific for N glycan structures One approach with the potential to bridge this technology deficit is the isolation of antibodies with exquisite specificity for distinct glycans or glycoconjugates such as glycoproteins and glycolipids However obtaining antibodies that recognize specific glycoforms can be challenging for a number of technical reasons thereby limiting the pace at which new orthogonal antibody glycotope pairs are developed To address these challenges the objective of this proposal is to create a robust integrated pipeline for producing renewable well defined glycotopes and corresponding high affinity glycan recognizing antibodies grAbs in a rapid and routine manner Under Specific Aim specially engineered Escherichia coli cells along with a new bioenzymatic synthesis strategy will be used to produce structurally uniform free reducing end N glycan structures The resulting glycans will be used for direct selection and characterization of grAbs A scalable method for discovering synthetic antibody fragments from phage displayed combinatorial libraries will be adapted for parallel in vitro selections of antibodies against immobilized E coli derived glycotopes Under Specific Aim all isolated grAbs will be characterized using ELISA surface plasmon resonance and glycan microarrays to determine affinity and specificity for the targeted glycan structure In addition grAbs will be used to probe the temporal and spatial expression of authentic glycan signatures on the surfaces of cell lines or in the context of glycoproteins Given its modularity and scalability this grAb glycotope pipeline has the potential to yield customized affinity reagents on a glycome wide scale providing a high quality and easy to use toolkit for glycan analysis that will be broadly accessible to the laboratories of specialists and non specialists alike Project Narrative Unraveling the `sugar codeandapos that underlies the structure and biological function of glycans could be accelerated by a comprehensive set of renewable well defined affinity reagents such as antibodies that can recognize and differentiate specific glycan structures For example the structures of N glycans are often altered during malignant transformation which is thought to impact survival and progression of cancer cells To bridge this gap the objective of the proposed studies is to create a scalable pipeline for generating glycan specific antibodies for probing the temporal and spatial expression of N glycans in cells and tissues