Forward and reverse degradomics defines the proteolytic landscape of human knee osteoarthritic cartilage and the role of the serine protease HtrA1.

Proteolytic destruction of articular cartilage, a major pathogenic mechanism in osteoarthritis (OA), was not previously investigated by terminomics strategies. We defined the degradome of human knee OA cartilage and the contribution therein of the protease HtrA1 using Terminal Amine Isotopic Labeling of Substrates (TAILS). Proteins from OA cartilage taken at knee arthroplasty (n = 6) or separately, from healthy cartilage incubated in triplicate with/without active HtrA1, were labeled at natural and proteolytically cleaved N-termini by reductive dimethylation, followed by trypsin digestion, enrichment of N-terminally labeled/blocked peptides, tandem mass spectrometry and positional peptide annotation to identify cleavage sites. Biglycan proteolysis by HtrA1 was validated biochemically and Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) was used to define the HtrA1 cleavage sites. We identified 10,155 unique internal peptides from 2,162 proteins, suggesting at least 10,797 cleavage sites in OA cartilage. 7,635 internal peptides originated in 371 extracellular matrix/secreted components, many undergoing extensive proteolysis. Rampant ragging of protein termini suggested pervasive exopeptidase activity. HtrA1, the most abundant protease in OA cartilage, experimentally generated 323 cleavages in 109 cartilage proteins, accounting for 171 observed cleavages in the OA degradome. ATOMS identified HtrA1 cleavage sites in a selected substrate, biglycan, whose direct cleavage by HtrA1 was thus orthogonally validated. OA cartilage demonstrates widespread proteolysis by endo- and exopeptidases. HtrA1 contributes broadly to cartilage proteolysis. Forward degradomics of OA cartilage together with reverse degradomics of proteases active in OA, e.g., HtrA1, can potentially fully annotate OA proteolytic pathways and provide new biomarkers.

Bhutada S ，Li L ，Willard B ，Muschler G ，Piuzzi N ，Apte SS ... -  《-》

Degradomics defines proteolysis information flow from human knee osteoarthritis cartilage to matched synovial fluid and the contributions of secreted proteases ADAMTS5, MMP13 and CMA1 to articular cartilage breakdown.

Bhutada S ，Hoyle A ，Piuzzi NS ，Apte SS ... -  《-》

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

Kleifeld O ，Doucet A ，Prudova A ，auf dem Keller U ，Gioia M ，Kizhakkedathu JN ，Overall CM ... -  《-》

Cell density-dependent proteolysis by HtrA1 induces translocation of zyxin to the nucleus and increased cell survival.

Sabino F ，Madzharova E ，Auf dem Keller U 《Cell Death & Disease》

Identification of proteolytic products and natural protein N-termini by Terminal Amine Isotopic Labeling of Substrates (TAILS).

Doucet A ，Kleifeld O ，Kizhakkedathu JN ，Overall CM ... -  《-》