Generation of antibodies to monitor Notch O-fucosylation in vivo

Orlando, Ron

Abstract

Notch1 is a cell surface protein that has diverse roles in human biology at all stages of life. Notch1 is modified by a number of sugar structures, including a number that are termed O-fucose. Alteration of the pattern of O- fucose structures found on Notch1 has significant effects on Notch1 function, which in turn has been linked to both developmental and adult pathologies, including cancer. To date the study of O-fucose structures on Notch1 has been severely hindered by a lack of research reagents that can specifically detect and monitor this structure. Currently, mass spectrometry is the method of choice for the analysis of O-fucose glycans on Notch1. Although an extremely powerful technique, it requires specialized instrumentation and expertise not available in most laboratories. Development of sensitive and specific antibodies for O-fucose glycans on Notch1 will provide many investigators with the tools to analyze O-fucose glycans in vivo, including clinical specimens. We will utilize a newly-developed methodology that enables reliable production of antibodies that can recognize distinct protein epitopes in the context of specific glycan modifications to produce antibodies to O- fucose on Notch1. If successful these O-fucose-Notch1-specific antibodies will facilitate research on Notch1 in many laboratories and possibly could lead to new diagnostic and therapeutic tools.

Public Health Relevance

Modification of the human Notch1 protein by a sugar structure termed O-fucose is now under scrutiny for its role in cell biology and human diseases such as cancer. There are currently no protein-specific reagents to study O-fucose. We propose to develop site-specific O-fucose antibodies to the human Notch1 protein thereby providing valuable tools for the study of O-fucosylation and Notch1 biology and their roles in human disease.