Letter pubs.acs.org/macroletters
Synthesis of an End-to-End Protein−Glycopolymer Conjugate via Bio-Orthogonal Chemistry Hailong Zhang, Jacob Weingart, Valentinas Gruzdys, and Xue-Long Sun* Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, Ohio 44115, United States S Supporting Information *
ABSTRACT: We report the synthesis of an end-to-end protein− glycopolymer conjugate, namely, site-specific modification of recombinant thrombomodulin at the C-terminus with a chainend-functionalized glycopolymer. Thrombomodulin (TM) is an endothelial membrane glycoprotein that acts as a major cofactor in the protein C anticoagulant pathway. To closely mimic the glycoprotein structural feature of native TM, we proposed a sitespecific glyco-engineering of recombinant TM with a glycopolymer. Briefly, recombinant TM containing the epidermal growth factor (EGF)-like domains 4, 5, and 6 (rTM456) and a C-terminal azidohomoalanine was modified with a dibenzylcyclooctyne (DBCO) chain-end-functionalized glycopolymer via copper-free click chemistry to afford the end-to-end TM−glycopolymer conjugate. The TM glycoconjugation was confirmed with SDS-PAGE, Western blot, and protein C activation assay, respectively. The reported site-specific end-to-end protein glycopolymer conjugation approach facilitates uniform glycoconjugate formation via biocompatible chemistry and in high efficiency providing a rational strategy for generating an rTM-based anticoagulant agent.
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coagulation factors Va and VIIIa, providing an essential feedback mechanism to prevent excessive coagulation. At the same time, TM-bound thrombin is unable to cleave fibrinogen or to activate platelets, diminishing its procoagulant activity. The structural domains of TM necessary for protein C activation have been resolved. The fifth and sixth epidermal growth factor (EGF)-like domains of TM represent the thrombin binding site, while the fourth EGF-like domain is responsible for protein C activation.10,11 Consequently, recombinant TM containing the EGF-like domains 4, 5, and 6 (rTM456) has been considered to be a potent anticoagulant candidate.13−16 However, rTM456 has a very short half-life time (6−9 min) in animals, as compared to that of 5 h for recombinant human soluble TM (rhsTM),17 which limits its usage as an anticoagulant. Native TM is a glycoprotein that contains O-glycans attached to the domain next to EGF domains, which contributes to TM and thrombin binding, protein stability, and plasminogen activation activities.18 Therefore, we hypothesized that modification of recombinant rTM456 with a glycopolymer may improve recombinant TM’s in vivo activity, such as increasing TM stability and extending its plasma half-life time. The great challenges for protein modification are to carry out biocompatible and site-specific reaction to a protein, to achieve uniform protein conjugate, and to avoid diminishing its
rotein modification with polymers has been a versatile approach for expanding the protein’s functional capacity, especially for enhancing its biological activity and stability. This has led to a wealth of applications, particularly in the area of biomedicine including biopharmaceuticals, drug delivery, and tissue engineering.1 For example, PEGylation, covalent attachment of polyethylene glycol (PEG) to proteins, has been a practical approach to prolong its serum half-life time and enhance the pharmacokinetics of therapeutic proteins.2 An alternative approach is glyco-engineering aimed at adding carbohydrates to proteins to alter their pharmacodynamics and pharmacokinetic properties, such as increasing in vivo activity and prolonging the duration of action of proteins, and it has become a developing field in regards to the enhancement of protein therapeutics.3,4 A successful example was the development of darbepoetin alfa, an erythropoietin analogue that contains additional carbohydrates, which resulted in a 3-fold increase in serum half-life time and increased in vivo activity.5 Synthetic glycopolymers with multiple copies of sugar moieties have shown very promising results when used to mimic natural oligosaccharides.6 Recently, variations of synthetic glycopolymers have been explored for therapeutic applications.7,8 Thrombomodulin (TM), a membrane glycoprotein predominately expressed on endothelium, plays essential roles in keeping local hemostatic balance by serving as a cofactor for the activation of anticoagulant proteins. Particularly, TM modulates the activity of thrombin from a procoagulant to an anticoagulant protease.9−12 When bound to TM on the endothelial surface, thrombin activates plasma protein C, and the activated form of protein C (APC) selectively inactivates © XXXX American Chemical Society
Received: November 10, 2015 Accepted: December 15, 2015
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DOI: 10.1021/acsmacrolett.5b00805 ACS Macro Lett. 2016, 5, 73−77
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ACS Macro Letters
Figure 1. Synthesis of rTM456−glycopolymer conjugate via recombinant expression and copper-free click chemistry conjugation. TM: thrombomodulin; rTM456: recombinant TM containing EGF-like domains 4, 5, and 6; IIa: thrombin; PC: Protein C.
Figure 2. Glyco-modification of recombinant TM456-azide through copper-free click chemistry and SDS-PAGE (12%) characterization of the glycocojugate: (A) Glyco-staining, (B) Coomassie blue staining: Lane 1: His-rTM456-N3 treated with DBCO-PEG4-CONH-Ph-β-Gal; Lane 2: HisrTM456 treated with DBCO-PEG4-CONH-Ph-β-Gal; and Lane 3: His-rTM456-N3 treated with pNH2-Ph-Gal.
derivative at the C-terminus with a chain-end-functionalized glycopolymer via copper-free click chemistry to afford an endto-end protein−glycopolymer conjugate that closely mimics the native TM structure (Figure 1). The proposed biomimetic rTM456−glycopolymer conjugate is expected to be a potential anticoagulant with enhanced pharmacokinetic properties. A truncated TM fragment containing EGF4−6 domains with the insertion of a C-terminal azide-containing non-natural methionine analogue (rTM456-Azide) was chosen as our target antithrombotic protein for site-specific conjugation (Figure 1). Specifically, the recombinant TM mutant-containing amino acids 349−492 (from full TM sequence) with a Met-388-Leu substitution and a C-terminal linker GlyGlyMet-N3 were constructed using site-directed mutagenesis.16 To test whether rTM456-Azide could be conjugated to a dibenzylcyclooctyne (DBCO) sugar derivative, our initial glyco-modification of the protein was carried out with DBCO-containing galactose via copper-free click chemistry (DBCO-PEG4-CONH-Ph-β-Gal, see Supporting Information) in PBS buffer (pH 7.4) for 12 h at room temperature, followed by glyco-staining and Coomassie blue staining to confirm the reaction product, respectively. As a negative control, rTM456 lacking an azide group was used in this experiment. The use of the glyco-staining for carbohydrate allowed us to detect the attached glycopolymer based on a carbohydrate-specific periodic acid Schiff (PAS) staining
biological activity, which are often the cases when employing conventional chemistry. Advanced protein engineering now permits the introduction of unique attachment sites within the protein for protein structure and functional studies. For example, the introduction of unique chemical groups into a protein by means of non-natural amino acids allows for sitespecific functionalization.19 Previously, an azido-containing rTM construct was reported for the site-specific conjugation with a methoxy-terminated polyethylene glycol (mPEG) derivative via Staudinger ligation.16 However, Staudinger ligation often suffers from oxidation risk of triphenylphospine, which leads to low conjugation yield. Site-specific immobilization of the rTM456 derivative through the C-terminus via Cu(I)catalyzed click chemistry was also demonstrated utilizing alkyne PEG-modified glass slides.20 Although click chemistry has become of great use in modifying proteins and other macromolecules, traditional click chemistry is at a disadvantage due to the potential presence of residual copper catalyst, which can be potentially toxic in the final product intended for biological application.21,22 Recently, copper-free click chemistry has emerged as an alternative bio-orthogonal ligation strategy to avoid the use of the potentially toxic copper as catalyst.23 It has been used for biomolecule24 and cell surface modification,25 as well as biomaterial applications.26 In this study, we explored site-specific chemoselective glyco-functionalization of a rTM456 74
DOI: 10.1021/acsmacrolett.5b00805 ACS Macro Lett. 2016, 5, 73−77
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ACS Macro Letters Scheme 1. Synthesis of DBCO Chain-End-Functionalized Lactose-Based Glycopolymer
Figure 3. 1H NMR spectrum of DBCO chain-end-functionalized glycopolymer (D2O).
method, in which the cis-diol sugar groups were oxidized to aldehydes and then subsequently reacted with Schiff reagent to yield a magenta band.27 As shown in Figure 2, only the sample containing rTM456 with azide and DBCO-PEG4-CONH-Ph-βGal (Lane 1) exhibited a magenta band, whereas a sample containing rTM456 without azide and DBCO-PEG4-CONH-Phβ-Gal (Lane 2, positive control) and sample of rTM456 with azide and phenyl-galactose without DBCO (Lane 3, negative control) showed no glyco-staining. Additionally, Coomassie blue staining did not show any new bands for all proteins. The molecular mass of the DBCO-PEG4-CONH-Ph-β-Gal is only 849 Da, which is too small to alter the protein migration pattern and to be detected on SDS-PAGE. These results indicated a successful modification of rTM456-azide with a galactose derivative via copper-free click chemistry. Synthetic glycopolymers containing multiple copies of sugar moieties have been widely used as natural oligosaccharide mimics.6,28 In this study, lactose-containing glycopolymer was
used as a model glycopolymer for the modification of rTM456. A DBCO chain-end-functionalized lactose-based glycopolymer was synthesized by chain-end modification of a lactose-based O-cyanate chain-end functionalized glycopolymer via isourea bond formation, which was synthesized from a lactose acrylamide derivative via cyanoxyl-mediated free radical polymerization in our previously reported method (Scheme 1).29,30 The DBCO chain-end-functionalized glycopolymer was characterized by 1H NMR, in which a terminal aromatic group allows for determination of the average molecular weight (about MW = 35 500) of the glycopolymer by comparing the integrated signal from aromatic protons, lactose anomeric protons, and methyl and methylene protons of the copolymer backbone of both lactosyl acrylamide and acrylamide (Figure 3). Next, site-specific glycopolymer modification of the recombinant TM456 at the C-terminal was investigated using the DBCO-PEG4 chain-end-functionalized glycopolymer via 75
DOI: 10.1021/acsmacrolett.5b00805 ACS Macro Lett. 2016, 5, 73−77
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ACS Macro Letters
Figure 4. Site-specific glycopolymer modification of rTM456-azide via copper-free click chemistry and its SDS-PAGE (12%) characterization: (A) glyco-staining, (B) Coomassie blue staining: Lane 1: His-rTM456-N3 treated with DBCO-PEG4-glycopolymer; Lane 2: His-rTM456 treated with DBCO-PEG4-glycopolymer; and Lane 3: His-rTM456-N3 treated with glycopolymer (without DBCO).
Table 1. Protein C Activation Activity of the rTM456 Derivative and Its Glycoconjugate Km (μM) kcat (min−1) kcat/Km (min−1·μM−1) a
full TMa
His-rTM-azide
His-rTM-Gal
His-rTM-GP
0.60 ± 0.15 0.20 ± 0.03 0.37 ± 0.14
0.80 ± 0.2 0.28 ± 0.05 0.40 ± 0.15
0.95 ± 0.22 0.22 ± 0.12 0.25 ± 0.14
0.87 ± 0.15 0.18 ± 0.17 0.18 ± 0.11
Commercial full TM of human. Gal: galactose. GP: glycopolymer.
defined as moles of produced activated protein C per minute by given amounts of rTM456 and rTM456 glycoconjugates in the presence of thrombin and calcium. All protein C activation assays by rTM456 and rTM456 glycoconjugate were performed for 60 min in a buffer solution of 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% BSA, and 5 mM Ca2+ at 37 °C, as previously reported.31 The rate of protein C activation was linear with time until ∼20% of the protein C was activated. Reaction conditions (TM concentration or incubation time) were adjusted so that less than 10% of the protein C was activated to ensure that the amount of activated protein C by TM was in the linear range. As shown in Table 1, there was no apparent activity change upon glyco-modification of rTM456 with either galactose monosaccharide or lactose-containing glycopolymer. The highly efficient and biocompatible method developed for end-to-end protein glycopolymer conjugation did not affect protein activity and thus provides a practical site-specific method for glyco-engineering of proteins of interests. In summary, we demonstrated a successful strategy to synthesize a bioinspired TM conjugate by site-specific conjugation of rTM456 at the C-terminus with a chain-endfunctionalized glycopolymer via copper-free click chemistry. The protein conjugation method presented here has distinct advantages over traditional methods. First, the site-specific, chemo- and bio-orthogonal conjugation provides a simple and convenient route to synthesize a uniform one-to-one protein− glycopolymer conjugate in short time and in good yield. Second, mild conjugation conditions minimize the chance of protein denaturation. Continued studies of in vitro and in vivo antithrombotic activity of these TM conjugates and their pharmacokinetic properties are under investigation. Overall, the proposed glyco-engineering of rTM456 with glycopolymer provides a rational design strategy for facilitating studies of TM functions and generating a TM-based antithrombotic agent.
copper-free click chemistry as indicated above. SDS-PAGE was used to separate and identify the conjugate formed, followed by glyco-staining and Coomassie blue staining, respectively. As shown in Figure 4, only the sample including rTM456-azide (Lane 1) showed a magenta band of high molecular weight (>130 kDa), while the rTM456 (without azide) treated with DBCO-PEG4-glycopolymer (Lane 2, positive control) and rTM456-azide treated with glycopolymer without DBCO (Lane 3, negative control) showed no glyco-staining (Figure 4A). Coomassie blue staining (Figure 4B) showed the same high molecular weight band that appeared on the glyco-staining gel on Lane 1 containing rTM456-azide and DBCO-PEG4glycopolymer and the protein band at ∼51 kDa corresponding to unreacted rTM456-azide. The molecular weight achieved for the glycopolymer−protein conjugate was higher than anticipated on the SDS-PAGE gel. This could be due to the fact that the attached glycopolymer is a very hydrophilic macromolecule consisting of lactose and amide side chains, which prevent SDS from interacting with the protein and introducing a homogeneous negative charge across the protein surface, thus hampering the protein conjugate’s ability to move through the gel during electrophoresis. This phenomenon has been observed in a previous report when using glycopolymer to modify streptavidin.29 Neither the positive nor the negative controls exhibited the formation of any new bands after Coomassie blue staining. About 75% conjugation yield was confirmed based on the band image of the rTM 456 glycopolymer and rTM456 (Figure 4B, Lane 1). These results indicated successful glycopolymer modification of the rTM456azide via copper-free click chemistry. Specifically, the reaction between chain end of both protein and glycopolymer facilitates the formation of a uniform one-to-one protein glycopolymer conjugate with a point of attachment that is site specific and chemoselective. This method will facilitate continuing study on rTM456-azide modification with glycopolymers of interest. In this study, protein C activation activity of the rTM456 glycoconjugate was also evaluated. The activity of rTM456 was 76
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(19) Strømgaard, A.; Jensen, A. A.; Strømgaard, K. ChemBioChem 2004, 5, 909−916. (20) Sun, X.-L.; Stabler, C.; Cazalis, C.; Chaikof, E. L. Bioconjugate Chem. 2006, 17, 52−57. (21) van Dijk, M.; Rijkers, D. T. S.; Liskamp, R. M. J.; van Nostrum, C. F.; Hennink, W. E. Bioconjugate Chem. 2009, 20, 2001−2016. (22) Gaetke, L. M.; Chow, C. K. Toxicology 2003, 189, 147−163. (23) Debets, M. F.; van Berkel, S. S.; Dommerholt, J.; Dirks, A. J.; Rutjes, F. P. J. T.; van Delft, F. L. Acc. Chem. Res. 2011, 44, 805−815. (24) Debets, M. F.; van der Doelen, C. W. J.; Rutjes, F. P. J. T.; van Delft, F. L. ChemBioChem 2010, 11, 1168−1184. (25) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008, 47, 2253−2255. (26) van Dongen, S. F. M.; Verdurmen, W. P. R.; Peters, R. J. R. W.; Nolte, R. J. M.; Brock, R.; van Hest, J. C. M. Angew. Chem., Int. Ed. 2010, 49, 7213−7216. (27) Carlsson, S. R. Glycobiology: A Practical Approach; Fukuda, M., Kobata, A., Eds.; Oxford University Press: Oxford, 1993; pp 14. (28) Kiessling, L. L.; Grim, J. C. Chem. Soc. Rev. 2013, 42, 4476− 4491. (29) Sun, X.-L.; Faucher, K. M.; Houston, M.; Grande, D.; Chaikof, E. L. J. Am. Chem. Soc. 2002, 124, 7258−7259. (30) Narla, S. N.; Sun, X.-L. Org. Biomol. Chem. 2011, 9, 845−850. (31) Esmon, N. L.; DeBault, L. E.; Esmon, C. T. J. Biol. Chem. 1983, 258, 5548−5553.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.5b00805. Syntheses and characterizations of DBCO-PEG4-CONHPh-galactose, DBCO chain-end-functionalized lactosebased glycopolymer, recombinant expression of rTM456azide and its site-specific glyco-modification with DBCOPEG4-CONH-Ph-galactose, DBCO chain-end-functionalized lactose-based glycopolymer, and protein C activation assays of rTM456 glycoconjugates were described (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by grants from the American Heart Association (14GRNT20290002, X.-L. Sun), NIH (1R01HL102604-04, X.-L. Sun), National Science Foundation (CHE-1126384, X.-L. Sun), and Cleveland State University Center for Gene Regulation in Health and Disease Fund.
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