Functional modification of matrix metalloproteinase (MMP) activity by post-translational modifications (PTMs)

Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases, functionally implicated in regulating physiological and pathological processes. Subsequently, MMPs are regulated by transcriptional and post-transcriptional control of their gene expression, zymogen activation, and extracellular inhibition. Additionally, almost all members of the MMP family and their substrates are modified by post-translational modifications (PTMs). Given the emerging evidence of PTMs' role in the structural and functional integrity of proteins, an important role of PTMs in modulating the MMPs activities has been suggested. However, the biological consequences of either MMP or substrate modifications are mostly unknown. In this thesis, we systematically assessed the fine-tuning of limited proteolysis in the extracellular space by glycosylation and phosphorylation, two of the most widely observed PTMs, using cells and animal models and applying the Terminal Amine Labeling of Substrates (TAILS) approach, a quantitative proteomics workflow for the system-wide assessment of protein N termini and protease cleavage events in complex biological samples. In a first study, we investigated the influence of O-glycosylation of extracellular substrate proteins on their general susceptibility to limited proteolysis and specific processing by MMP9. Initially, we glycoengineered human breast adenocarcinoma cells to get access to substrate proteomes expressing truncated O-glycans. Next, we incubated the wild-type (wt) and glycoengineered cell culture supernatant (secretomes) for different time periods with recombinant MMP9 and recorded their proteomes and N-terminomes by 16plex-TMT TAILS analysis. From this analysis, we revealed a significant impact of O-glycosylation on general proteome stability, identified novel glycol-dependent processing events, including functional modifications such as ectodomain shedding, and found an inhibitory role for O-glycosylation on MMP9-dependent cleavages. In a second project, we performed an in-depth characterization of MMP9 glycosylation. We used advanced mass spectrometry (MS)–based techniques, glycomics, and glycoproteomics, to characterize the structure and site-specific location of the N- and O-glycan repertoire of recombinant human MMP9 (rhMMP9) expressed in human embryonic kidney cells and native
neutrophil-derived human MMP9. We identified that MMP9 has two N-glycosylation sites, Asn³⁸ and Asn¹²⁰, in the propeptide and catalytic domain and characterized the rhMMP9 N-glycans as partially sialylated, core-fucosylated multi-antennary complex-type glycans. The rhMMP9 Oglycans were identified as a mixture of mono- and disialylated core 1 (Galβ1-3GalNAc) and core 2 (Galβ1-3[GlcNAcβ1-6] GalNAc) glycan structures. The human neutrophil-derived MMP9 carried core-fucosylated biantennary structures N-glycans, elongated with LacNAc repeats with Lex (Galβ1(Fucα1-3)4GlcNAc) and sialyl Lex (NeuAcα2-3Galβ1(Fucα1-3)4GlcNAc) epitopes. Comparing both glycomes revealed the MMP9 glycosylation dynamics between the different cell types. In a third study, we explored the regulatory crosstalk of tyrosine phosphorylation by a vertebrate lonesome kinase (Vlk) and limited proteolysis in the extracellular space using samples from genetically modified mice and 8plex-TMT-TAILS analysis. We compared the proteome and N-terminome of embryonic lung tissues from wt animals and mice with conditional deletion of Vlk in fibroblasts. Thereby, we identified a change in the abundance of proteins involved in lung organogenesis. We detected an upregulation of TGF-β2, a pleiotropic cytokine necessary for lung organogenesis and homeostasis, and fibromodulin, an extracellular collagen-binding protein, involved in lung morphogenesis. Additionally, the extracellular matrix proteins collagen alpha-1 and syndecan-1 were also increased in abundance. Furthermore, we observed a downregulation of blood coagulation cascade proteins, including plasminogen, fibrinogen, antithrombin-III; histidinerich glycoprotein, a plasma protein recognized to have anticoagulant and antifibrinolytic properties; and fibronectin (FN), typically highly expressed in mouse embryonic lung. Moreover, we detected increased proteolytic activity in processes associated with the cell cycle, protein synthesis, and extracellular matrix-related processes. Lastly, we identified a subset of differentially abundant proteolytic events between wt and Vlk deficient mice, which contained a tyrosine residue in P1 position of the cleavage site. Among them, the most interesting were collagen alpha-1(VI) chain (ALHY↓⁹³SDEV), laminin subunit gamma-1 (VEIY↓¹²⁶⁷ASVA), and basement membrane-specific heparan sulfate proteoglycan core protein (HSPG, HLVF↓³⁵⁸⁸SYQL). These results indicated important functions of Vlk in development and tissue homeostasis and demonstrated the influence of tyrosine phosphorylation on proteolytic shaping of the extracellular matrix proteome.