Pickering, I. J. | bioRxiv Science

bioRxiv2025

Arylsulfatase I is a novel lysosomal chondroitin sulfatase 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, 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 functional assays revealed that ARSI impacts lysosome homeostasis in chondrocytes. Biochemical analyses of ARSI gain and loss of function cell lines and isolated cell-free systems revealed that ARSI is a novel chondroitin sulfatase, specifically desulfating GalNAc4S at the nonreducing terminal of CS/DS. 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.

cell biology↗

bioRxiv2021

Spike protein disulfide disruption as a potential treatment for SARS-CoV-2

The coronaviral pandemic is exerting a tremendously detrimental impact on global health, quality of life and the world economy, emphasizing the need for effective medications for current and future coronaviral outbreaks as a complementary approach to vaccines. The Spike protein, responsible for cell receptor binding and viral internalization, possesses multiple disulfide bonds raising the possibility that disulfide-reducing agents might disrupt Spike function, prevent viral entry and serve as effective drugs against SARS-CoV-2. Here we show the first experimental evidence that reagents capable of reducing disulfide bonds can inhibit viral infection in cell-based assays. Molecular dynamics simulations of the Spike receptor-binding domain (RBD) predict increased domain flexibility when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the surface loop, comprised of residues 456-490, which interacts with the Spike cell receptor ACE2. Consistent with this finding, the addition of exogenous disulfide bond reducing agents affects the RBD secondary structure, lowers its melting temperature from 52 to 36-39{degrees}C and decreases its binding affinity to ACE2 by two orders of magnitude at 37{degrees}C. Finally, the reducing agents dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) inhibit viral replication at high {micro}M - low mM levels with a negligible effect on cell viability at these concentrations. The antiviral effect of monothiol-based reductants N-Acetyl-L-cysteine (NAC) and reduced glutathione (GSH) was not observed due to decreases in cell viability. Our research demonstrates the clear potential for medications that disrupt Spike disulfides as broad-spectrum anticoronaviral agents and as a first-line defense against current and future outbreaks.

microbiology↗