Chondroitin sulfate (CS) is a long, highly sulfated polysaccharide found at the cell surface and in the extracellular matrix. CS plays important roles in numerous biological processes, including cell signaling and morphogenesis. The chain elongation step, during which the long polysaccharide backbone is generated, is a critical step in CS biosynthesis. Four chondroitin sulfate synthase proteins (CHSY1, CHSY3, CHPF, and CHPF2) have been previously identified in humans. However, their individual roles and cooperative mechanisms in CS chain polymerization have remained unclear.
In this study, published in Nature Communications, researchers from the SAGAG group identified four heterodimeric complexes that carry out CS chain polymerization in humans : CHSY1–CHPF, CHSY1–CHPF2, CHSY3–CHPF, and CHSY3–CHPF2. The team recombinantly purified the four enzyme complexes and demonstrated that they can catalyze chain polymerization. Using cryo-electron microscopy, the researchers determined a high-resolution structure of the CHSY3–CHPF complex, revealing its three-dimensional architecture and the spatial organization of its catalytic domains.
Next, the team generated CHSY3–CHPF mutant complexes, each harboring single amino acid substitutions in the putative catalytic sites. Using functional assays, they demonstrated that the N-terminal domain of CHSY3 catalyzes glucuronic acid transfer, while the C-terminal domain is responsible for N-acetylgalactosamine transfer. Surprisingly, CHPF lacks catalytic activity. Cellular assays using human CRISPR/Cas9 double-knockout cell lines confirmed that only CHSY1 and CHSY3 exhibit bifunctional glycosyltransferase activity, while CHPF and CHPF2 are essential for complex formation and stability.
Based on the spatial arrangement of the catalytic sites, the authors propose that CS chain polymerization follows a non-processive, distributive mechanism. These results support a molecular model for CS chain elongation, which may help the future development of drugs that modulate CS levels for the treatment of diseases such as cancer.
Structural basis for human chondroitin sulfate chain polymerization. Dutta P, Cordeiro RL, Friedel-Arboleas M, Bourgeais M, Vallet SD, Weber M, Molinas M, Shu H, Grønset MNN, Miller RL, Boeri Erba E, Wild R. Nat Commun. 2025 Nov 26 ;16(1):11663.
Contact : Rebekka Wild (IBS/Structure and Activity of Glycosaminoglycans Group)