Vallet, S. D. | Search bioRxiv

bioRxiv2025

Structural basis for human chondroitin sulfate chain polymerization

Chondroitin sulfates are complex polysaccharide chains that regulate various biological processes at the cell surface and within the extracellular matrix. Here, we identify four heterodimeric complexes responsible for chondroitin sulfate chain polymerization in humans: CHSY1-CHPF, CHSY1-CHPF2, CHSY3-CHPF and CHSY3-CHPF2. Using a novel in vitro glycosylation assay based on chemo-enzymatically synthesized fluorescent substrates, we demonstrate that all four complexes exhibit chain polymerization activity. The cryo-EM structure of the CHSY3-CHPF complex provides, for the first time, molecular insights into the chondroitin sulfate chain polymerization reaction. The architecture of the catalytic sites suggests that CHSY1 and CHSY3 are enzymatically active, while CHPF and CHPF2 primarily play a stabilizing role. Mutational analysis of purified enzyme complexes, combined with an in cellulo complementation assay, confirms that only CHSY1 and CHSY3 have bifunctional glycosyltransferase activities. Based on the spatial arrangement of the catalytic sites, we propose that chondroitin sulfates chain polymerization follows a non-processive, disruptive mechanism.

molecular biology↗

bioRxiv2020

Computational and experimental characterization of the novel ECM glycoprotein SNED1 and prediction of its interactome

The extracellular matrix (ECM) protein SNED1 has been shown to promote breast cancer metastasis and control neural crest cell-specific craniofacial development, but the cellular and molecular mechanisms by which it does so remain unknown. ECM proteins exert their functions by binding to cell surface receptors, sequestering growth factors, and interacting with other ECM proteins, actions that can be predicted using knowledge of proteins sequence, structure and post-translational modifications. Here, we combined in-silico and in-vitro approaches to characterize the physico-chemical properties of SNED1 and infer its putative functions. To do so, we established a mammalian cell system to produce and purify SNED1 and its N-terminal fragment, which contains a NIDO domain. We have determined experimentally SNED1s potential to be glycosylated, phosphorylated, and incorporated into insoluble ECM produced by cells. In addition, we used biophysical and computational methods to determine the secondary and tertiary structures of SNED1 and its N-terminal fragment. The tentative ab-initio model we built of SNED1 suggests that it is an elongated protein presumably able to bind multiple partners. Using computational predictions, we identified 114 proteins as putative SNED1 interactors. Pathway analysis of the newly-predicted SNED1 interactome further revealed that binding partners of SNED1 contribute to signaling through cell surface receptors, such as integrins, and participate in the regulation of ECM organization and developmental processes. Altogether, we provide a wealth of information on an understudied yet important ECM protein with the potential to decipher its functions in physiology and diseases.

biochemistry↗