Defining the Structural Cues within Heparan Sulfate that Direct Heparanase Cleavage Public Deposited

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  • March 22, 2019
Creator
  • Peterson, Sherket Breshon
    • Affiliation: Eshelman School of Pharmacy
Abstract
  • Heparanase is a glucuronidase that cleaves heparan sulfate in a variety of cells and tissues. The depolymerization of HS by heparanase results in smaller fragments of variable sizes that bind and modulate the functions of a multitude of proteins, including growth factors and their receptors, chemokines, enzymes, and extracellular matrix proteins. Identification of precise structural features within HS that direct heparanase cleavage has been hindered by the heterogeneity of substrates, the lack of techniques to synthesize structurally defined oligosaccharides or polysaccharides with site-specific sulfation, and a lack of large quantities of pure heparanase. Here, we report how advances in the methodology for synthesizing substrates with defined sulfation types has allowed us to identify specific sulfation types as well as residues that may direct heparanase cleavage. Utilizing chemo-enzymatically synthesized polysaccharide substrates with unique sulfation patterns, we were able to dissect that heparanase cleaves the linkage between a GlcUA unit and an N-sulfo glucosamine unit carrying either a 3-O-sulfo or a 6-O-sulfo group. In addition, heparanase cleaves the linkage of a GlcUA unit and N-sulfo glucosamine unit with a 2-O-sulfated GlcUA residue, not a 2-O-sulfated IdoUA residue, in proximity. We also discovered that the polysaccharide with repeating disaccharide units of IdoUA2S-GlcNS inhibits the activity of heparanase. Moreover, the utilization of a selective library of oligosaccharides coupled with ESI-MS analysis and large quantities of a pure enzyme provided the opportunity to examine the substrate specificity of heparanase in greater detail. We determined that heparanase utilizes the GlcNAc6S/GlcNS6S at the nonreducing end to direct the cleavage pattern. Moreover, heparanase depolymerizes the oligosaccharides utilizing a non-processive method. Our data reveals for the first time that heparanase is regulated by two main factors; the primary factor is heparanase recognition of different sulfation types with its non-processive mode of action having a secondary role. These findings advance the understanding of the substrate specificity of heparanase and reveal a unique control mechanism for the action of heparanase. Moreover, our result advances the understanding of the regulation of HS biosynthesis and the intimate relationship between HS and heparanase.
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  • In Copyright
Advisor
  • Liu, Jian
Degree
  • Doctor of Philosophy
Graduation year
  • 2012
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