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ADAM15 is a member of the disintegrin-metalloproteinase family of sheddases, which plays a role in several biological processes including cartilage homeostasis. In contrast with well-characterized ADAMs, such as the canonical sheddases ADAM17 and ADAM10, little is known about substrates of ADAM15 or how the enzyme exerts its biological functions. Herein, we used “surface-spanning enrichment with click-sugars (SUSPECS)” proteomics to identify ADAM15 substrates and/or proteins regulated by the proteinase at the cell surface of chondrocyte-like cells. Silencing of ADAM15 by siRNAs significantly altered membrane levels of 13 proteins, all previously not known to be regulated by ADAM15. We used orthogonal techniques to validate ADAM15 effects on 3 of these proteins which have known roles in cartilage homeostasis. This confirmed that ADAM15-silencing increased cell surface levels of the programmed cell death 1 ligand 2 (PDCD1LG2) and reduced cell surface levels of vasorin and the sulfate transporter SLC26A2 through an unknown post-translational mechanism. The increase of PDCD1LG2 by ADAM15 knockdown, a single-pass type I transmembrane protein, suggested it could be a proteinase substrate. However, shed PDCD1LG2 could not be detected even by a data-independent acquisition mass spectrometry, a highly sensitive method for identification and quantification of proteins in complex protein samples, suggesting that ADAM15 regulates PDCD1LG2 membrane levels by a mechanism different from ectodomain shedding.

A disintegrin and metalloproteinase 15 (ADAM15) is a member of the ADAM family of sheddases. Its genetic ablation in mice suggests that ADAM15 plays an important role in a wide variety of biological functions, including cartilage homeostasis. Nevertheless, while the substrate repertoire of other members of the ADAM family, including ADAM10 and ADAM17, is largely established, little is known about the substrates of ADAM15 and how it exerts its biological functions. Herein, we used unbiased proteomics to identify ADAM15 substrates and proteins regulated by the proteinase in chondrocyte-like HTB94 cells. ADAM15 silencing did not induce major changes in the secretome composition of HTB94 cells, as revealed by two different proteomic approaches. Conversely, overexpression of ADAM15 remodeled the secretome, with levels of several secreted proteins being altered compared to GFP-overexpressing controls. However, the analysis did not identify potential substrates of the sheddase, i.e., transmembrane proteins released by ADAM15 in the extracellular milieu. Intriguingly, secretome analysis and immunoblotting demonstrated that ADAM15 overexpression increased secreted levels of tissue inhibitor of metalloproteinases 3 (TIMP-3), a major regulator of extracellular matrix turnover. An inactive form of ADAM15 led to a similar increase in the inhibitor, indicating that ADAM15 regulates TIMP-3 secretion by an unknown mechanism independent of its catalytic activity. In conclusion, high-resolution quantitative proteomics of HTB94 cells manipulated to have increased or decreased ADAM15 expression did not identify canonical substrates of the proteinase in the steady state, but it revealed that ADAM15 can modulate the secretome in a catalytically-independent manner.