Glycosyltransferases EXTL2 and EXTL3 cellular balance dictates heparan sulfate biosynthesis and shapes gastric cancer cell motility and invasion

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells’ glycocalyx and extracellular matrix. By acting as scaffolds for protein–protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell–extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4 cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.