Single Molecule Glycosaminoglycan Sequencing using Lysosomal Enzymes - Glycosaminoglycans (GAG) are a family of large, linear, sulfated polysaccharides produced in mammals and other organisms. GAGs play diverse roles in tissue development/growth, inflammation, blood coagulation, viral infection, and amyloid plaque formation. As a result, GAGs have been used as biomarkers for many diseases. They are also the most widely used anticoagulant. Because of their biological activities, interest in structure- activity relationships of GAGs has always been high. However, due to their size, complexity, and heterogeneity, the analysis of GAG structures using conventional ensemble techniques is challenging. There is currently no method to sequence these important polysaccharides. We have been exploring single-molecule techniques for determining GAG structures for several years. Although work from us and other groups demonstrate that solid- state and protein nanopores can obtain some structural information on GAG polysaccharides, full sequencing of GAG by nanopore has not been realized because of a lack of translocation speed control. In this proposal, we want to develop a new single-molecule GAG sequencing method using fluorophore-labeled lysosomal GAG degradation enzymes. Our idea is inspired by fluorescence-based single molecule sequencing techniques for other biopolymers. These techniques utilize processive enzymes whose action on the polymer produces fluo- rescent signals that can be used to infer the sequence. We think lysosomal GAG degradation enzymes are perfect for this method. In particular, these enzymes processively degrade GAG from the non-reducing end (NRE) and each enzyme recognizes a specific feature of the NRE. As a result, the degradation of GAG proceeds through a set of enzymatic steps determined by the GAG sequence. This means the GAG sequence can be determined by the order in which enzymes bind the polymer. In this proposal, we want to develop such a se- quencing method by producing a set of lysosomal GAG degradation enzymes each with a unique fluorescent signature for identification. These enzymes will be used to degrade immobilized GAG chains while the unique fluorescence of each enzyme can be used to determine these enzymes’ binding order. This information can then be used to infer the GAG sequence. Because such a method requires no homogeneous samples, can sequence longer GAG polymers, and can provide high-resolution information, we think its realization will be a dramatic improvement over existing techniques. In particular, we want to complete the following two aims: 1) Design and produce fluorophore-labeled GAG degradation enzymes and use TIRFM to characterize the fluorescent signa- ture these enzymes produce when binding the correct immobilized substrates. 2) We will prepare a library of structurally defined GAG ligands and use the data obtained from aim 1 to determine their structures using these fluorophores labeled enzymes. Completing these aims will provide the crucial foundation for developing a gen- eral method for sequencing GAGs.
