Exploring glycan function. I. Intrinsic effects of N -glycans on beta-sheet protein folding. II. Development of glycoproteomic tools for monitoring glycan transformations

I. Intrinsic effects of glycosylation on beta-sheet folding . N-glycans are known to stabilize protein structures and influence protein folding in vivo by interacting with folding chaperones in the endoplasmic reticulum. Evidence also suggests that N-glycans can directly promote protein folding through an intrinsic chemical mechanism, although the biophysical details regarding their energetic and structural contributions remain undefined. Herein, the hypothesis that N-glycosylation can significantly affect the kinetics and thermodynamics of beta-sheet folding was tested in vitro by studying the folding energetics of the mono-N-glycosylated adhesion domain of the human immune cell receptor CD2 (hCD2ad). To unravel the biophysical effects of the N-linked oligosaccharide and its substructures, unglycosylated and several glycosylated hCD2ad variants were prepared and tested. The glycoforms ranged from full heterogeneous N-glycan structures to those which were truncated down to a single asparagine-linked N-acetylglucosamine (GlcNAc) via enzymatic remodeling. Variant folding studies revealed that the N-glycan significantly hastens folding and stabilizes the hCD2ad structure. These pro-folding attributes arise from the highly conserved core triose structure, Man3GlcNAc2. Within the trios, the proximal GlcNAc provides the kinetic impetus and contributes to thermodynamic stabilization, while the additional saccharides further enhance folding and thermodynamic stabilization. Such pro-folding attributes provided by N-glycans likely enhance protein secretion and function, and might explain the evolutionary conservation of the triose unit among virtually all eukaryotic N-glycan structures.;B. Glycan labeling, visualization, and glycoproteomics with alkynyl sugar reporters. Developing tools to investigate the cellular activity of glycans will help to delineate the molecular basis for their physiological and pathophysiological roles, including how aberrant glycosylation is involved in cancer. Metabolic oligosaccharide engineering, which inserts sugar-reporting groups into cellular glycoconjugates via promiscuous glycan biosynthetic machinery, represents a powerful method for imaging the localization, trafficking, and dynamics of glycans and isolating them for glycoproteomic analysis. The alkyne group was investigated as a reporting group for cellular glycans since it is a small, inert, bio-orthogonal handle that can be chemoselectively labeled using the Cu(I) catalyzed azide-alkyne [3+2] cycloaddition (CuAAC). Alkynyl sugar monomers, based on fucose (Fuc) and N-acetylmannosamine (ManNAc), a biosynthetic sialic acid precursor, were incorporated into fucosylated and sialylated glycans in cancer cells. These alkynyl-tagged glycans were labeled with CuAAC-competent, detectable probes allowing for cell surface and intracellular visualization of glycoconjugates, as well as observation of individual alkyne bearing glycoproteins. Click-activated fluorogenic probes, which become fluorescent only after CuAAC, were investigated as practical tools for efficient and selective labeling of alkynyl-tagged glycans. A glycoproteomic identification and glycan site mapping (GIDmap) protocol was developed, wherein alkynylated glycoproteins were selectively isolated via biotin probe labeling and then analyzed by enzymatic digestions and tandem liquid chromatography mass spectrometry. Glycoproteins and associated sites of N-glycosylation were identified from prostate cancer proteomes, hundreds of which represent a novel mapping of glycosylation sites. The method is saccharide specific, which will allow for investigations into why fucose and sialic acid are notably increased in many cancers.;II. Developing glycoproteomic tools for monitoring important glycan transformations. A. Exploring mechanism-based inhibition of sulfatases . Sulfatases are an interesting class of enzymes with emerging biological relevance in the fields of cancer, developmental cell signaling, and pathogenesis. Their involvement in cleaving sulfate esters in the heparan sulfate proteoglycans makes them particularly attractive as chemoenzymatic tools to manipulate and study the myriad of intricate sulfate-dependent binding events that occur at the cell surface. An interest in discovering and monitoring the activity of sulfatases led us to investigate mechanism-based inhibitors (MbIs) that could also function as useful enzyme labels. The MbIs were designed as simple aromatic sulfates, a commonly accepted substrate motif across the enzyme class, so that they might be generally useful for sulfatase labeling and capture. Cyclic sulfamates (CySAs) demonstrated inhibition profiles consistent with an active-site directed mode of action. These molecules represent a novel scaffold for labeling sulfatases and for probing their catalytic mechanism.