Heparan sulfate modifications of betaglycan promote TIMP3-dependent ectodomain shedding to fine-tune TGF-ß signaling.

Betaglycan/type III TGF-ß receptor (TGFBR3) is an established co-receptor for the TGF-ß superfamily with direct binding demonstrated for TGF-ß 1-3 and inhibin A. Betaglycan can be membrane-bound or have its ectodomain cleaved/ shed to produce soluble-betaglycan that has been demonstrated to sequester ligands. Extracellular domain of betaglycan is modified with glycosaminoglycan chains at Ser residues S534 and S545, to which heparan sulfate and chondroitin sulfate chains are covalently attached. To delineate the contribution of the heparan and chondroitin sulfate modifications on betaglycan on its shedding and thereby on TGF-ß signaling in ovarian cancer biology, we used mutants of betaglycan with alterations to the different glycosaminoglycan modifications. We made the unexpected discovery that the heparan sulfate modifications are essential for maximum ectodomain shedding of betaglycan, which is further essential for the ability of betaglycan to suppress TGF-ß signaling and the tumor cells responses to exogenous TGF-ß ligand. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-ß signaling. Taken together, these studies are the first to demonstrate a critical link between the well-known modifications on betaglycan and TGF-ß signaling responses. Overall design: To investigate the role of specific glycosaminoglycan modifications on betaglycan shedding and its impact on TGF-ß signaling, we generated HEYA8 cells expressing serine 534 to alanine and Ser 545 to alanine mutants of betaglycan either as single mutations or double mutants ( delat GAG). We then performed RNA-SEQ collected from the mutant BG expressing cells in three biological replicates and performed gene expression profiling analysis.