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Site-specific reversible protein and peptide modification: transglutaminasecatalyzed glutamine conjugation and bioorthogonal light-mediated removal kevin moulton, Amissi Sadiki, Bilyana Koleva, Lincoln Ombelets, Tina Tran, Shanshan Liu, Bryan Wang, Hongyan Chen, Emily Micheloni, Penny Beuning, GEORGE O’DOHERTY, and Zhaohui Sunny Zhou Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00145 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019
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Bioconjugate Chemistry
Site-specific reversible protein and peptide modification: transglutaminase-catalyzed glutamine conjugation and bioorthogonal lightmediated removal Kevin R. Moulton‡,†,§, Amissi Sadiki‡,†,§, Bilyana N. Koleva§, Lincoln J. Ombelets†,§, Tina H. Tran†,§, Shanshan Liu†,§, Bryan Wang†,§, Hongyan Chen†,§, Emily Micheloni†,§, Penny J. Beuning§, George A. O’Doherty§, and Zhaohui Sunny Zhou*,†,§ †
Barnet Institute of Chemical and Biological Analysis, §Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts, 02115-5000, United States KEYWORDS: bioconjugation, hybrid modality engineering, photolysis, protein modifications
ABSTRACT: Dynamic photoswitches in proteins that impart spatial and temporal control are important to manipulate and study biotic and abiotic processes. Nonetheless, approaches to install these switches into proteins site-specifically are limited. Herein we describe a novel site-specific method to generate photo-removable protein conjugates. Amine-containing chromophores (e.g., venerable o-nitrobenzyl and less-explored onitrophenylethyl groups) were incorporated via transamidation into glutamine side-chain of alpha-gliadin, LCMV and TAT peptides, as well as beta-casein and UmuD proteins by transglutaminase (TGase, EC 2.3.2.13). Subsequently, photolysis regenerated the native peptides and proteins. When this modification leads to the reduction or abolishment of certain activities, the process is referred to as caging, as in the case for E. coli polymerase manager protein UmuD. Importantly, this method is simple, robust and easily adaptable, e.g., all components are commercially available.
INTRODUCTION Spatial and temporal controls are hallmarks of biological and other complex systems. Due to its intrinsic physical properties, light serves as an ideal stimulus for such controls. For most wavelengths, light is essentially bioorthogonal, i.e., the vast majority of biological processes do not directly involve light; and conversely, most biological processes function similarly in the presence and absence of light. As such, light-controlled processes have broad and diverse applications in biology, chemistry, engineering and medicine. A conceptually straightforward approach is to chemically modify a molecule with a chromophore (e.g., o-nitrobenzyl derivatives) that can be removed by light with the corresponding wavelength (e.g., ultraviolet). When the modification reduces or abolishes certain activities, this modification process is referred to as caging and the molecule is caged. In practice, both site specificity for modification and chemical diversity of the chromophores can be generally achieved for small molecules and peptides (e.g., via solid phase peptide synthesis1-2). The seminal work by Givens, Lawrence and others3-7 of these light-responsive reagents illustrates their chemistry and applications. For instance, photocaged small molecule neurotransmitters such as glutamic
Scheme 1. Transglutaminase (TGase)-catalyzed transamidation (bioconjugation) of glutamine by amine-containing chromophores and light-mediated removal of the chromophore (conjugate) to regenerate the native peptide or protein. acid and gamma-amino butyric acid (GABA), are widely used and commercially available. However, methods for modifications of proteins with removable photo-switches are limited, particularly in a sitespecific manner. The common chemical conjugation approaches (e.g., acylation of lysyl amines8 and alkylation of cysteinyl thiols9) are not site-specific10,11. Some of these chemical modifications are also prone to side reactions which are often underappreciated12. Furthermore, the few that are sitespecific (e.g., unnatural amino acids by Schultz, Chatterjee and others13-15) are laborious, and more importantly, lack chemical diversity (i.e., many chromophores are not compatible). To achieve both site-specificity and chemical diversity, we have developed a chemo-enzymatic approach that targets glutamine residues, as illustrated in Scheme 1. In the forward process, an amine-containing chromophore is covalently attached to the side-chain of glutaminyl residue of proteins using transglutaminase. In the reverse process, the photolabile group is cleaved from the modified amide to regenerate the native peptide or protein (i.e., glutamine) using light.
RESULTS AND DISCUSSION Accepting various amines, the enzyme transglutaminase (TGase, EC 2.3.2.13) converts the unsubstituted amide of the side chain of glutamine into substituted amides. Keillor and others16,17 have shown how powerful and versatile tool TGase is for bioconjugation18, particularly for a few noteworthy
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Bioconjugate Chemistry
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Figure 1. MALDI mass spectra of tryptic peptides of native UmuD (top), nitrobenzylamine-modified UmuD by TGase (middle), and modified UmuD after photolysis (bottom).
characteristics. First, high site specificity toward the target proteins can be generally achieved by quickly experimenting with different TGases. Numerous TGase isoforms are available, such as microbial (mTGase)19,20,46, mammalian guinea pig (TG2)21,22, and recently, newly-discovered or engineered TGase with various degree of specificity24, ranging from very broad to extremely narrow. In addition, activity of transglutaminases can be modulated by inhibitors23. Second, for the amine substrates, broad specificity has been observed for most TGases, albeit with two limitations: only primary amines are accepted and substituents at the alpha position may slow down the reaction16,17. It is worth noting that few methods exist for the modification of protein amides, due to their intrinsic chemical inertness; as such, our method significantly expands the repertoire and adds to the novelty of reversible photomodification of proteins. Table 1. Transglutaminase (TGase) specificity and photoremoval of nitrobenzyl and nitrophenylethyl chromophores. A. o-Nitrobenzyl amines
substrate for TGase
yes
no
photo-removal
yes
n/a
substrate for TGase
yes
yes
photo-removal
yes
yes
B. o-Nitrophenylethyl amines
While the site-specificity is controlled by TGases, the photo-chemical reactivities of the substituted glutaminyl amides are determined by the chromophores. The venerable orthonitrobenzyl system is still commonly used and serves as a reference for other new systems, and thus was tested first. As illustrated in Figures 1 and S.4.1., S.5.1., S.5.5. and S.5.6., onitrobenzylamine (compound 1 in Table 1) was an excellent substrate of TGases for all peptides and proteins we tested, e.g., quantitative transformation could be achieved readily. For photo-removal, as expected, clean and quantitative conversion was also observed (Figures 1, S.4.1 and S.5.6.). To illustrate the modulation of TGase’s activity, calcium-dependence was observed for TG2-mediated modification (Figure S.5.13). Next, we explored photolytic yield of the chromophores as this is a key structure-activity element. Substituents at the benzylic position impact the quantum yield25. Therefore, we evaluated a methyl substituted o-nitrobenzylamine (compound 2 in Table 1), which turned out not to be a substrate for either mTGase or TG2 (Figures S.5.1. and S.5.5., respectively). These results were consistent with the substrate specificity observed by Keillor and others16,17. Specifically, substituents at the acarbon next to the amine significantly lower the reaction rates, but primary amines with an adjacent methylene (e.g., R-CH2NH2) are generally good substrates; as such, based on our results, we expect many commonly used chromophores, such as coumarins and quinolones should be good substrates for TGases. To further expand photo-chemical diversity, we explored an alternative system based on the o-nitrophenylethyl groups, which are known to undergo photo-enolization/elimination (see compounds 3 and 4 in Table 1). Because of the extra methylene between the amine and chromophore, both amines (either substituted at the benzyl position or not) were excellent substrates of TGases (e.g., quantitative transamidation, Figures S.5.1., S.5.5., S.5.7., S.5.9., S.5.11.). For the reverse direction, quantitative photo-regeneration of glutamine was observed as well; and moreover, photolysis of methyl substituted 4 was
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Bioconjugate Chemistry UmuD TGase RecA Light kDa 37
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Figure 2. Site-specific photo-caging of protein. (A) SDS-PAGE gel showed self-cleavage activity of UmuD, caged UmuD, and photolysis of caged UmuD, (B) Highly flexible and dynamic Nterminal region (in red, shown on one monomer) determined by hydrogen-exchange mass spectrometry and EPR spectroscopy27,28, (C) Space-filling model depicts buried Gln23 in the active site, (D) UmuD cleaves between Cys24 (P1) and Gly25 (P1’), and steric perturbation of modified Gln23 block its activity.
faster than its unsubstituted counterpart 3, as shown in Figures S.6.1., S.6.2., and S.6.3., illustrating the effects of substituents at the benzyl position26. Notwithstanding the structural similarity to o-nitrobenzyl group, photolysis of o-nitrophenylethyl derivatives proceed through a different beta-elimination mechanism to generate o-nitrostyrene by-products6,53 (Figure S1, bottom), and are distinct from that of the o-nitrobenzyl systems; the latter involves a hydrogen transfer of an aci-nitro intermediate, followed by cyclization, ring-opening and hydrolysis6 (Figure S1, top). To the best of our knowledge, onitrophenylethyl groups have not been reported on glutaminyl residues of peptides and proteins thereby presenting a novel addition to photo-reversible modifications of proteins. Furthermore, due to their mechanistic differences, this new onitrophenylethyl system is likely to offer additional benefits to the venerable o-nitrobenzyl chemistry. To illustrate the utility and scope of our method, particularly the modulation of biological activities, our rationales and results for the UmuD protein are described herein. UmuD is a homodimer that regulates mutagenesis as part of the SOS response in Escherichia coli and many other bacteria29. First, we examined the structure of UmuD for potential selective bioconjugation by TGase. Its N-terminal arm is highly dynamic and flexible as shown by both hydrogen-deuterium exchange mass spectrometry (HDX-MS, see Figure 2.B) and electron paramagnetic (EPR) spectroscopy27,28; and moreover, contains two glutamine residues (Gln23 and Gln37). As aforementioned, while the substrate specificity of TGase is not fully understood, solvent-accessible and dynamic areas of proteins are likely to be modified by TGase. Indeed, we found both Gln23 and Gln37 were modified by o-nitrobenzylamine in high yield (e.g., no native form was left, see Figure 1 middle). Conversely, no modification was observed for the four additional glutamine residues in the less dynamic and flexible region. Next, the effects of modifications of Gln23 and Gln37 on the activity of UmuD was investigated. In the presence of RecA/ssDNA nucleoprotein filament, UmuD (139 amino acids)
undergoes self-cleavage that removes its N-terminal peptide (24 amino acids), with the scissile bond between Cys24 and Gly2530-32 (Figure 2.D). The resulting 115-amino-acid cleaved protein, referred to as UmuD’, can be fully resolved from UmuD by SDS-PAGE (see Figure 2.A lane 4). Because Gln23 is adjacent to one of the scissile bond residues Cys24 and is also buried inside the active site (see Figure 2.C and 2.D), we expected modification of Gln23 would lead to significant perturbation of the cleavage site and thus reduction of activity; indeed, little self-cleavage was observed for modified UmuD (Figure 2.A lane 5), thus being “caged”. It is worth noting that the modified residue does not have to be located within the active site or the center of binding site in order to abolish activity of the target protein, as long as the modification can sufficiently perturb the interactions. For instance, in the work by Lawrence and others, a photoactivatable enzyme was constructed by propinquity covalent labelling of a non-active site residue33. Furthermore, by increasing the size and/or binding properties of the conjugates introduced by TGase, modification of a remote site may be sufficient to abolish activity. Finally, photo-removal of the chromophore and recovery of the activity of UmuD was investigated. As shown in Figure 1 (bottom), the chromophore was readily and quantitatively removed by light, showing that modification of multiple sites is not an issue; and as shown in Figure 2.A lane 6, full recovery and activity of UmuD was observed after photolysis under these conditions. Altogether, the UmuD protein was “uncaged” or “decaged”. Furthermore, as shown in Figure 1, the native and “uncaged” UmuD were analytically indistinguishable, demonstrating that neither TGase bioconjugation nor photolysis cause any side reactions to the protein. These results were consistent with the lack of photo-damage under typical photolysis conditions, as previously reported34. Similar results were observed for all other peptides and proteins tested as detailed in the supplementary materials. Both onitrobenzyl and o-nitrophenylethyl chemistries work for alphagliadin, LCMV and TAT peptides, as well as the beta-casein and UmuD proteins that were evaluated (Table S1), i.e., they could be selectively modified and the resulting substituted amides could be photo-cleaved in quantitative yields, so the chemistries are robust as expected35,36. Altogether, our approach can be applied to most if not all proteins.
CONCLUSION Several features are noteworthy for our novel methods of generating site-specific and reversible by light (i.e., photocleavable) protein conjugates. First and foremost, each component of the process is tunable including the protein substrates, amine-containing chromophores, the TGase enzyme isoforms, light wavelengths and delivery modes. Based on the structure of the protein (e.g., flexibility and dynamic) and rapid experimentation, one can quickly establish whether certain glutamine residues can be modified by a particular TGase isoform. In parallel, for the enzymes, different isoforms and engineered TGase can be explored for broader and/or tailored specificity, as shown by our group37,38 and others39. In addition, reactive glutamine(s) can be introduced or removed via mutagenesis. Given that TGases can accept large amines (e.g., polyethylene glycol, PEG), highly decorated amine chromophores (e.g., for payload release) can be used directly or constructed via stepwise conjugation after the initial conjugation to the proteins, e.g., via click chemistry40. Furthermore, for photo-removal, amine-containing chromophores compatible
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with different wavelengths41 or multiphoton photolysis42-44 can be screened for lessened biological damage and deeper light penetration; alternatively, upconverting nanoparticles and irradiation at near-infrared (NIR) light45 that achieve deeper penetration can also be coupled to our system. From a practical point of view, all reagents used are commercially available, and the procedure is simple and straightforward enough to be carried out by many scientists in their own laboratories. Last but not least, following the same concepts, aside from light, other chemical or environmental stimuli can be utilized for the reversible bioconjugation, which is ongoing in our laboratory.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, supporting table, and supporting figures (PDF)
AUTHOR INFORMATION Corresponding Author * Email:
[email protected] Author Contributions ‡
K. Moulton and A. Sadiki contributed equally.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT The authors thank NSF (NSF MCB-1615946 to P.J.B and NSF/CHE-1565788 to G.A.O) and NIH (NIH NIGMS 1R01GM101396 to Z.S.Z) for funding, Professor Roman Manetsch for photolysis instrument access, and Professor David Lawrence, SunnyLanders and reviewers for helpful discussion.
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(10) Marriott, G., Ottl, J., Heidecker, M., and Gabriel, D. (1998) Light-directed activation of protein activity from caged protein conjugates. Methods Enzymol. 291, 95-116. (11) Thompson, S., Spoors, J. A., Fawcett, M. C., and Self, C. H. (1994) Photocleavable nitrobenzyl-protein conjugates. Biochem. Biophys. Res. Commun. 201, 1213-1219. (12) Yang, L. H., Chumsae, C., Kaplan, J. B., Moulton, K. R., Wang, D. D., Lee, D. H., and Zhou, Z. S. (2017) Detection of Alkynes via Click Chemistry with a Brominated Coumarin Azide by Simultaneous Fluorescence and Isotopic Signatures in Mass Spectrometry. Bioconjugate Chem. 28, 2302-2309. (13) Erickson, S. B., Mukherjee, R., Kelemen, R. E., Wrobel, C. J., Cao, X., and Chatterjee, A. (2017) Precise Photoremovable Perturbation of a Virus-Host Interaction. Angew. Chem. Int. Ed. 56, 42344237. (14) Peters, F. B., Brock, A., Wang, J., and Schultz, P. G. (2009) Construction of a Light-Activated Protein by Unnatural Amino-Acid Mutagenesis. Chem. Biol. 16, 148-152. (15) Mendel, D., Ellman, J. A., and Schultz, P. G. (1991) Photocleavage of the polypeptide backbone by 2-nitrophenylalanine. J. Am. Chem. Soc. 113, 2758-2760. (16) Gnaccarini, C., Ben-Tahar, W., Mulani, A., Roy, I., Lubell, W. D., Pelletier, J. N., and Keillor, J. W. (2012) Site-specific protein propargylation using tissue transglutaminase. Org. Biomol. Chem. 10, 5258-5265. (17) Gundersen, M. T., Keillor, J. W., and Pelletier, J. N. (2014) Microbial transglutaminase displays broad acyl-acceptor substrate specificity. Appl. Microbiol. Biotechnol. 98, 219-230. (18) Strop, P. (2014) Versatility of microbial transglutaminase. Bioconjugate Chem. 25, 855-862. (19) Sugimura, Y., Yokoyama, K., Nio, N., Maki, M., and Hitomi, K. (2008) Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phagedisplayed peptide library. Arch. Biochem. Biophys. 477, 379-383. (20) Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H., and Motoki, M. (1989) Purification and characterization of a novel transglutaminase derived from microrganisms. Agric. Biol. Chem. 53, 2613-2617. (21) Folk, J. E., and Cole, P. W. (1966) Mechanism of action of guinea pig liver transglutaminase. I. Purification and properties of the enzyme: identification of a functional cysteine essential for activity J. Biol. Chem. 241, 5518-5525. (22) Folk, J. E. (1980) Tranglutaminases. Ann. Rev. Biochem. 49, 517-531. (23) Keillor, J. W., Chica, R. A., Chabot, N., Vinci, V., Pardin, C., Fortin, E., Gillet, M. F. G., Nakano, Y., Kaartinen, M. T., V., Pelletier, J. N., et al. (2008) The bioorganic chemistry of transglutaminase — from mechanism to inhibition and engineering. Can. J. Chem. 86, 271-276. (24) Steffen, W., Ko, F. C., Patel, J., Lyamichev, V., Albert, T. J., Benz, J., Rudolph, M. G., Bergmann, F., Streidl, T., Kratzsch, P., et al. (2017) Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins. J. Biol. Chem. 292, 15622-15635. (25) Ramesh, D., Wieboldt, R., Billington, A. P., Carpenter, B. K., and Hess, G. P. (1993) Photolabile precursors of biological amides: synthesis and characterization of caged o-nitrobenzyl derivatives of glutamine, asparagine, glycinamide, and gamma-aminobutyramide. J. Org. Chem. 58, 4599-4605. (26) Hasan, A., Stengele, K. P., Giegrich, H., Cornwell, P., Isham, K. R., Sachleben, R. A., Pfleiderer, W., and Foote, R. S. (1997) Photolabile protecting groups for nucleosides: synthesis and photodeprotection rates. Tetrahedron 53, 4247-4264. (27) Fang, J., Rand, K. D., Silva, M. C., Wales, T. E., Engen, J. R., and Beuning, P. J. (2010) Conformational Dynamics of the Escherichia coli DNA Polymerase Manager Proteins UmuD and UmuD '. J. Mol. Biol. 398, 40-53. (28) Ollivierre, J. N., Budil, D. E., and Beuning, P. J. (2011) Electron spin labeling reveals the highly dynamic N-terminal arms of the SOS mutagenesis protein UmuD. Mol. Biosyst. 7, 3183-3186. (29) Ollivierre, J. N., Fang, J., and Beuning, P. J. (2010) The Roles of UmuD in Regulating Mutagenesis. J. Nucleic Acids 2010, Article ID 947680.
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