Dataset Information

Plasminogen proteoform profiling

ABSTRACT: Plasminogen, the zymogen of plasmin, is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plasminogen dysregulation has been implicated in a range of diseases. Classically, human plasminogen is categorized into two types based on the presence (type I) or absence (type II) of a single N-linked glycan, supposedly having different functional features. Using high-resolution native mass spectrometry (native MS), we uncover that the proteoform profiles of plasminogen (and plasmin) are substantially more extensive than this simple binary classification. In samples derived from human plasma, we identified up to 14 distinct proteoforms of human plasminogen, including a novel, highly abundant phosphorylation site at Ser339. To elucidate potential functional effects of these post-translational modifications, we performed proteoform-resolved kinetic analyses of the plasminogen-to-plasmin conversion using a canonical activator. This conversion is thought to involve at least two independent cleavage events: one to remove the N-terminal peptide, and another to form the active catalytic site. Our analyses reveal that these processes are not independent but are instead tightly regulated and occur in a step-wise order. Notably, N-terminal cleavage at the canonical site (Lys77) does not occur directly from intact plasminogen. Instead, an activation intermediate corresponding to cleavage at Arg68 is initially produced, which only then is further processed to the canonical Lys77 product. Based on our results, we propose a new categorization system for human plasminogen isoforms. This work highlights the capacity of high-resolution native MS to reveal new aspects of structure, even in frequently-studied serum glycoproteins.

INSTRUMENT(S):

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human)

TISSUE(S): Blood Plasma, Blood Serum

SUBMITTER: Dario Cramer

LAB HEAD: Albert J. R. Heck

PROVIDER: PXD042225 | Pride | 2024-05-24

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Fusion_bottomup_female_1_gluC_HCD.raw	Raw
	Fusion_bottomup_female_1_gluC_HCD_peaks.mgf	Mgf
	Fusion_bottomup_female_1_gluC_HCD_search.byrslt	Other
	Fusion_bottomup_female_1_gluC_HCD_search.byspec2	Other
	Fusion_bottomup_female_1_trypsin_EtHCd.raw	Raw

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Publications

Proteoform-Resolved Profiling of Plasminogen Activation Reveals Novel Abundant Phosphorylation Site and Primary N-Terminal Cleavage Site.

Cramer Dario A T DAT Yin Victor V Caval Tomislav T Franc Vojtech V Yu Dingyi D Wu Guojie G Lloyd Gordon G Langendorf Christopher C Whisstock James C JC Law Ruby H P RHP Heck Albert J R AJR

Molecular & cellular proteomics : MCP 20231213 1

Plasminogen (Plg), the zymogen of plasmin (Plm), is a glycoprotein involved in fibrinolysis and a wide variety of other physiological processes. Plg dysregulation has been implicated in a range of diseases. Classically, human Plg is categorized into two types, supposedly having different functional features, based on the presence (type I) or absence (type II) of a single N-linked glycan. Using high-resolution native mass spectrometry, we uncovered that the proteoform profiles of human Plg (and P ...[more]

PMID: 38101751

Similar Datasets

Project description:Plasminogen (Plg) is a highly abundant, liver-secreted plasma protein and zymogen precursor of plasmin, a key serine-protease in fibrinolysis. Zymogen Plg (Glu-Plg) has been reported to transport extracellular RNA, cholesterol, and oxidized phospholipids (oxPL) cargo which likely defines the biological functions of non-enzymatic Plg particles, including immunogenicity towards innate immune cells. Here, we demonstrate that native Plg dose-dependently activates NF-kB transactivity, pro-inflammatory gene expression and cytokine secretion in primary macrophages through toll-like receptors, i.e., toll-like receptor 2 (TLR2), as identified through a TLR screen. Plg stimulation of macrophages and NF-kB transactivity reporter cells were sensitive to small molecule inhibitors of TLR2, and Tlr2 and Myd88 genetic-deficiency. Plg treatments were found to stimulate both mouse and human primary macrophages but failed to activate microglial cells and vascular smooth muscle cells. Both C29, and its derivative ortho-vanillin, were found to inhibit macrophage TLR2 signaling and cytokine secretion in vitro, as profiled by proximity extension assays (PEA). Plg activation of TLR2 was observed to be independent of Plg’s conversion to plasmin or down-stream plasmin protease activity, as Plg stimulation of TLR2 were not affected by active-site mutagenesis, lysine analogues ε-aminocaproic acid and tranexamic acid, VPLCK peptide inhibitor, α2-anti-plasmin, α2-macroglobin, or urokinase plasminogen activator (uPA, Plau-/-) and tissue plasminogen activator (tPA, Plat-/-) macrophage deficiency. Mass spectrometry-based untargeted lipidomics demonstrated the full profile of bioactive lipids covalently bound or loosely associated with human Plg particles. Plg’s immunogenicity towards macrophages was found to be dependent, in part, through lipid cargo, as lipid removal with lipases and organic lipid extractions were found to reduce Plg’s capacity to activate macrophages. Plg-deficiency in hypercholesterolemic mice were observed to be associated with reduced atherosclerosis and intravenous injections of ortho-vanillin were found to significantly reduce lesion area and alter macrophage sub-phenotypes in mouse lesions, as per single cell RNA sequencing. Results from these studies support that Glu-Plg’s lipid cargo and TLR2 may be viable drug targets to reduce chronic inflammation without compromising fibrinolysis.

Project description:Venkatraman2011 - PLS-UPA behaviour in the presence of substrate competition The posibility of ultrasensitivity and bistable activation of PLS (Plasmin) and UPA (Urokinase-type plasminogen activator) in the presence of substrate competition is explained here using a mathematical model. This model is described in the article: Steady states and dynamics of urokinase-mediated plasmin activation in silico and in vitro. Venkatraman L, Li H, Dewey CF Jr, White JK, Bhowmick SS, Yu H, Tucker-Kellogg L. Biophys. J. 2011 Oct; 101(8): 1825-1834 Abstract: Plasmin (PLS) and urokinase-type plasminogen activator (UPA) are ubiquitous proteases that regulate the extracellular environment. Although they are secreted in inactive forms, they can activate each other through proteolytic cleavage. This mutual interplay creates the potential for complex dynamics, which we investigated using mathematical modeling and in vitro experiments. We constructed ordinary differential equations to model the conversion of precursor plasminogen into active PLS, and precursor urokinase (scUPA) into active urokinase (tcUPA). Although neither PLS nor UPA exhibits allosteric cooperativity, modeling showed that cooperativity occurred at the system level because of substrate competition. Computational simulations and bifurcation analysis predicted that the system would be bistable over a range of parameters for cooperativity and positive feedback. Cell-free experiments with recombinant proteins tested key predictions of the model. PLS activation in response to scUPA stimulus was found to be cooperative in vitro. Finally, bistability was demonstrated in vitro by the presence of two significantly different steady-state levels of PLS activation for the same levels of stimulus. We conclude that ultrasensitive, bistable activation of UPA-PLS is possible in the presence of substrate competition. An ultrasensitive threshold for activation of PLS and UPA would have ramifications for normal and disease processes, including angiogenesis, metastasis, wound healing, and fibrosis. The cooperativity parameter "ci" was missing in the original model. The parameter "ci" has been added to the added. This model is hosted on BioModels Database and identified by: BIOMD0000000630. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Venkatraman2012 - Interplay between PLS and TSP1 in TGF-β1 activation The interplay between PLS (Plasmin) and TSP1 (Thrombospondin-1) in TGF-β1 (Transforming growth factor-β1)is shown using mathematical modelling and in vitro experimentents. This model is described in the article: Plasmin triggers a switch-like decrease in thrombospondin-dependent activation of TGF-β1. Venkatraman L, Chia SM, Narmada BC, White JK, Bhowmick SS, Forbes Dewey C Jr, So PT, Tucker-Kellogg L, Yu H. Biophys J. 2012 Sep 5;103(5):1060-8. Abstract: Transforming growth factor-β1 (TGF-β1) is a potent regulator of extracellular matrix production, wound healing, differentiation, and immune response, and is implicated in the progression of fibrotic diseases and cancer. Extracellular activation of TGF-β1 from its latent form provides spatiotemporal control over TGF-β1 signaling, but the current understanding of TGF-β1 activation does not emphasize cross talk between activators. Plasmin (PLS) and thrombospondin-1 (TSP1) have been studied individually as activators of TGF-β1, and in this work we used a systems-level approach with mathematical modeling and in vitro experiments to study the interplay between PLS and TSP1 in TGF-β1 activation. Simulations and steady-state analysis predicted a switch-like bistable transition between two levels of active TGF-β1, with an inverse correlation between PLS and TSP1. In particular, the model predicted that increasing PLS breaks a TSP1-TGF-β1 positive feedback loop and causes an unexpected net decrease in TGF-β1 activation. To test these predictions in vitro, we treated rat hepatocytes and hepatic stellate cells with PLS, which caused proteolytic cleavage of TSP1 and decreased activation of TGF-β1. The TGF-β1 activation levels showed a cooperative dose response, and a test of hysteresis in the cocultured cells validated that TGF-β1 activation is bistable. We conclude that switch-like behavior arises from natural competition between two distinct modes of TGF-β1 activation: a TSP1-mediated mode of high activation and a PLS-mediated mode of low activation. This switch suggests an explanation for the unexpected effects of the plasminogen activation system on TGF-β1 in fibrotic diseases in vivo, as well as novel prognostic and therapeutic approaches for diseases with TGF-β dysregulation. This model is hosted on BioModels Database and identified by: MODEL1303130000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.