Table2_Identification of membrane proteins regulated by ADAM15 by SUSPECS proteomics.XLSX

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.

ADAM15是解整联蛋白-金属蛋白酶（disintegrin-metalloproteinase）家族脱落酶（sheddases）的一员，参与包括软骨稳态（cartilage homeostasis）在内的多种生物学过程。与研究较为透彻的ADAM家族经典成员（如经典脱落酶ADAM17与ADAM10）相比，目前学界对ADAM15的底物或该酶发挥生物学功能的具体机制仍知之甚少。本研究采用糖点击法表面富集（surface-spanning enrichment with click-sugars, SUSPECS）蛋白质组学技术，在软骨细胞样细胞的细胞表面层面鉴定ADAM15的底物或受该蛋白酶调控的蛋白。通过小干扰RNA（small interfering RNA, siRNA）沉默ADAM15后，13种蛋白的膜表达水平发生显著改变，且此前均未被报道受ADAM15调控。我们采用正交验证技术，对其中3种在软骨稳态中具有已知功能的蛋白开展ADAM15调控效应的验证实验。结果证实，ADAM15沉默可通过未知的翻译后机制，提升程序性细胞死亡1配体2（programmed cell death 1 ligand 2, PDCD1LG2）的细胞表面水平，并降低血管蛋白（vasorin）与硫酸盐转运蛋白SLC26A2的细胞表面水平。作为单次跨膜I型蛋白，ADAM15敲低所导致的PDCD1LG2水平升高，提示其可能为该蛋白酶的底物。然而，即便采用对复杂蛋白样品中蛋白鉴定与定量灵敏度极高的数据非依赖采集质谱（data-independent acquisition mass spectrometry）技术，也未能检测到脱落的PDCD1LG2，这表明ADAM15调控PDCD1LG2膜水平的机制不同于胞外域脱落（ectodomain shedding）。