Abstract 197: Molecular interactions of Globo H ceramide with translin-associated factor X (TRAX) underlie its proangiogenic activities

Abstract

Abstract Glycosphingolipids (GSLs) are ubiquitous glycolipids in eukaryotic plasma membranes that consist of a polar glycan chain and a hydrophobic sphingosine- containing ceramide tail. Although GSLs display various diversities and functions, the mechanistic details of GSL interactions with membranous or intracellular components are poorly understood. Globo H ceramide (GHCer), a tumor-associated GSL overexpressed in most types of epithelial cancers, is a potential target of cancer immunotherapy. Previously, we showed that GHCer is shed by cancer cells to mediate immune suppression and promote angiogenesis in the tumor microenvironment. At the cellular level, GHCer from cancer cells binds to translin-associated factor (TRAX), with consequent release of PLCB1 from TRAX to trigger Ca2+ mobilization. Herein, we found that Gb5-ceramide (Gb5Cer), the pentasaccharide precursor of GHCer, could not promote angiogenesis nor Ca2+ mobilization. At the molecular level, ELISA and biacore analysis revealed that GHCer binds to TRAX with a distinct dissociation constant of 4.09×10-8 M, while its precursors, galactosyl-, lactosyl-, Gb4-, and Gb5Cer- showed negligible binding to TRAX. Molecular docking and molecular dynamics indicated that the fucose residue of Globo H is an important contributor to a favorable glycan conformation for interaction with TRAX, consistent with the finding that Gb5Cer had no proangiogenic activities. In addition, GHCer competed with recombinant C-terminus region of phospholipase C β1 (PLCB1) for binding to TRAX, thus providing a molecular mechanism for the proangiogenic effect of GHCer on tumor microenvironment. This study provides the first evidence for interaction of TRAX with a non-protein GSL, deciphered distinct regions of TRAX for interacting with glycan and lipid portions of GHCer, and illustrated the crucial contribution of fucose moiety on GHCer for its interaction with TRAX and angiogenic activity. Citation Format: John Yu, Sheng-Hung Wang, Jing-Yan Cheng, Jung-Tung Hung, Chun-Cheng Lin, Fei-Yun Lo, Chien-Wei Lee, Alice L. Yu. Molecular interactions of Globo H ceramide with translin-associated factor X (TRAX) underlie its proangiogenic activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 197.