Fig 1.
Schematic representation of tissue plasminogen activators (PA) mode of action and functional domains.
Plasminogen activators cleave the zymogen plasminogen to form the serine protease, plasmin. The fibrinolytic enzyme plasmin breaks the cross-links between fibrin molecules, which are the structural support of the blood clot generating fibrin degradation products (FDP). Alteplase (tPA) has 5 domains: A finger domain, an epidermal growth factor (EGF)-like domain, 2 kringle domains, and a serine protease proteolytic domain. Reteplase (rPA) is a deletion of tPA consisting in only the kringle 2 domain and the serine protease proteolytic domain. Protein glycosylation sites are indicated.
Fig 2.
Schematic representation of the expression vectors used in this investigation.
The binary vector (p31) with a barley α-amylase signal peptide for apoplast targeting was used to clone rPA fused to the monomeric red fluorescent protein (mRFP). The modified TMV-based MagnICON vector (pICHα26211α) was used to clone rPA-Fc-fusion proteins. Common features are: g7T: Agrobacterium gene 7 terminator; P35S: Cauliflower mosaic virus (CaMV) 35S promoter; PAct2: Arabidopsis actin 2 promoter; RdRpTVCV: RNA-dependent RNA polymerase from Turnip vein clearing virus; MP: Movement protein from the tobacco mosaic virus; SP: Barley α-amylase signal peptide for apoplast targeting; Tnos: Nopalin synthase gene terminator; 3’UTR: TVCV Pol. 3’-untranslated region; LB: Left border; RB: Right border. The expected protein molecular weight is shown for each fusion (for full peptide sequence see also S1 Fig). Inset show a schematic representation of the oligomerization of the rPA fusions.
Fig 3.
Subcellular localization of rPA-mRFP.
Subcellular localization of rPA-mRFP was determined by live-cell imaging of infiltrated N. benthamiana leaf epidermal cells. rPA-mRFP was transiently co-expressed in N. benthamiana with EGFP-LTI6b (PM-GFP), a plasma membrane marker, and analyzed at 2dpi. The fluorescence (magenta) signal is typical of secreted proteins. Superposed images (merged) show significant co-localization of rPA-mRFP and PM-GFP. Scale bar is indicated.
Fig 4.
Expression of dimeric rPA-Fc fusions in N. benthamiana.
(A) Total soluble proteins extracts (TSP) from N. benthamiana ΔXF leaves expressing rPA-HFc co-expressed with (+) or without (-) p19 were analysed in 12% SDS-PAGE under reducing conditions with anti-hIgG antibodies. (B) TSP and apoplastic fluids (AF) from N. benthamiana ΔXF leaves expressing rPA-LFc were analysed in 12% SDS-PAGE under reducing conditions with anti-hIgG antibodies (left panel). rPA-LFc and tPA (Alteplase) were also analysed with anti-tPA antibodies (right panel). (C) Protein A purified rPA-LFc was incubated with acidic AF isolated from N. benthamiana wild type plants. Samples were analysed at different time points with anti-hIgG antibodies. Incubation with AF previously boiled to inactivate plant proteases served as a negative control. (D) TSP from N. benthamiana ΔXF leaves expressing rPA-LFc were analysed in 8% native PAGE with anti-hIgG antibodies (left panel) and protein A purified rPA-LFc was stained with Commassie brilliant blue under reducing conditions (CBB) (right panel). Protein size markers are shown in kilo Dalton (kDa).
Fig 5.
Expression of monomeric rPA-Fc fusions in N. benthamiana.
(A) Total soluble proteins extracts (TSP) from N. benthamiana ΔXF leaves expressing mFc (non-fused) were analysed in 12% SDS-PAGE under reducing (R) and non-reducing (NR) conditions and in native PAGE with anti-hIgG antibodies. (B) TSP from N. benthamiana ΔXF leaves expressing rPA-mFc co-expressed with (+) or without (-) p19 were analysed in 12% SDS-PAGE under reducing conditions with anti-hIgG antibodies at different days post infiltration (dpi). Ponceau staining shows similar amounts of protein loaded. (C) rPA-mFc was expressed in N. benthamiana ΔXF with (+) or without (-) co-expression of an oligosaccharyltransferase from Leishmania major (OST). TSP were analysed in 12% SDS-PAGE under reducing conditions with anti-hIgG antibodies. (D) rPA-mFc purified out of TSP with Protein G was stained with Commassie brilliant blue under reducing conditions (CBB) (left panel) and analysed in 8% SDS-PAGE under reducing (R) and non-reducing (NR) conditions with anti-hIgG antibodies (right panel). Protein size markers are shown in kilo Dalton (kDa).
Fig 6.
Glycosylation status of plant-derived rPA-LFc.
(A) Purified rPA-LFc expressed in N. benthamiana ΔXF plants was digested with Endo H and PNGase F and analysed with anti-hIgG antibodies. Undigested rPA-LFc served as control (C). (B) Purified rPA-LFc expressed in N. benthamiana wild type (WT), in ΔXF; in ΔXF overexpressing the human β1,4-galactosyltransferase (ΔXFGalT) and in ΔXF overexpressing the entire protein sialylation pathway (ΔXFSIA) were analysed for the presence of Lewis-A epitopes (JIM84), terminal β1,4-galactose (RCA) and terminal α2,6-sialic acid (SNA). tPA (Alteplase) was used as a control.
Fig 7.
Glycan analysis of CHO-derived tPA and plant-derived rPA-Fc fusions.
Tryptic glycopeptides of tPA (Alteplase) and ΔXF-rPA-LFc were analysed by LC-ESI-MS. N-glycosylation profiles ([M+3H]3+ and [M+4H]4+) are shown for tPA glycosites Asn184: YSSEFCSTPACSEGNSDCYFGNGSAYR and Asn448: CTSQHLLNR and for ΔXF-rPA-LFc glycosites Asn12: SYQGNSDCYFGNGSAYR and Asn278: CTSQHLLNR. Glycosylation of tPA at Asn117 glycosite is shown in S6B Fig. Total glycans released from ΔXFSIA-rPA-mFc co-expressed with mammalian α1,6-fucosyltransferase (FUT8) were analysed by MALDI-MS. The major glycosylated peaks are depicted. The assigned N-glycan structures were labelled according to the ProGlycAn nomenclature (http://www.proglycan.com/). A cartoon illustration highlights the main glycan structure detected for each peptide (http://www.functionalglycomics.org/).
Fig 8.
Functional assays of rPA-Fc fusions expressed in ΔXF plants.
(A) The proteolytic activity of rPA-LFc was assessed by clot lysis assay. Clotted blood plasma was poured on Petri dishes (left) or in glass tubes (right) and purified rPA-LFc was spotted at different concentrations (μg). tPA (Alteplase) was used as a positive control and Fc as a negative control. Fibrin digestion can be visualized by grey spots (left panel) or solubilized plasma (right panel). (B) Plasminogen zymography of purified plant-derived rPA-Fc. Staining of 8% SDS-PAGE with Coomassie brilliant blue shows the proteolytically cleaved sites as white clear smears on a dark blue background. 1. rPA-mFc and 2. rPA-LFc. Proteolytic activity is also observed on the positive control, (C+, Alteplase) but not on the negative control (C-, IgG1). Protein size markers are shown in kilo Dalton (kDa).