Extracellular tyrosyl-tRNA synthetase cleaved by plasma proteinases and stored in platelet α-granules: Potential role in monocyte activation

Abstract

Background: Tyrosyl-tRNA synthetase (YRS) belongs to the family of enzymes that catalyzes the tRNA aminoacylation reaction for protein synthesis, and it has been recently shown to exert noncanonical functions. Although database results indicate extremely low levels of YRS mRNA in platelets, YRS protein is abundantly present. The source of YRS in platelets, as well as the physiological role of platelet-stored YRS, remains largely unknown.

Objectives: To clarify how YRS accumulates in platelets and determine the potential role of platelet-stored YRS.

Methods: Recombinant YRS proteins with epitope tags were prepared and tested in vitro for proteolytic cleavage in human plasma. Fluorescent-labeled YRS was examined for uptake by platelets, as demonstrated by western blotting and confocal microscopy analysis. Using RAW-Dual reporter cells, Toll-like receptor and type I interferon activation pathways were analyzed after treatment with YRS.

Results: Full-length YRS was cleaved by both elastase and matrix metalloproteinases in the plasma. The cleaved, N-terminal YRS fragment corresponds to the endogenous YRS detected in platelet lysate by western blotting. Both full-length and cleaved forms of YRS were taken up by platelets in vitro and stored in the α-granules. The N-terminal YRS fragment generated by proteolytic cleavage had monocyte activation comparable to that of the constitutive-active mutant YRS (YRS^Y341A) previously reported.

Conclusion: Platelets take up both full-length YRS and the active form of cleaved YRS fragment from the plasma. The cleaved, N-terminal YRS fragment stored in α-granules may have potential to activate monocytes.

Keywords: elastase; matrix metalloproteinases (MMPs); monocytes; platelets; tyrosyl‐tRNA synthetase (YRS); α‐granules.

FIGURE 1

Western blot analysis of endogenous tyrosyl‐tRNA synthetase (YRS) in circulating plasma and platelets. (A) Plasma samples collected from healthy donors were analyzed by SDS‐PAGE under non reduced conditions and blotted with anti‐YRS monoclonal antibody (A‐10). Recombinant YRS^WT was loaded as a positive control. The asterisk represents oligomerized YRS and the arrowhead points to the truncated form of YRS. In the lower panel, the same human plasma samples (N1‐N3) were analyzed by SDS‐PAGE under reduced conditions and blotted with the same antibody (A‐10). The arrow indicates full‐length YRS. (B) Platelet lysates obtained from healthy human donors and C57BL/6J mice were loaded onto SDS‐PAGE, together with whole cell lysates from B16 melanoma cell line and RAW cells. The arrow corresponds with full‐length YRS. The arrowhead represents the truncated form of YRS

FIGURE 2

YRS can be cleaved by matrix metalloproteinase (MMP) and elastase present in plasma. (A) Schematic representation of YRS and its various forms. YRS^Mini and YRS^Mini+ are truncated, active forms. (B) Recombinant dual‐tagged YRS (dt‐YRS) was incubated in plasma with or without inhibitors against elastase, MMP, or both for 1 h. Samples were analyzed by Western blotting and the band intensity was quantified using LI‐COR ImageStudio. The intensity of recombinant dt‐YRS that was promptly denatured without incubation was used as a reference for normalization. The experiment was repeated 4 times using plasma derived from different donors, and the mean value with standard deviation is shown. *P < .05 by one‐way ANOVA with Dunn’s multiple comparison test

FIGURE 3

Recombinant tyrosyl‐tRNA synthetase (YRS) can be taken up by platelets when exogenously added to plasma. (A) Recombinant dual‐tagged YRS (dt‐YRS) was incubated in platelet rich plasma for 17 h. Platelets and plasma were separated by centrifugation as described in materials and methods. The samples were analyzed under reduced conditions, and the membrane was blotted with rat anti‐FLAG antibody. (B) The percentage of dt‐YRS taken up by platelets and dt‐YRS remaining in plasma was calculated by quantifying the signal using LI‐COR ImageStudio. The same amount of dt‐YRS incubated in plasma in the presence of inhibitors against elastase and matrix metalloproteinase (MMP) was used as reference. The results quantified from blotting with anti‐FLAG and anti‐V5 antibodies are shown. The experiment was repeated 3 times using plasma derived from different donors, and the mean value with standard deviation are shown. (C) Recombinant YRS^WT, YRS^Mini, YRS^Mini+, and BSA labeled with AlexaFluor647 were incubated with platelet‐rich plasma (PRP) at a concentration of 500 nmol/L for 17 h. After the incubation, platelets were analyzed by flow cytometry. (D) After loading, platelets were lysed and loaded onto SDS‐PAGE under nonreduced conditions, transferred to polyvinylidene difluoride membrane, and AlexaFluor647 signals were detected with LI‐COR Odyssey imaging system. Oligomeric YRS^Mini+ is shown with an asterisk

FIGURE 4

Tyrosyl‐tRNA synthetase (YRS) taken up by platelets is partially stored in the α‐granules. (A) AlexaFluor647‐labeled dt‐YRS and AlexaFluor488‐labeled fibrinogen were incubated with platelet‐rich plasma (PRP) for 17 h. After washing, platelets were resuspended and visualized using confocal microscopy (LSM 880; Carl Zeiss). Scale bar = 5 µm. (B) To evaluate the localization of dual‐tagged YRS (dt‐YRS) in the α‐granules, the colocalized area of dt‐YRS and fibrinogen was divided by the total area of dt‐YRS area using Fiji (n = 8)

FIGURE 5

Tyrosyl‐tRNA synthetase (YRS)^Mini+ activates monocyte via Toll‐like receptors (TLRs)–NF‐κB and type I interferon (IFN‐I) pathways. (A) RAW‐Dual reporter cells (InvivoGen) were incubated with 500 nmol/L of YRS^Mini+, and the activation of TLR‐NFκB pathway and IFN‐I pathway were measured by secreted embryonic alkaline phosphatase (SEAP) and Lucia luciferase (Luc), respectively. The mean and standard deviation of 5 experiments are shown. **P < .01 by unpaired t test. (B) Platelets were pre‐incubated with bovine serum albumin (BSA) or YRS^Mini+ (500 nmol/L) at 37˚C for 17 h. After washing, platelets (2 × 10⁷ platelets) were added onto RAW‐Dual reporter cells seeded at 1 × 10⁵ cells per well in a 96‐well plate, incubated for 21 h and assayed for the SEAP and Luc reporter activities. The experiment was repeated three times using platelets derived from different donors, and the representative result is shown. *P < .05 by unpaired t test

FIGURE 6

Schematic representation of tyrosyl‐tRNA synthetase (YRS) cleavage, storage in platelets, and potential function to activate monocytes. Circulating YRS can be cleaved by elastase or matrix metalloproteinases (MMPs) in plasma, producing a constitutively active form of YRS. YRS^WT and its cleaved forms, YRS^Mini and YRS^Mini+, can be taken up by platelets, mediated by integrins or other receptors, and then partially stored in α‐granules where they are protected from further degradation. Upon activation, platelets may release the stored YRS, thereby activating monocytes/macrophages. Monocyte activation leads to MMP upregulation which may enhance YRS cleavage, producing more active YRS and feed‐forward loop