Chondroitin sulfate (CS), from the Greek word ‘chondros’ (cartilage), is a complex sugar chain found on the cell surface and in the extracellular space. It is crucial in various processes, including tissue repair, brain development, and tumor formation. However, how CS chains are synthesized in our cells remains a mystery. Our expression tests and computational studies uncovered how the four human enzymes form specific heterodimers. We demonstrate that these four pairs of enzymes can be purified in a functional form. We applied cryo-electron microscopy to see the architecture of one of these enzyme pairs at near-atomic resolution, identifying key amino acids for CS chain elongation. Mutational analysis of purified enzymes and in-cellulo complementation assays revealed that only one enzyme within the pair is catalytically active, while the other helps to stabilize the complex. Our mechanistic insights might enable the development of new treatments for diseases like arthritis and cancer.​
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