Arylsulfatase I is a novel lysosomal chondroitin endosulfatase regulating endochondral ossification

During endochondral ossification, chondrocytes undergo maturation and biochemically modify the collagenous extracellular matrix of cartilage. Similar modifications to cartilage proteoglycans (PGs), which are predominantly chondroitin sulfate PGs, have not been characterized. Using synchrotron X-ray fluorescence imaging, we demonstrated that PG sulfation significantly decreased during cartilage maturation of chick embryos. Laser-capture microdissection and RNAseq revealed upregulation of Arylsulfatase I (Arsi) in mature cartilage of mouse. ARSI protein also increased in mature cartilage of mouse and chick in vivo and during maturation of ATDC5 chondrocytes in vitro, whereas expression of the two known chondroitin sulfate PG sulfatases (ARSB and GALNS) was not specific to mature cartilage. Colocalization studies suggested that ARSI is lysosomal, and lysosome homeostasis was altered in ARSI loss of function chondrocytes. Biochemical analyses of ARSI gain and loss of function cell lines and isolated cell-free systems revealed that ARSI is a novel chondroitin endosulfatase, specifically desulfating chondroitin-4-sulfate at pH 4.5. Finally, Arsi knockout in RCS chondrocytes caused increased expression of maturation genes, such as Col10a1 and Mmp13. In total, these data identify ARSI as a novel PG sulfatase regulating endochondral ossification.