Glycosulfopeptides (GSP) represent a novel type of complex glycoconjugate, which contain a recognition motif consisting of clustered tyrosine sulfates (Tyr-SO3H) and a core 2 O-glycan that bears a sialyl LewisX-containing hexasaccharide epitope (C2-O-sLeX). GSPs are present in PSGL-1, GPIba, endoglycan, VWF, Factor VIII, and CCR5, among many other biomolecules and play critical roles in innate immunity, thrombosis, and cancer. Despite the potential of sLeX-GSPs to serve as tools for biological studies and their promise as therapeutic agents, their utility has been limited by low yield and limited stability. Recent innovations by the investigative team in chemical and molecular genetic techniques have established an important new framework for the design of sLeX-GSPs and related multicomponent conjugates. Specifically, we intend to: (1) Define efficient schemes for scalable generation of GSPs containing tri-member recognition motifs (Tyr-SO3H, C2-O-sLeX, peptide backbone). Poor regio- and stereoselectivity and low yield continues to pose a significant challenge for the synthesis of C2-O-sLeX hexasaccharides and related variants. To overcome these limitations, we will design convergent glycosylation schemes in association with low temperature pre-activation, building blocks that require minimal protecting group manipulation, as well as one-pot schemes for glycans containing sLeX. To expand the diversity of novel GSP structures, new analogues will be generated in which tyrosine sulfates are replaced by sulfonates, Glu or Asp, and sulfated sugars incorporated in the glycan. (2) Identify orthogonal bioconjugation strategies for the construction of GSP bearing microarrays, as well as microspheres and liposomes as cellular `avatars'. Chemical and genetic orthogonal bioconjugation schemes will be investigated for the construction of microarrays containing printed GSPs, as well as GSP bearing microspheres and liposomes as cellular `avatars' to mimic cell-like presentation of ligands. In addition to standard bioorthogonal approaches, such as click chemistry and native chemical ligation, mutant sortase variants that exhibit orthogonal substrate specificity for peptide sequences LPETG, LAETG, and LPESG will be used to construct highly divergent GSP supramolecular systems. In the process, we will also define complementary approaches for the generation of GSP multimers and macrocycles. (3) Construct libraries of native and non-natural sLeX-bearing GSPs for the identification of highly potent selectin, integrin, and chemokine antagonists. We will generate microarrays, microspheres, and liposomes presenting GSPs modeled after human and murine PSGL-1, GPIba, endoglycan, and CCR5. Candidate high affinity selectin, integrin, and chemokine antagonists will be identified through a combination of microarray-based screens, Biacore analysis, flow cytometry, and microfluidic cell binding assays. GSP-derivatized microspheres and liposomes, as cellular `avatars', will define the density- and presentation-dependent features that influence molecular recognition.
'Glycosulfopeptides' (GSPs) represent a novel type of complex glycoconjugate present in P-selectin glycoprotein ligand-1, GPIba, endoglycan, and CCR5, among many other biomacromolecules, which contain a recognition motif consisting of clustered tyrosine sulfates (Tyr-SO3H) and a core 2 O-glycan that bears a sialyl LewisX-containing hexasaccharide epitope (C2-O-sLeX). GSPs play critical roles in innate immunity, thrombosis, and cancer, but despite their potential to serve as tools for biological studies and their promise as therapeutic agents, the utility of GSPs has been limited by low yield and limited stability. Recent innovations by the investigative team in chemical and molecular genetic techniques have established an important new framework for the design of sLeX-GSPs and related multicomponent glycoconjugates.
|Patel, Madhukar S; Miranda-Nieves, David; Chen, Jiaxuan et al. (2017) Targeting P-selectin glycoprotein ligand-1/P-selectin interactions as a novel therapy for metabolic syndrome. Transl Res 183:1-13|
|Morikis, Vasilios A; Chase, Shannon; Wun, Ted et al. (2017) Selectin catch-bonds mechanotransduce integrin activation and neutrophil arrest on inflamed endothelium under shear flow. Blood 130:2101-2110|
|Sardar, Mohammed Y R; Krishnamurthy, Venkata R; Park, Simon et al. (2017) Synthesis of LewisX-O-Core-1 threonine: A building block for O-linked LewisX glycopeptides. Carbohydr Res 452:47-53|
|Krishnamurthy, Venkata R; Sardar, Mohammed Y R; Ying, Yu et al. (2015) Glycopeptide analogues of PSGL-1 inhibit P-selectin in vitro and in vivo. Nat Commun 6:6387|