Identification of membrane proteins regulated by ADAM15 by SUSPECS proteomics

Abstract

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.

Digital spatial profiling of segmental outflow regions in trabecular meshwork reveals a role for ADAM15

“…ADAM15 is a member of the disintegrin-metalloproteinase family and is best known for its role in cartilage homeostasis and tissue remodeling in vascular and cardiac tissues [72][73][74]. It has recently been suggested to orchestrate a novel pro-inflammatory mechanosensing pathway in synovial fibroblasts [75] and to play a role in regulating TGFβ signaling in cartilage [76]. Furthermore, it may modulate the activity of SLC26A2, which is a sulfate transporter responsible for maintaining adequate sulfation of proteoglycans [77].…”