Heparan sulfate (HS), a class of polysaccharides that includes the well-known drug heparin, plays essential roles in cell growth and development. The many structural variants of HS observed in mammals have been hypothesized to differentially modulate growth factor-mediated signaling and regulate hemostasis during health and disease. Our project will explore how the HS structural variation is generated during biosynthesis/catabolism as well as map routes to prepare more defined molecules having greater selectivity and more potent desired bioactivities. A favored hypothesis is that the pattern of HS domains (comprised of both N-sulfo (NS) and N-acetyl (NA) domains) encodes information that modulates HS interaction with proteins like growth factors, cytokines, and clotting factors. Currently, it is very difficult to understand how HS biosynthetic modification an domain placement is controlled and which HS domain structures possess the highest activities in different biological systems. We will apply our newly developed synthetic methodology to answer key questions in the field. A small library of HS polymers having size-defined and placement-defined NS and NA domains will be generated. These HS polymers will then be modified by biosynthetic enzymes, including O-sulfotransferases, and C5-epimerase and/or catabolic enzymes including endo-6- Oendosulfatases and heparanase. A focused combinatorial approach will be used to produce defined HS polymers to test two opposing models of growth factor signaling, a key event in cell proliferation, embryonic development, and cancer. In this project our specific aims are:
Aim 1 : To chemoenzymatically synthesize a small library of HS polymers having defined NS and NA domains.
Aim 2. To analyze the modification patterns generated through the action of various enzymes on the HS polysaccharide library.
Aim 3 : To characterize the structure/function relationship of HS activity as the co-receptor fibroblast growth factor receptor signaling.
Heparan sulfate (HS), a heterogeneous class of polysaccharides that includes the well-known drug heparin, plays essential roles in the human body. The many sulfation isomers of HS observed in mammals have been hypothesized to differentially modulate growth factor- and cytokine-mediated signaling and regulate hemostasis/coagulation during health and disease. The HS structural diversity is generated by the action of at least several classes of modification enzymes in the Golgi as well as extracellular remodeling enzymes;as with other glycoconjugates such as N-linked glycoproteins, many similar but distinct sugar polymers are created by cells. HS-binding proteins are observed to exhibit preferences for some HS isoforms over others thus it is theorized that HS complexity is employed as an exquisite control system. Our project will (i) explore how the HS structural variation is initially generated during its biosynthesis, (ii) elucidate which HS polymers possess the most selectivity and/or highest potency in growth factor systems, and (iii) map a synthetic route to prepare more defined molecules with potential as therapeutics. Currently, it is very difficult to understand (a) how the HS modification pattern first is generate and (b) which HS forms possess the highest biological activities. As purification of the individual HS forms in their native state from cells (even with knockout or transgenic organisms) in larger amounts is virtually impossible, we will use our new synthetic methodology to generate a series of the most defined N- sulfated HS polymers to date. Our strategy is to make polymer backbones in vitro so that we can bypass the initial key step, de-N-acetylation and N-sulfonation usually catalyzed by Golgi enzymes in vivo, that is hypothesized to set the stage for later HS modification and remodeling steps that create a panoply of HS molecules with various biological activities. In this project, a small library of sugar polymers having size-defined and placement-defined sulfated and nonsulfated domains will be generated by chemoenzymatic synthesis using recombinant heparosan synthases and UDP-sugar analogs with two types of protected amino groups (base- or acid-sensitive) developed during the previous grant period. There is currently no other reported method to create such defined polymers. These backbone polymers will then be further modified by biosynthetic enzymes in vitro including O-sulfotransferases, and C5-epimerase and/or catabolic enzymes including O-endosulfatases and heparanase using a focused combinatorial approach. The resulting modified molecules will be analyzed by (1) electrophoretic, chromatographic, and mass spectrometric methods to determine their structures thus learn the rules governing HS biosynthesis, and (2) biophysical and cell-based bioassays to study HS/growth factor/receptor interactions. Our goals include: (a) to understand the roles of domains in the biosynthesis of HS, (b) to produce defined HS polymers to test two opposing models of growth factor signaling, a key event in proliferation, development, and cancer, and (c) to explore the potential for synthesis of next- generation sugar-based therapeutics.
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