Discovery of a Functional Sequon for Chondroitin Sulfate Glycosylation

The amino acid sequence requirements that instruct the modification of proteins with chondroitin sulfate (CS) have been unknown, precluding predictions or opportunities for precise protein engineering. This study identified an essential amino acid motif for CS addition, the "CS-sequon" (EDQDDKDGGDFSGWGG), by comparing the secreted sulfatases SULF1 and SULF2, where only SULF2 is CS-modified. A cluster of seven amino acids plus a nearby tryptophan are critical for CS attachment; inserting the CS-sequon into SULF1 enabled it to gain CS modification. This sequon recruits the activity of the CS-initiating xylosyltransferase to the peptide modification site and CS addition enhances SULF1/2 extracellular abundance and enzymatic activity. These findings were validated across human and non-human cells and in Drosophila, providing a foundational resource for engineering CS modifications into proteins.