High-specificity affinity reagents for N-glycosylation site mapping and glycomics

DESCRIPTION Glycans have several distinct properties that make them excellent targets for disease biomarkers Firstly the location of the glycans on the cell surface makes them the first point of contact of cellular interactions and thus crucial in the control of normal metabolic processes Cell surface molecules are also strategically exposed for surveillance by the immune system allowing for the potential of immune recognition of abnormal cells Secondly specific glycan structures that are not present or are in low amounts in normal states proliferate in disease states And lastly changes in glycosylation involve many proteins including those that are highly abundant Therefore a single change in a cellandapos s glycosylation machinery can affect many different glycoconjugates To effectively employ and discover glycan disease markers new glycan specific reagents are urgently needed Using computational methods to guide molecular evolution carbohydrate processing enzymes will be converted into high affinity receptor proteins that retain the native specificity of the enzyme but which no longer have enzyme activity Because such a protein has lectin like properties but is derived from an enzyme we are calling them andquot Lectenz r andquot Lectenz r have several potential advantages over lectins and antibodies as glycomics reagents including precise definition of specificity ease of preparation in a monovalent form and for human homologues minimal in vivo toxicity In this proposal the peptide N glycanase F PNGase F carbohydrate processing enzyme will be converted into a high specificity affinity reagent for peptides and proteins that contain asparagine linked carbohydrate chains The Lectenz r based on PNGase F may be employed directly to address the needs of glycomics proteomics analysis through sample enrichment thus facilitating glycosylation site mapping Glycosylation site mapping is currently extremely tedious to perform and yet is essential in fully characterizing and exploiting glycans as markers of specific disease states The principle advantages of an engineered Lectenz r are that the Lectenz r is specific to a defined carbohydrate sequence and in contrast to antibodies will recognize that sequence in a broad range of glycan contexts Further in contrast to plant lectins engineered Lectenz r are derived from enzymes that have exquisite substrate specificities and low toxicities Lastly as there is an abundance of carbohydrate processing enzymes known it is possible to employ the Lectenz r technology patent pending to assemble panels of affinity reagents tailor made for characterizing monitoring or detecting specific glycans PUBLIC HEALTH RELEVANCE We are converting carbohydrate processing enzymes into high affinity receptor proteins called Lectenz r that can be used to help discover detect and monitor glycan based disease markers The principle advantages of Lectenz r over other reagents such as antibodies or plant lectins is that they have known sequence specificity that is not context dependent they may be produced as monomeric proteins and for human homologues have minimal in vivo toxicity Here a Lectenz r will be derived from the PNGase F carbohydrate processing enzyme for use as a high specificity affinity reagent for peptides and proteins that contain asparagine linked carbohydrate chains which will find use in glycomics analyses aimed at disease marker discovery