The polysaccharides attached to proteoglycans (glycosaminoglycans – GAGs) feature anionic sulfate groups installed during GAG biosynthesis. The unique presentations of anionic sulfate groups are used as recognition motifs for cytokines and growth factors. The “sulfation code” therefore regulates numerous cellular functions such as differentiation, motility, and proliferation by sequestering growth factors and disrupting signaling pathways. In the extracellular environment, the sulfation code is modified by two human ECM 6-O-endosulfatases (hSulf1 and hSulf2). The removal of sulfate groups from the 6-O-position of glycans in the GAG heparin sulfate (HS), mobilizes growth factors and indirectly activates signaling pathways. A highly charged region in the protein, termed the hydrophilic domain (HD), is thought to be the main site for GAG recognition. The HD, which spans both subunits is unique to the ECM sulfatases. Interestingly, deletion of the HD impairs endosulfatase catalytic activity, but doesn’t affect the enzyme’s ability to desulfate arylsulfates, suggesting necessary regulatory elements within the HD that influence the conformation and some activity of the catalytic domain (CD). Our group seeks to develop molecules and methods that will aid in the imaging and characterization of the hSulfs. Our first step is the design of a synthetic chemical probe that enables visualization of enzyme activity through enzyme-mediated fluorescence turn-on. Other structural features of our probes interrogate the enzyme’s HD and the effect of specific sugar chemical moieties on enzyme-substrate affinity. We demonstrate work on the fabrication of these tools, and solutions to unique challenges that hinder studying a highly charged and complicated network of GAGs and their associated proteins. Future work will use the information gathered to guide structural studies of the enzyme’s recognition domain and the design of potential inhibitors.

Novel strategy for probing the activity of the human 6-O-endosulfatases

2019 Fall National Meeting

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