Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Eur J Pharm Sci. 2019 Sep 9;139:105066. doi: 10.1016/j.ejps.2019.105066

End-point Modification of Recombinant Thrombomodulin with Enhanced Stability and Anticoagulant Activity

Xia Liu 1,2, Mallorie Boron 1, Yu Zhao 1, Xue-Long Sun 1
PMCID: PMC6767613  NIHMSID: NIHMS1539876  PMID: 31513922

Abstract

Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance. The 4, 5, 6 EGF-like domain of TM (TM456) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM456 is a promising anticoagulant candidate but has a very short half-life. Ligation of poly (ethylene glycol) to a bioactive protein (PEGylation) is a practical choice to improve stability, extend circulating life, and reduce immunogenicity of the protein. Site-specific PEGylation is preferred as it could avoid the loss of protein activity resulting from nonspecific modification. We report herein two site-specific PEGylation strategies, enzymatic ligation and copper-free click chemistry (CFCC), for rTM456 modification. Recombinant TM456 with a C-terminal LPETG tag (rTM456-LPETG) was expressed in Escherichia coli for its end-point modification with NH2-diglycine-PEG5000-OMe via Sortase A-mediated ligation (SML). Similarly, an azide functionality was easily introduced at the C-terminus of rTM456-LPETG via SML with NH2-diglycine-PEG3-azide, which facilitates a site-specific PEGylation of rTM456 via CFCC. Both PEGylated rTM456 conjugates retained protein C activation activity as that of rTM456. Also, they were more stable than rTM456 in Trypsin digestion assay. Further, both PEGylated rTM456 conjugates showed a concentration-dependent prolongation of thrombin clotting time (TCT) compared to non-modified protein, which confirms the effectiveness of these two site-specific PEGylation schemes.

Keywords: copper-free click chemistry, Protein C, PEGylation, Sortase A-mediated ligation, thrombin clotting time, Thrombomodulin

Graphical Abstract

graphic file with name nihms-1539876-f0001.jpg

1. Introduction

Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance by activating anticoagulant and anti-inflammatory proteins and simultaneously inactivating pro-coagulant and pro-inflammatory proteins (Dittman and Majerus, 1990). TM serves as a receptor for thrombin and directly inhibits its pro-coagulant activity (direct anticoagulant activity). So far, various forms of TM have been cloned and recombinant expressed for structural and functional study of TM (Wang et al., 2014). Actually, various recombinant TMs show promising antithrombotic and anti-inflammatory activity in vitro and in vivo (Ma et al., 2015). Particularly, recombinant human soluble TM (ART-123) has been approved in Japan for DIC treatment (Ito et al., 2016). However, a phase III trial of ART-123 for DIC in USA was withdrawn in 2019 (van der Poll et al., 2019). It is speculated that recombinant human soluble TM has many domains that have different activities. The last three consecutive EGF-like domains (E456) are responsible for the anticoagulant activity of TM (Zushi et al., 1989). Specifically, the E56 domains of TM bind thrombin, while promoting protein C binding to the E45 domains of TM, together converting protein C to activated protein C (APC) (Fuentes-Prior et al., 2000). Meanwhile, TM binding prevents the binding of thrombin to its pro-coagulant substrates, such as fibrinogen and coagulation factors V and VIII. Therefore, recombinant EGF-like domains 4, 5, 6 of TM (TM456) is a promising anticoagulant candidate. However, its half-life is very short (6-9 min) in animal models (Suzuki et al., 1998).

Modification of protein can expand the protein’s pharmacodynamic and pharmacokinetic properties, such as functional capacity and stability (Pelegri-O'Day et al., 2014). Covalent conjugation of polyethylene glycol (PEG) molecule to protein has been used successfully in approved protein drugs (Turecek et al., 2016). The key point for a practical protein modification is to carry out site-specific chemistry to avoid upsetting the protein’s activity due to random modification (Dozier and Distefano, 2015; Nischan and Hackenberger, 2014). Bio-orthogonal chemistry has been developed for selective and efficient modification of proteins (Willems et al., 2011). The most widely used approaches are azide-reactive Staudinger ligation (Cazalis et al., 2004) and click chemistry (Agard et al., 2006). These approaches benefit from the azide functionality since it is small, bears no overall charge and can be introduced with relative ease to a wide variety of structures, often without disruption to biological behavior of proteins. On the other hand, enzymatic ligation has been explored for selective protein modification in native linkages as well (Wu and Guo, 2012). For example, Sortase A-mediated ligation (SML) has been utilized to site-specifically modify proteins (Tsukiji and Nagamune, 2009). Specifically, Sortase A (SrtA) recognizes the unique pentapeptide LPXTG, where X is variable, of the C-terminal domain of target proteins and transfers the carboxylic group of Thr to a substrate carrying an N-terminal glycine to afford the transpeptidation products. Therefore, SML could be used for direct PEGylation of protein containing LPXTG with glycine-functionalized PEG molecule (Popp et al., 2011).

In this report, we expressed a recombinant rTM456-LPETG with a FLAG fusion tag at the N-terminus and a His-tag at the C-terminus, in which the LPETG motif facilities end-point modification of rTM456 with diglycine chain-end functionalized PEG catalyzed by SrtA, which also has a His-tag at the C-terminus. The His-tag on both rTM456-LPETG and SrtA allows for an easy cleanup of the unreacted rTM456-LPETG and SrtA by nickel affinity column after the SML reaction. In our previous study, we expressed recombinant TM containing an azido-functionalized methionine analog at the C-terminus (rTM456-Azide) for site-specific rTM456 modification (Zhang et al., 2013). However, the low expression yield of the rTM456-Azide with the unnatural amino acid limits its further functional investigation and practical application as well. Alternatively, rTM456 containing a LPETG pentapeptide at C-terminus was expressed in higher yield, through which diGly-PEG3-azide could be conjugated via SML to incorporate azide functionality into the C-terminus of rTM456 (Jiang et al., 2012; Wang et al., 2016). This approach affords the higher overall yield for preparing recombinant TM456 with azide functionality at its C-terminus and thus facilitates a site-selective PEGylation of rTM456 with DBCO chain-end functionalized PEG via Copper-free click chemistry (CFCC) (Fig. 1). Overall, both enzymatic and chemical modification aimed to add PEG to rTM456 at a specific site generated homogenous rTM456 conjugates. These site-specific modification strategies will lay the foundation for developing TM-based antithrombotic agents with enhanced antithrombotic activity and pharmacokinetic properties.

Fig. 1.

Fig. 1.

Open in a new tab

Domain structures of recombinant TM456 fragment and its end-point PEGylation strategy via SML and CFCC.

2. Materials and methods

2.1. Materials and reagents

All solvents, chemicals and reagents were purchased from commercial sources and were used unless otherwise noted. pET39b vector, pET28b vector and competent cells were purchased from EMD Chemicals. Kanamycin sulfate and IPTG (Isopropyl-beta-D-thiogalactopyranoside) were purchased from Calbiochem. The mouse monoclonal antibody specific to human TM, rabbit monoclonal antibody specific to Polyethylene glycol (PEG), goat anti-mouse IgG H&L (HRP) and goat anti-rabbit IgM mu chain (HRP) were purchased from Abcam. Human protein C, human thrombin and human antithrombin III were obtained from Haematologic Technologies Inc. Chromogenic thrombin substrate BIOHPEN CS-01(38) and bovine serum albumin (BSA) were from Fisher Scientific. Heparin, Aprotinin and Trypsin were purchased from Sigma. Pooled normal human plasma was from Innovative Research. NH2-Gly-Gly-PEG3-azide and NH2-Gly-Gly-NH-PEG5000-OMe were purchased from CarboSynUSA. DBCO-Cy5 and DBCO-mPEG5000-OMe were purchased from Click Chemistry Tools.

2.2. Expression and purification of rTM456-LPETG

The recombinant TM contained the EGF domain 4-6 of human thrombomodulin with a FLAG tag on N-terminal, a C-terminal LPETG sequence and a His tag (rTM456) was expressed and purified as previously described with some modification (Jiang et al., 2012). The expression plasmid, pET39b-rTM456 was transformed into E. coli. B834 (DE3) cells for expression. The E. coli. B834 (DE3) cells were incubated in LB medium with 35 μg/mL kanamycin at 37°C, shaking at 150 rpm until an OD600 of 0.8 was reached, and then IPTG was added to a final concentration of 1 mM to induce the overexpression of rTM456 by incubation for 5 h at 37°C. The bacteria were then centrifuged at 8,000g for 5 min, and the cell pellets were collected and purified or stored in −80 °C.

The bacteria pellets were resuspended in 10 mL of ice-cold lysis buffer (20 mM Tris and 150 mM NaCl, pH 8.0) with 1 mM PMSF (phenylmethanesulfonyl fluoride) and lysed by sonication. The extract was collected by centrifugation at 20,000g for 15 min, then loaded into HisTrap FF column (GE Healthcare) charged with Ni2+ ions and eluted with buffer (20 mM Tris, 0.5 M NaCl and 250 mM imidazole, pH 8.0). Pooled fractions containing rTM456-LPETG were collected and then dialyzed by Amicon Ultra Centrifugal Filter (Millipore) with a cutoff molecular weight of 10,000 Da to afford rTM456-LPETG. Sterilized glycerol was added to the rTM456-LPETG solution to a final concentration is 10% to keep rTM456-LPETG activity. After detection of the concentration of this rTM456-LPETG solution by Bradford method, the rTM456-LPETG was aliquoted and stored in −80 °C.

2.3. Expression and purification of soluble SrtA

SrtA with a C-terminal His tag was expressed and purified from E. coli BL21 as previously described (Tsukiji and Nagamune, 2009). Briefly, the E. coli BL21 transformed with the plasmid pET28b-SrtA were grown in LB medium to an OD600 of 0.8 at 37 °C, and protein expression was induced by the addition of IPTG to a final concentration of 1 mM for 5 h. Then the bacteria cells were harvested by centrifugation and resuspended in 10 mL of ice-cold lysis buffer (20 mM Tris and 150 mM NaCl, pH 8.0) with 1mM PMSF, and lysed by sonication. The extract was centrifuged at 20,000g for 15 min, and supernatant was applied onto HisTrap FF column charged with Ni2+ ions and eluted with buffer (20 mM Tris, 0.5 M NaCl and 250 mM imidazole, pH 8.0). The pooled fractions containing SrtA were collected and dialyzed, then kept in 10% glycerol and stored in −80°C until use.

2.4. C-terminal covalent PEGylation of rTM456-LPETG via Sortase A-Mediated Ligation (SML)

rTM456-LPETG (14 μM) was mixed with SrtA (2 μM) and NH2-Gly-Gly-NH-PEG5000-OMe (133 μM) in reaction buffer (20 mM Tris and 150 mM NaCl, pH 8.0). The reaction mixture was then incubated at 37°C for 3 h to obtain the C-terminal PEGylated rTM456 (rTM456-PEG5000-OMe). The obtained rTM456-PEG5000-OMe was purified from the remaining reaction mixture by collecting the pass-through fraction of His SpinTrap column (GE Healthcare). Excess NH2-Gly-Gly-NH-PEG5000-OMe was then removed by Amicon Ultra Centrifugal Filter (Millipore) with a cutoff molecular weight of 10,000 Da and the rTM456-PEG5000-OMe was concentrated. The total amount of pure rTM456-PEG5000-OMe was measured by Bradford assay.

2.5. Incorporation of azide functionality to the C-terminal of rTM456-LPETG and its covalent PEGylation via Copper-free Click Chemistry (CFCC)

rTM456-LPETG (14 μM) was mixed with SrtA (2 μM) and NH2-Gly-Gly-PEG3-azide (3 mM) in reaction buffer (20 mM Tris and 150 mM NaCl, pH 8.0) to first prepare the product of rTM-PEG3-azide. The rTM456-LPETG-PEG3-azide was purified from the remaining reaction mixture by collecting the pass-through fraction of His SpinTrap column (GE Healthcare), and dialyzed by Amicon Ultra Centrifugal Filter (Millipore) with a cutoff molecular weight of 10,000 Da to obtain the pure product. Then, the pure rTM456-LPETG-PEG3-azide (8 μM) and DBCO-PEG5000-OMe (64 μM) were incubated and shaken at room temperature overnight in reaction buffer (20 mM Tris and 150 mM NaCl, pH 8.0). The excess unreacted DBCO-PEG5000 was removed by dialyzing via Amicon Ultra Centrifugal Filter (Millipore) with a cutoff molecular weight of 10,000 Da. The successful formation of rTM456-PEG3-PEG5000-OMe was measured and stored in −20 °C until use.

2.6. SDS-PAGE Coomassie Blue staining and Western blot analysis

The rTM456-LPETG, SrtA, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe were detected in 15% SDS-polyacrylamide gels under reducing conditions by Coomassie Blue staining. Also, Western blot was conducted by transferring the gel onto polyvinylidene difluoride membranes by Trans-Blot Turbo Transfer system (Bio-Rad). The membranes were blocked in 5% non-fat milk in TBST (contained 0.05% of Tween 20) for anti-TM and in 10% BSA in TBST for anti-PEG at room temperature for 1 h with gentle shaking. Primary anti-TM monoclonal antibody (1:2000) or anti-PEG monoclonal antibody (1:1000) in 5% non-fat milk or in 10% BSA was incubated with the membrane at 4 °C overnight. The membranes were then washed with TBST (3 × 10 min) and then incubated with a secondary antibody (goat anti-mouse IgG HRP or goat anti-rabbit IgM HRP 1:5000) in 5% non-fat milk for 1 h at room temperature. After washing with TBST (3 × 10 min), protein bands on the membrane were visualized using an enhanced chemiluminescence (ECL) solution (Thermo fisher).

2.7. Stability of rTM456-LPETG and PEGylated rTM456-LPETG conjugates under proteolytic digestion by Trypsin

Briefly, 10 μg of rTM456-LPETG, rTM456-PEG5000-OMe or rTM456-PEG3-PEG5000-OMe, was incubated with 20 μg of Trypsin in the reaction buffer (20 mM Tris and 150 mM NaCl, pH 8.0) at 37 °C for 0, 1, 2, 3, 4, 5 and 6 h, respectively. Then the reaction mixture was supplemented with SDS-PAGE reducing sample buffer, heated to 100 °C for 10 min, and then electrophoresed on SDS-PAGE (15% acrylamide) gels. The amount of undigested protein in a given lane was determined by Western blot by anti-TM and anti-PEG antibodies, respectively. Image J software was employed to calculate the band intensity of rTM456-LPETG, PEGylated rTMs and their remains after Trypsin digestion in three independent experiments.

2.8. Protein C activation activity assay of rTM456-LPETG and PEGylated rTM456-LPETG conjugates with or without Trypsin digestion

The cofactor activities of rTM456-LPETG, rTM456-PEG5000-OMe, rTM456-PEG3-PEG5000-OMe and their Trypsin digestion products were assessed by protein C activation assay as previously described with some modification (Popp et al., 2011). Briefly, rTM456-LPETG and PEGylated rTMs obtained from the above conjugation were added into assay buffer (20 mM Tris, 150 mM NaCl, 5 mM CaCl2 and 0.1% BSA, pH 8.0) containing 200 nM of human protein C (PC) to a final concentration of 50 nM, and the reaction volumes were adjusted to 100 μL. The PC activation was initiated with the addition of human α-thrombin to a final concentration of 10 nM. After incubation for 1 h at 37 °C while shaking, the PC activation was terminated by addition of 30 μL human antithrombin III (1 mg/mL) and 2 μL heparin (10 U/mL) for 10 min at 37 °C while shaking. The activated PC was measured by incubation with chromogenic thrombin substrate BIOHPEN CS-01(38) (0.5 mM) for 20 min at 37 °C to determine its enzymatic activity. The UV absorbance at 405 nm was measured.

For protein C activation assays, Trypsin digestion reaction of rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe were stopped by adding 10 μg/mL of Aprotinin in the reaction buffer to avoid the effect of Trypsin on protein C activation. All protein C activation assays were conducted as above with the presence of Aprotinin.

2.9. Thrombin clotting time (TCT) assay of PEGylated rTM456-LPETG conjugates

rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe (0.002 mg/mL, 0.004 mg/mL and 0.005 mg/mL) in normal human plasma were prepared, and similar concentrations of NH2-Gly-Gly-NH-PEG5000-OMe and DBCO-PEG5000-OMe were prepared as controls. The clotting of each sample (100 μL) was triggered by adding 200 μL of human thrombin (5 U/mL). The TCT was measured by BBL fibrometer (Becton-Dickinson). The initial point was obtained with buffer (20 mM Tris and 150 mM NaCl, pH 8.0) alone.

2.10. Statistical analysis

All data is presented as the means ± the standard error of the mean. Differences between experimental groups were analyzed using one-way ANOVA with an independent t-test. All reported p values were two-tailed, and p values of less than 0.05 were considered statistically (*, p <0.05; **, p < 0.01; ***, p < 0.001). Statistical analyses were performed with SPSS 16.0 software.

3. Results and Discussion

Translational incorporation of functional amino acids has been a very promising strategy to improve protein physicochemical and biological properties and create functional groups for site-specific protein modification applications (Boutureira and Bernardes, 2015; Hendrickson et al., 2004).Azidohomolananine can serve as a methionine surrogate and can be incorporated into proteins, providing an azidohomolananine-containing protein for chemo-selective modification (Kiick et al., 2002). In our preliminary studies, we have expressed recombinant TM456 construct containing an azido-functionalized methionine analog at the C-terminus for site-specific immobilization (Sun et al., 2006) and modification (Zhang et al., 2013) via bio-orthogonal chemistry. However, low translational efficiency of the unnatural amino acid azidohomolananine is the limiting factor for larger scale preparation, modification and functional study of the protein. Enzymatic modification provides another robust tool for site-specific protein modification (Sunbul and Yin, 2009). In our preliminary study, we expressed recombinant TM456 containing a LPETG pentapeptide at the C-terminus (rTM456-LPETG) for enzymatic end-point immobilization (Jiang et al., 2012) and modification (Jiang et al., 2014) via SML. Therefore, we speculated that rTM456-LPETG is versatile for site-specific PEGylation of rTM456 directly with glycine chain-end functionalized PEG via SML. In this study, the rTM456-LPETG was prepared by expression in E. coli B834 (DE3) cells with TM456GGMLPETG and all tags plasmid. After purification, the protein integrity was confirmed by SDS-PAGE (Fig. 3A). Optimization for the large-scale expression and efficient purification was conducted to provide sufficient amount of rTM456-LPETG for its PEG conjugate preparation and stability and activity studies. In addition, an azide functionality was easily incorporated to rTM456-LPETG with a glycine molecule containing azide via SML, which facilitates a site-specific PEGylation via CFCC.

Fig. 3.

Fig. 3.

Open in a new tab

Characterization of PEGylated rTM456 conjugates obtained from site-specific PEGylation of rTM456-LPETG via Click Chemistry (CC) and Srtase A-mediated Ligation (SML). (A) SDS-PAGE from Coomassie blue staining and Western blot with both anti-TM antibody (B) and anti-PEG antibody (C).

3.1. C-terminal covalent PEGylation of rTM456-LPETG via SML

It has been known that a diglycine at the N-terminus of a nucleophile shows the highest yield for Sortase-catalyzed transpeptidation and the addition of any more glycines has no significant effect on the reaction rate (Tsukiji and Nagamune, 2009). Therefore, diglycine was chosen as the nucleophile of the PEG molecule in our study. Specifically, N-terminal diglycine-containing PEG5000-OMe (NH2-Gly-Gly-NH-PEG5000-OMe) was used for site-specific PEGylation of rTM456-LPETG via SML. In this study, the His-tag at the C-terminus of rTM456-LPETG and SrtA allows for an easy removal of the unreacted rTM456-LPETG-His-tag and SrtA-His-tag by a nickel affinity column, while the PEGylated product was obtained directly by passing the reaction mixture through the nickel column as it does not have the His-tag due to the cleavage of T/G in LPETG motif after the SML reaction (Fig. 2). Briefly, rTM456-LPETG was incubated with SrtA and NH2-Gly-Gly-NH-PEG5000-OMe in reaction buffer for 3 h at 37°C followed by passing through the nickel affinity column to afford the C-terminal PEGylated rTM456-LPETG (rTM456-PEG5000-OMe). The resultant rTM456-PEG5000-OMe was analyzed by SDS-PAGE and Western blot (Fig. 3). As shown in the SDS-PAGE gel resulting from Coomassie blue staining (Fig. 3A), a major band with larger molecular weight formed, indicating the successful PEGylation via SML. Also, Western blot with mouse monoclonal antibody specific to human TM (Fig. 3B) and rabbit monoclonal antibody specific to PEG molecule (Fig. 3C) confirmed the PEGylated rTM456-LPETG conjugates. From the Western blot analysis with anti-TM antibody, two extra bands with much higher molecular weight were observed for the SML reaction products (Fig. 3B). The top band might be the dimer of the rTM456 formed during the reaction or purification step. Another one might be the rTM456-LPETG-acyl-SrtA intermediate that stays unreacted with the Gly2-PEG5000-OMe nucleophile. This indicates that the larger substrate may have steric issue that prevents it reaching and reacting with the acyl-SrtA intermediate, which is in agreement with our previous finding (Sun et al., 2006). This might be the drawback of SML with larger molecules in conjugation.

Fig. 2.

Fig. 2.

Open in a new tab

Site-Specific PEGylation via SML and rapid purification flowchart of SML product via Nickel affinity column.

3.2. C-terminal covalent PEGylation of rTM456-LPETG via CFCC

Next, we investigated an alternative PEGylation of rTM456 containing an azide at the C-terminus with DBCO-PEG5000-OMe via CFCC, which is very efficient in protein modification (Jang et al., 2012). First, an azide functionality was incorporated into rTM456-LPETG with diglycine-PEG3-azide via SML by an efficient purification flow as shown in Figure 2. Then, the pure rTM456-PEG3-azide was incubated with DBCO-PEG5000-OMe in the same reaction buffer for 12 h at room temperature. The PEGylated rTM456 product was confirmed by SDS-PAGE from Coomassie blue staining (Fig. 3A) and Western blot with both anti-TM (Fig. 3B) and anti-PEG antibodies (Fig. 3C). These results demonstrate that CFCC-mediated PEGylation provides a site-specific protein modification. There is an extra band around 16 kDa observed from the CFCC-mediated PEGylation on SDS-PAGE. This may be the degraded fragment from rTM456 as it is unstable during 12 h long time of reaction. This extra band was not confirmed in the Western blot (Supporting Information, S1). This may be due to that the anti-TM antibody does not recognize this small fragment. Therefore, we conducted the reaction for 3 h at room temperature. As a result, there was no this extra band formed in addition to the PEGylated product (Supporting Information, S2). In addition, the unreacted rTM456 was observed in SDS-PAGE but not in Western blot because that only one sixth amounts of the proteins were used in Western blot. However, it was observed when larger amount of the protein was used (Supporting Information, S3).

3.3. Stability of PEGylated rTM456-LPETG conjugates under proteolytic digestion by Trypsin

As described above, PEGylation could enhance the protein’s stability. In this study, a Trypsin digestion assay was conducted to evaluate the PEGylation effect on the stability of rTM456-LPETG, which is often used to test the resistance to proteolytic degradation (Guan et al., 2004; Veronese et al., 1996). Briefly, rTM456-LPETG, rTM456-PEG5000-OMe or rTM456-PEG3-PEG5000-OMe was incubated with Trypsin in the reaction buffer at 37 °C for 0, 1, 2, 3, 4, 5 and 6 h, respectively. Then, all samples were electrophoresed on a SDS-PAGE gel and the undigested protein were determined by Western blot. As a result, unmodified rTM456-LPETG was completely digested at 4 h, while rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe were almost unchanged until 6 h after Trypsin treatment (Fig. 4B and 5A). Interestingly, both PEGylated rTM456-LPETG conjugates showed an immediate breakdown at 0 h upon Trypsin treatment, which cleaved a small part from the N-terminal as confirmed by Western blot with both anti-TM antibody and anti-PEG antibody. Both SML-mediated and CCFC-mediated PEGylation products showed the same trend of protein stability against Trypsin digestion as shown in Western blot with both anti-TM antibody (Fig. 4B) and anti-PEG antibody (Fig. 5). rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe were stable in the reaction buffer (Fig. 4A). All these results indicate that PEGylation of rTM456-LPETG enhanced protein stability.

Fig. 4.

Fig. 4.

Open in a new tab

Stability of rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe. (A) rTM456-LPETG, rTM-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe without Trypsin, (B) rTM456-LPETG, rTM-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe with Trypsin examined by Western blot with mouse monoclonal anti-TM antibody. Gel intensities are normalized to 0 h time of Trypsin digestion.

Fig. 5.

Fig. 5.

Open in a new tab

Stability of rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe upon Trypsin digestion. (A) Western blot with rabbit monoclonal anti-PEG antibody, (B) gel intensity of western blot, gel intensities are normalized to 0 h time of Trypsin digestion.

3.4. Protein C activation activity assay of PEGylated rTM456-LPETG conjugates with or without Trypsin digestion

In order to know whether PEGylation of rTM456 affects rTM456’s anticoagulant activity, protein C activation activities of rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe were measured. Briefly, rTM456-LPETG, rTM456-PEG5000-OMe or rTM456-PEG3-PEG5000-OMe was first incubated with thrombin and protein C to generate activated protein C (APC). Then, APC was measured by quantifying the hydrolysis product of the chromogenic substrate at 405 nm. As shown in Figure 6A, rTM-PEG5000-OMe and rTM-PEG3-PEG5000-OMe conjugates showed a similar level of protein C activation activity to rTM456-LPETG, indicating that the PEGylation didn’t hamper the rTM456-LPETG activity. These results demonstrate that the site specific PEGylation of rTM456 did not affect its anticoagulant activity, even using a two-step reaction involving SML and CFCC. In addition, protein C activation capability was monitored for all Trypsin digestion sample solutions as described above. As a result, unmodified rTM456-LPETG lost 50% protein C activation activity upon 4 h Trypsin digestion (Fig. 6B), while PEGylated rTM456-LPETG rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe maintained protein C activation activity after 6 h Trypsin treatment (Fig. 6C and 6D), which are well agreement with the Western blot results above (Fig. 4 and 5). These results further confirm the enhanced stability of rTM456 through PEGylation.

Fig. 6.

Fig. 6.

Open in a new tab

Protein C activation activity. (A) rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe without Trypsin (activities are normalized to rTM456), (B) rTM456-LPETG, (C) rTM456-PEG5000-OMe and (D) rTM456-PEG3-PEG5000-OMe with Trypsin (activities are normalized to 0 h time of Trypsin digestion). p Values of less than 0.05 were considered statistically (*, p <0.05; **, p < 0.01; ***, p < 0.001)

3.5. Thrombin clotting time (TCT) assay of PEGylated rTM456-LPETG conjugates

The thrombin clotting time (TCT) was measured for rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe, respectively, in order to determine the effect of PEGylation on their anticoagulation activity. Different concentrations of rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe were mixed with human plasma, and then human thrombin was added to trigger the clotting. rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe showed a concentration-dependent clotting time prolongation (Fig. 6), while the control PEG molecules did not show any activity. There was a clear difference in clotting time for rTM456-LPETG and PEGylated rTM456-LPETG conjugates. At higher concentrations, PEGylated rTM456-LPETG conjugates showed a longer TCT compared to unmodified rTM456-LPETG. This result is different from the in vitro protein C activation assay above, in which rTM456-LPETG and PEGylated rTM456-LPETG conjugates showed the same level of APC generation activity. This can be explained that the TCT assay was conducted in human plasma, in which un-modified rTM456-LPETG may be unstable due to enzymatic degradation. However, PEGylated rTM456-LPETG may be resistant to the enzymatic degradation. This result indicates possible in vivo stability of the PEGylated rTM456-LPETG conjugates, which is expected in a future animal study.

4. Conclusions

Protein modification warrants improved biological activity and pharmacokinetic properties. Two successful site-specific strategies for PEGylation of rTM456 were demonstrated via Sortase A-mediated ligation (SML) and copper-free click chemistry (CFCC). The PEGylation by both chemistries did not upset the rTM456 biological activities, such as protein C activation activity and anti-thrombin activity. Both reactions have advantages of mild reaction conditions and high yields. In addition, the His-tag of both rTM456 and Sortase A allows for a quick purification of the PEGylated product via a nickel affinity column. We anticipate that the PEGylated rTM456 will show an enhanced pharmacokinetic property in vivo. These strategies are viable for modification of a variety of proteins with different molecules for elevated activity and stability, novel functions, and other specific properties of interest as well.

Supplementary Material

1

Fig. 7.

Fig. 7.

Open in a new tab

Thrombin clotting time (TCT) of rTM456-LPETG, rTM456-PEG5000-OMe and rTM456-PEG3-PEG5000-OMe.

Acknowledgments

Funding

This work was supported by grants from the NIH (1R15HL138544-01, X.-L. Sun), National Science Foundation MRI Grant (CHE-1126384, X.-L. Sun) and Faculty Research Development Grant and the Research Fund from the Center for Gene Regulation in Health and Disease (GRHD) at Cleveland State University. X. Liu appreciates the China Oversea Scholar Award from China Scholarship Council.

Footnotes

Declaration of interest

None

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Agard NJ, Baskin JM, Prescher JA, Lo A, Bertozzi CR, 2006. A comparative study of bioorthogonal reactions with azides. ACS Chem Biol. 1, 644–648. [DOI] [PubMed] [Google Scholar]
  2. Boutureira O, Bernardes GJ, 2015. Advances in chemical protein modification. Chem Rev. 115, 2174–2195. [DOI] [PubMed] [Google Scholar]
  3. Cazalis CS, Haller CA, Sease-Cargo L, Chaikof EL, 2004. C-terminal site-specific PEGylation of a truncated thrombomodulin mutant with retention of full bioactivity. Bioconjug Chem. 15, 1005–1009. [DOI] [PubMed] [Google Scholar]
  4. Dittman WA, Majerus PW, 1990. Structure and function of thrombomodulin: a natural anticoagulant. Blood. 75, 329–336. [PubMed] [Google Scholar]
  5. Dozier JK, Distefano MD, 2015. Site-Specific PEGylation of Therapeutic Proteins. Int J Mol Sci. 16, 25831–25864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fuentes-Prior P, Iwanaga Y, Huber R, Pagila R, Rumennik G, Seto M, Morser J, Light DR, Bode W, 2000. Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex. Nature. 404, 518–525. [DOI] [PubMed] [Google Scholar]
  7. Guan R, Sun XL, Hou S, Wu P, Chaikof EL, 2004. A glycopolymer chaperone for fibroblast growth factor-2. Bioconjug Chem. 15, 145–151. [DOI] [PubMed] [Google Scholar]
  8. Hendrickson TL, de Crecy-Lagard V, Schimmel P, 2004. Incorporation of nonnatural amino acids into proteins. Annu Rev Biochem. 73, 147–176. [DOI] [PubMed] [Google Scholar]
  9. Itoh S, Shirabe K, Kohnoe S, Sadanaga N, Kajiyama K, Yamagata M, Anai H, Harimoto N, Ikegami T, Yoshizumi T, Maehara Y 2016. Impact of Recombinant Human Soluble Thrombomodulin for Disseminated Intravascular Coagulation. Anticancer Res. 36, 2493–2496. [PubMed] [Google Scholar]
  10. Jang S, Sachin K, Lee HJ, Kim DW, Lee HS, 2012. Development of a simple method for protein conjugation by copper-free click reaction and its application to antibody-free Western blot analysis. Bioconjug Chem. 23, 2256–2261. [DOI] [PubMed] [Google Scholar]
  11. Jiang R, Wang L, Weingart J, Sun XL, 2014. Chemoenzymatic bio-orthogonal chemistry for site-specific double modification of recombinant thrombomodulin. Chembiochem. 15, 42–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jiang R, Weingart J, Zhang H, Ma Y, Sun XL, 2012. End-point immobilization of recombinant thrombomodulin via sortase-mediated ligation. Bioconjug Chem. 23, 643–649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kiick KL, Saxon E, Tirrell DA, Bertozzi CR, 2002. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc Natl Acad Sci U S A. 99, 19–24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ma CY, Chang WE, Shi GY, Chang BY, Cheng SE, Shih YT, Wu HL 2015. Recombinant thrombomodulin inhibits lipopolysaccharide-induced inflammatory response by blocking the functions of CD14. J Immunol. 194, 1905–1915. [DOI] [PubMed] [Google Scholar]
  15. Nischan N, Hackenberger CP, 2014. Site-specific PEGylation of proteins: recent developments. J Org Chem. 79, 10727–10733. [DOI] [PubMed] [Google Scholar]
  16. Pelegri-O'Day EM, Lin EW, Maynard HD, 2014. Therapeutic protein-polymer conjugates: advancing beyond PEGylation. J Am Chem Soc. 136, 14323–14332. [DOI] [PubMed] [Google Scholar]
  17. Popp MW, Dougan SK, Chuang TY, Spooner E, Ploegh HL, 2011. Sortase-catalyzed transformations that improve the properties of cytokines. Proc Natl Acad Sci U S A. 108, 3169–3174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sun XL, Stabler CL, Cazalis CS, Chaikof EL, 2006. Carbohydrate and protein immobilization onto solid surfaces by sequential Diels-Alder and azide-alkyne cycloadditions. Bioconjug Chem. 17, 52–57. [DOI] [PubMed] [Google Scholar]
  19. Sunbul M, Yin J, 2009. Site specific protein labeling by enzymatic posttranslational modification. Org Biomol Chem. 7, 3361–3371. [DOI] [PubMed] [Google Scholar]
  20. Suzuki M, Mohri M, Yamamoto S, 1998. In vitro anticoagulant properties of a minimum functional fragment of human thrombomodulin and in vivo demonstration of its benefit as an anticoagulant in extracorporeal circulation using a monkey model. Thromb Haemost. 79, 417–422. [PubMed] [Google Scholar]
  21. Tsukiji S, Nagamune T, 2009. Sortase-mediated ligation: a gift from Gram-positive bacteria to protein engineering. Chembiochem. 10, 787–798. [DOI] [PubMed] [Google Scholar]
  22. Turecek PL, Bossard MJ, Schoetens F, Ivens IA, 2016. PEGylation of Biopharmaceuticals: A Review of Chemistry and Nonclinical Safety Information of Approved Drugs. J Pharm Sci. 105, 460–475. [DOI] [PubMed] [Google Scholar]
  23. van der Poll T 2019. Recombinant Human Soluble Thrombomodulin in Patients With Sepsis-Associated CoagulopathyAnother Negative Sepsis Trial? JAMA. 321, 1978–1980. [DOI] [PubMed] [Google Scholar]
  24. Veronese FM, Monfardini C, Caliceti P, Schiavon O, Scrawen MD, Beer D, 1996. Improvement of pharmacokinetic, immunological and stability properties of asparaginase by conjugation to linear and branched monomethoxy poly(ethylene glycol). J Control Release. 40, 199–209. [Google Scholar]
  25. Wang L, Jiang R, Sun XL, 2014. Recombinant thrombomodulin of different domains for pharmaceutical, biomedical, and cell transplantation applications. Med Res Rev. 34, 479–502. [DOI] [PubMed] [Google Scholar]
  26. Wang L, Jiang R, Wang L, Liu Y, Sun XL, 2016. Preparation of chain-end clickable recombinant protein and its bio-orthogonal modification. Bioorg Chem. 65, 159–166. [DOI] [PubMed] [Google Scholar]
  27. Willems LI, van der Linden WA, Li N, Li KY, Liu N, Hoogendoorn S, van der Marel GA, Florea BI, Overkleeft HS, 2011. Bioorthogonal chemistry: applications in activity-based protein profiling. Acc Chem Res. 44, 718–729. [DOI] [PubMed] [Google Scholar]
  28. Wu Z, Guo Z, 2012. Sortase-Mediated Transpeptidation for Site-Specific Modification of Peptides, Glycopeptides, and Proteins. J Carbohydr Chem. 31, 48–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhang H, Weingart J, Jiang R, Peng J, Wu Q, Sun XL, 2013. Bio-inspired liposomal thrombomodulin conjugate through bio-orthogonal chemistry. Bioconjug Chem. 24, 550–559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zushi M, Gomi K, Yamamoto S, Maruyama I, Hayashi T, Suzuki K, 1989. The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity. J Biol Chem. 264, 10351–10353. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1539876-supplement-1.pdf (203KB, pdf)

RESOURCES