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ScienceDirect Semi-synthesis of chemokines Annette G Beck-Sickinger and Nydia Panitz Protein ligation allows the introduction of a wide range of modifications into proteins that are not accessible by mutagenesis. This includes non-proteinogenic amino acids and even backbone modification. This review summarizes recent reports on modified chemokine variants by ligation technologies and includes the development of the first protein with a full secondary structure motif exchanged by a helix that exclusively consists of b-amino acids. Furthermore the first protein activatable by light by rearrangement of a depsipeptide bond is described. Combining different ligation methods, immobilization and specific release of chemokines were achieved, which is of major importance for the gradient forming activity of chemokines. Examples are shown for CXCL8 (interleukin 8, IL-8) and CXCL12 (stromal derived factor 1, SDF 1) including their chemical and structural characterization as well as the most frequently used assays. Addresses Institute of Biochemistry, Faculty of Bioscience, Pharmacy and Psychology, Leipzig University, Bruederstrasse 34, 04103 Leipzig, Germany Corresponding author: Beck-Sickinger, Annette G ([email protected])
Current Opinion in Chemical Biology 2014, 22:100–107 This review comes from a themed issue on Synthetic biomolecules Edited by Paul F Alewood and Stephen BH Kent
acid except C [2,5]. CXC-class and CC-class form the two major chemokine classes, while C-chemokines and CX3C-chemokines contain only lymphotactin (Lptn) C-chemokine, and fractalkine and neuroalkine for the CX3C-subgroup [5]. The activity of chemokines is mediated by at least 20 different heptahelix receptors, of which most are coupled to G-proteins [2]. It has been demonstrated that the chemokine–chemokine receptor system is a multiligand/multi-receptor system, for which individual chemokines can activate different receptors and one receptor can be activated by different chemokines (Figure 1c) [6]. Because of various signaling pathways chemokines exhibit multifaceted functions and play an important role in the regulation of immune surveillance, wound healing, tissue repair and inflammation [1,7]. Accordingly, the chemokine network is a highly complex system that influences various biological functions and is far from being fully understood. Most interesting are the chemotactic properties of chemokines. Understanding how cells are guided in the body to different sites of action and accordingly via different pathways, would allow the modulation of major functions including embryogenesis and immune responses, but also tumor metastasis and spreading of tumor cells [7]. Such knowledge at a molecular level could lead to the development of new therapeutic molecules with significant importance in immunology, regenerative medicine as well as oncology.
http://dx.doi.org/10.1016/j.cbpa.2014.09.024 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction Chemokines are chemotactic cytokines with a molecular weight between 8 and 12 kDa [1,2]. Up to now more than 50 proteins belong to this class, which show a highly conserved tertiary structure that consists of a free Nterminus followed by a three stranded b-sheet with an embedded C-terminal a-helix (Figure 1a,b) [3]. Chemokines can be devided into inflammatory and homeostatic cytokines according to their role in the immune system [4]. Structural classification is based on the pattern of cysteine residues (C) within the N-terminal segment of the proteins, which are termed as the CC-class, CXC-class, C-class and CX3C-class [2] where C refers to conserved cysteine residues and X may be any other amino Current Opinion in Chemical Biology 2014, 22:100–107
Synthesis of chemokines by native chemical ligation Native chemical ligation (NCL) is a chemical method that allows the covalent linkage of two unprotected peptide segments under mild conditions. One peptide includes a C-terminal a-thioester and the second peptide contains an N-terminal Cys residue. Mechanistically the first step is a trans-thioesterification leading to a thioester intermediate followed by the rapid second step, the spontaneous intramolecular S ! N acyl shift, which results in a stable amide bond between the original segments (Figure 2a) [8]. Although this reaction was originally described by T Wieland on the level of amino acids in 1953 [9], it was only with the development of NCL by Dawson et al. in the 1990s that it was applied to chemical protein synthesis [10,11]. This opened a completely new field as chemical modifications easily achievable for peptides could now be introduced into larger peptides and small proteins. The reaction is selective for the N-terminal Cys and does not influence internal Cys residues, which allows the application also for peptides www.sciencedirect.com
Semi-synthesis of chemokines Beck-Sickinger and Panitz 101
Figure 1
(a)
(c) extra
NH2
intra HOOC
CCL1 XCL1 CXCL2 (b)
XCL2 CXCL5 CXCL6 CXCL8 CXCL11 CXCL12 CXCL14 CCL18
CXCR1 XCR1 CXCR2 CXCR4 CXCR7 CCR8 native receptor? Current Opinion in Chemical Biology
Secondary and tertiary structure of chemokines with a short overview demonstrating multi-ligand–multi-receptor system. (a) and (b) Shows an overview of the chemokine CCL1 (red; PDB: 1EL0) from CC-class and fractalkine (green; PDB: 1B2T) from CX3C-class and the structure of CXCL14 (yellow; PDB: 2HDL) from CXC-class and lymphotactin (blue; PDB: 2HDM) from C-class, which confirms the similar structure of all chemokines independent of the chemokine class. All proteins are comprised of a free N-terminus followed by a three-strand b-sheet with an overlaid C-terminal ahelix. (c) Demonstrates the interaction of chemokines and their receptors as a multi-ligand–multi-receptor system.
containing multiple Cys residues. Thus no side-products are obtained and protecting groups are unnecessary. Already in the first years after the introduction NCL was used to obtain chemokines, like interleukin-8 or vMIP [10,12]. In more recent studies NCL is used to obtain chemically modified chemokines, for example, glycosylated or lipidated variants. Marcaurelle et al. synthesized the eightfold glycosylated Lptn in a one-step NCL and compared it to the non-glycosylated Lptn (Figure 2a) [13]. A similar method was used by Okamoto et al. who were able to chemically synthesize glycosylated and non-glycosylated CCL1 as well as Ser-CCL1 by using two-step NCL (Figure 2b) [14]. Characterization of the variants by chemotaxis assays demonstrated that the glycosylation at position Asn29 and the addition of N-terminal Ser (Ser0) reduces receptor binding of CCL1. Furthermore lipidated CXCL12 was produced by two chemically synthesized segments. This variant showed a significantly longer half-life compared to native CXCL12 [15]. A new variant of NCL was published by Tsuji et al. who synthesized analogs of CXCL14 in N–C-directed sequential NCL and not in the usual C–N-directed way [16]. The authors could demonstrate that the C-terminus facilitated the introduction of modifications without influencing the chemoattractant activity of CXCL14 [16]. www.sciencedirect.com
Semisynthesis of chemokines by expressed protein ligation Long proteins are often obtained by either multistep NCL [17,18] or expressed protein ligation (EPL) [19,20] a technique first described by Muir et al. in 1998 that combines NCL with intein-mediated protein splicing [20]. One or more peptide or protein segments are generated as fusion proteins to a specifically modified intein that is linked to the chitin binding domain (CBD) to form the C-terminal thioester by using the IMPACTTM-system (intein-mediated purification with an affinity chitin binding tag) (Figure 3a) [21]. Different inteins like Mxe GyrA, Sce VMA and Mth RIR1 are often used [22], whereas new intein variants are consistently engineered [23,24,25]. The CBD binds to chitin beads to remove impurities from protein expression by simple washing steps. The cleavage is induced by an excess of thiol and is most frequently performed at the N-terminus of the intein, however other approaches like thermosensitive inteins or the TWIN systems exist as well [26]. Finally the target protein thioester can be eluted from the intein-CBD tag still bound to the chitin beads (Figure 3a). For cleavage the thiol 2-mercaptoethanesulfonate (MESNa) is often applied, because of its good stability and high reactivity in NCL [27], though other thioesters have been used as well [28]. The segment with the Nterminal Cys can be synthesized by SPPS or expressed [8]. After purification of the segment containing the Current Opinion in Chemical Biology 2014, 22:100–107
102 Synthetic biomolecules
Figure 2
(a)
(b)
HS H2N
O Lptn 1-47
glycosylated Lptn 49-93
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spontaneous intramolecular S→N acyl shift O HS Lptn 1-47
or Lptn 1-47
N H
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Lptn 49-93 O Current Opinion in Chemical Biology
Reaction mechanism of native chemical ligation. On the left side the reaction mechanism for the native chemical ligation is demonstrated. The first step is a reversible trans-thioesterification, whereas the thiol-sulfur of the Cys attacks the carboxyl-carbon of the Ca-thioester. The formed intermediate undergoes a spontaneous intramolecular S ! N acyl shift and forms a native peptide bond. The mechanism on the left side is demonstrated for the Lptn [13]. The right side of the figure features a short version for the two-step NCL for CCL1 [14].
Ca-thioester and the fragment with the N-terminal Cys, both segments are ligated by NCL to a native peptide bond (Figure 3b) [29]. EPL has been applied to chemokines with remarkable success. In contrast to NCL where usually the third Cys is used and two segments with 30–40 residues are synthesized, EPL allows using the fourth Cys for ligation and subsequently a shorter C-terminal synthetic segment. Recent examples include CXCL8 (IL-8, interleukin 8) and CXCL12 (SDF-1a, stromal derived factor 1 alpha) for which the most advanced analogs have been achieved. Both, CXCL8 and CXCL12 belong to the CXC-chemokines. CXCL8 has been described as 72 and 77 aa variants [30–32]. It is able to activate CXCR1 and CXCR2, two rhodopsin-like GPCRs and mainly plays an important role in inflammation [33]. In order to perform structure– activity studies two segments of the chemokine were designed. CXCL8(1–54) was expressed as a fusion protein with a C-terminal intein-CBD and purified by Current Opinion in Chemical Biology 2014, 22:100–107
using the IMPACTTM-system. The second segment was synthesized, whereas Lys69 was coupled to carboxyfluorescein (CF) according to Weber et al. and ligation was performed (Figures 3 and 4b) [34]. The biological activity of CXCL8(1–77) and [K69(CF)]CXCL8(1–77) was analyzed and the CF-labeled CXCL8 revealed the same agonistic activity as CXCL8 [35]. The labeled chemokine was used for time-dependent internalization experiments and confirmed earlier studies [36]. In addition, the role of CXCL8 in sialylation of CXCR2 biology could be identified by the CF labeled analog of CXCL8 [37]. A similar approach was used to generate photo-activatable analogs of CXCL8 to map the dimerization interface of CXCL8 by introducing the non-proteinogenic amino acid benzoyl-phenylalanine at Arg65 and Ala74, respectively (Table 1) [38]. It could be demonstrated that Arg65 plays no important role for dimerization, which is in agreement with previous studies from Jin et al. [39] but that Ala74 was identified as a key residue within the dimerization interface (Table 1). www.sciencedirect.com
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Figure 3
(b)
HS
O
Mxe GyrA CBD intein
N H
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O
CXCL8(1-54)
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(a)
S CXCL8(1-54)
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loading
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Mxe GyrA CBD intein
N H
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O N → S acyl shift
CXCL8(1-54)
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Mxe GyrA CBD intein
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CXCL8(56-77) or [K69(CF)]CXCL8 (56-77)
S S
O–
H 2N
Mxe GyrA CBD intein Current Opinion in Chemical Biology
Overview of the mechanism for the expressed protein ligation. The mechanism is demonstrated for the CXCL8, which was used for many subsequent investigations. On the left side the IMPACTTM-system is illustrated. One part of CXCL8 was expressed as a fusion protein together with the Mxe GyrA intein and the CBD, whereas the target protein could be purified from impurities through binding to chitin beads. The cleavage of the CXCL8 fragment was induced by MESNa, whereby the CXCL8(1–54)-thioester was formed. After generation of the second segment by SPPS both fragments were coupled by NCL, shown on the right side, leading to a native peptide bond.
Table 1 Summary of modified chemokines formed by EPL Modification
Chemokine Human CXCL8
Carboxyfluorescein
Human CXCL8
CP18(105–121) (GGLRKRLRKFRNKIKEK) b-Peptide 1 (SDYIKQLASFP) b-Peptide 2 (WRQIKEFRAVKEAN) Benzoyl-phenylalanine
Human CXCL8 Human CXCL8 Human CXCL12 Human CXCL12 Human CXCL12 Human CXCL12
15
Selective N-labeling 6-Nitroveratryl (Nvoc) L-Serine and L-homoserine with and without Nvoc-protection group Carboxyfluorescein Introduction of MMP-9 cleavage site (CG(PLSLRS)3-Ahx-GK-Ahx-Pra-NH2)
Results Same agonistic activity like CXCL8 New hypothesis of internalization process [35] Side chains and their orientation important for activity of CXCL8 [36]
Identification of A74 as one residue for dimerization interface Influence of CXCL8 a-helix for receptor binding [38] Importance of E75 for selection of GAG binding pose [40] Nvoc-group and K56 are opportunities to study effects in vivo and in vitro [52] Improved stability through L-homoserine of at K56 and I58 Controlled activation of CXCL12 including Nvoc by UV radiation [53] Lower migration potency of CF-CXCL12 Specific binding of CF-CXCL12 to CXCR4 [51] Immobilization on azided PEG/macromer films Digestion only on cleavage site Biologically active like CXCL12 [55]
EPL offers the possibility to introduce unnatural amino acids, fluorophores, protecting groups and labels into the sequence of CXCL8 and CXCL12, and even allows the replacement of full segments. Furthermore specific immobilization of CXCL12 on macromer films was possible.
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104 Synthetic biomolecules
Specific structural investigation is often undertaken by NMR. EPL provides the unique possibility to produce large parts of the protein by recombinant expression of the 15N, 13C-modified version of a protein, whereas peptide synthesis can only introduce labeled amino acids one by one. Using EPL CXCL8 was obtained, in which residues 1–54 and 75 were labeled with 15N, 13C, residues 55–74, 76 and 77 contained no isotopes (Table 1) [40]. This unique variant allowed the clarification of the role of specific amino acids in the glycosaminoglycan (GAG) interaction [41,42]. One of the most striking analogs of CXCL8 was the first protein that replaced a full secondary structure element by a helix that solely consisted of b-amino acids. b-Amino acids have been reported to be able to
form stable structural elements with different properties compared to those consisting of a-amino acids [43]. This includes dipole moment, handedness of the helix and orientation of the side chains. The C-terminal helix of CXCL8 was fully replaced by a specially designed segment that solely consists of b-amino acids (Figure 4a and Table 1). Amino acids were selected to resemble the position of the native sequence. The protein was shown to be correctly folded and its activity maintained. Furthermore due to the different properties of helices consisting of a-amino acid or bamino acid it could be concluded that the orientation of the side chains is more important for the activity of CXCL8 than the dipole orientation and the handedness [44].
Figure 4
(a)
(b)
(c)
MMP-9
CG
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SR
S)
3 -A
hx
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CXCL12 (57-68)
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- Nvoc Nvoc protected depsipeptide form
H2N CXCL12 (1-49)
CXCL12 (50-55)
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O CH2
CXCL12 (57-68)
O
pH ≥ 7 CXCL12 (1-49)
H N
CXCL12 (50-55)
CH2
O
uncaged depsipeptide form
O CXCL12 (57-68)
HO
all-amide form, active Current Opinion in Chemical Biology
Overview of modified chemokines prepared by EPL. Protein parts, colored in green, represents fragments obtained by protein expression, whereas the segments which are stained in blue, are formed by peptide synthesis. (a) Shows CXCL8 with a modified C-terminal b-helix formed by the introduction of b-amino acid sequence SDYIKQLASFP. (b) Represents a modified CXCL8, which carries the fluorophore carboxyfluorescein at position Lys69, whereby interaction-trafficking studies with its receptors CXCR1 and CXCR2 can be investigated. (c) Demonstrates the controlled release of modified CXCL12 by MMP-9 using an additional linker peptide with specific MMP-9 cleavage site, in which the CXCL12 position Ser4 was mutated to a Val to prevent MMP-9 cleavage within the protein. Thus [S4V]-CXCL12 remains active and can be immobilized on specific biomaterials or polymer films. (d) Shows the possibility to control the activity of CXCL12 by UV-cleavable Nvoc-group. Therefore Lys56 was mutated to Ser and the amino group of Ser was coupled with Nvoc leading to an O-acyl bond (depsipeptide) between the side chain and the followed amino acid. The removal of Nvoc by irradiation with UV light promotes the formation of a native peptide bond (all-amide form) by spontaneous O ! N acyl rearrangement. Only the allamide form was active, thus activity was controlled by light. Current Opinion in Chemical Biology 2014, 22:100–107
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Semi-synthesis of chemokines Beck-Sickinger and Panitz 105
One of the most important chemokines is CXCL12. It consists of 68 amino acids and displays various essential functions including angiogenesis, organogenesis and immune surveillance [45–47]. Knockout mice lacking CXCL12 or its receptor CXCR4 die in utero suggesting an important role in embryogenesis [48,49]. Furthermore its role in tumor metastasis is considered to be essential. CXCL12 interacts with two heptahelix receptors, CXCR4 and CXCR7. Whereas CXCR4 transmits classical Gprotein signaling pathways, the role of CXCR7 is not fully understood [50]. EPL has been applied to produce various analogs of CXCL12 for in vitro and in vivo investigation. These, include fluorescent analogs [51] or analogs labeled with nitroveratryl (Nvoc) [52]. The N-terminal segment M[A49]-CXCL12(1–49) was recombinantly expressed and purified with the IMPACTTM-system and the C-terminal fragments based on CXCL12(50–68) were synthesized and contained chemical modifications like CF. A detailed protocol for the preparation of CXCL12 by EPL was published recently [29]. How do we control protein activity in a time-dependent and space dependent manner by light? As the simple addition of Nvoc-groups did not lead to an inactive protein and therefore did not fulfill these criteria a more significant change had to be introduced into the protein. We hypothesized that with a depsi-peptide segment, in which the peptide chain is continued via the side chain of a Ser residue, the C-terminal helix should be destroyed. The Nterminus was protected with the photolabile protecting group Nvoc and after removal the free N-terminus should attack the ester bond and lead to a rearrangement, and hopefully to an active peptide. In this case, the removal of the Nvoc-group would trigger the activity of the protein. EPL was used to produce the first protein that contains such a depsi-peptide bond, which was converted into the amide-form after photolysis (Figure 4d). Either Lys56 or Ile58 was replaced by L-serine or L-homoserine that contain the following N-terminal segment via depsi-peptide bonding, and both were coupled with Nvoc leading to an inactive chemokine [53]. The exposure to UV light led to the removal of Nvoc, the in situ generated free a-amino group reacted indeed with the ester carbonyl carbon atom and finally rearranged by an intramolecular O ! N acyl shift to the desired peptide bond (Figure 4d). The CXCL12 variants were tested for their chemotactic potential before and after UV radiation. Analogs bearing the Nvoc group showed no activity, but after UV exposure high activity was restored. This fascinating example demonstrates how EPL can open the door for new protein variants with inducible activities that are not achievable by any other methods. Chemokines usually form gradients to attract cells. CXCL12 is an important mediator in wound-healing as www.sciencedirect.com
it can guide progenitor cells that follow the CXCL12 gradient. Accordingly, immobilization on surfaces followed by an induced and controlled release to form a gradient would be a great advantage in biomaterials. This however, is still challenging. Again, EPL provides the best answer. Whereas the proof of the relevance of gradient formation could be achieved with unspecific interaction with heparin [54] a more advanced approach was reported recently. Steinhagen et al. generated CXCL12 which was C-terminally extended by a peptide that contains a matrix metalloprotease cleavage site and propargylglycine for a click reaction to polylactid acid films (Figure 4c and Table 1) [55]. Immobilization was performed in a one-pot reaction as described for enzymes combining EPL and copper(I)-catalyzed azide/alkyne cycloaddition [56]. CXCL12 was released from the film in a controlled, metalloprotease-dependent manner, active and able to fully control migration of Jurkat cells.
Conclusion The semisynthesis of chemokines by EPL opens unique possibilities for a deeper insight into the different biological processes and structural analyses of this important class of proteins. Modified chemokines carrying unnatural amino acids, fluorescent dyes or other specific probes have been generated in high yield, sufficient amount and great purity. Clearly EPL offers a new approach to insert a wide range of modifications into chemokines, which can be used as a repertoire for future studies and give new directions in biological, biophysical and medical research.
Acknowledgements Parts of the work discussed in this review were kindly supported by the Deutsche Forschungsgemeinschaft (Be1264-5/1-2, TRR67). The financial support of the graduate school ‘Building with Molecules and Nano-objects’ (BuildMoNa), the EU and the Free State of Saxony is kindly acknowledged.
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25. Shah NH, Dann GP, Vila-Perello M, Liu Z, Muir TW: Ultrafast protein splicing iscommon among cyanobacterial split inteins: implications for protein engineering. J Am Chem Soc 2012, 134:11338-11341. First study that systematically describes the family of split DnaE inteins from cyanobacteria in more detail showing their splicing effiency and kinetics.
10. Dawson PE, Muir TW, Clark-Lewis I, Kent SB: Synthesis of proteins by native chemical ligation. Science 1994, 266: 776-779. 11. Dawson PE, Kent SB: Synthesis of native proteins by chemical ligation. Annu Rev Biochem 2000, 69:923-960. 12. Brik A, Keinan E, Dawson PE: Protein synthesis by solid-phase chemical ligation using a safety catch linker. J Org Chem 2000, 65:3829-3835. 13. Marcaurelle LA, Mizoue LS, Wilken J, Oldham L, Kent SB, Handel TM, Bertozzi CR: Chemical synthesis of lymphotactin: a glycosylated chemokine with a C-terminal mucin-like domain. Chemistry 2001, 7:1129-1132. 14. Okamoto R, Mandal K, Ling M, Luster AD, Kajihara Y, Kent SB: Total chemical synthesis and biological activities of glycosylated and non-glycosylated forms of the chemokines CCL1 and Ser-CCL1. Angew Chem Int Ed Engl 2014, 53: 5188-5193. Nice description for the chemical peptide synthesis of glycosylated chemokine segments performed for CCL1 and first study analysing the influence of glycosylation on the chemotactic potential of CCL1. 15. Bellmann-Sickert K, Beck-Sickinger AG: Palmitoylated SDF1alpha shows increased resistance against proteolytic degradation in liver homogenates. ChemMedChem 2011, 6:193-200. 16. Tsuji K, Shigenaga A, Sumikawa Y, Tanegashima K, Sato K, Aihara K, Hara T, Otaka A: Application of N–C- or C–N-directed sequential native chemical ligation to the preparation of CXCL14 analogs and their biological evaluation. Bioorg Med Chem 2011, 19:4014-4020. 17. Pentelute BL, Gates ZP, Dashnau JL, Vanderkooi JM, Kent SBH: Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities. J Am Chem Soc 2008, 130:9702-9707. 18. Becker CF, Hunter CL, Seidel R, Kent SB, Goody RS, Engelhard M: Total chemical synthesis of a functional interacting protein pair: the protooncogene H-Ras and the Ras-binding domain of its effector c-Raf1. Proc Natl Acad Sci U S A 2003, 100: 5075-5080. 19. Muir TW: Semisynthesis of proteins by expressed protein ligation. Annu Rev Biochem 2003, 72:249-289. 20. Muir TW, Sondhi D, Cole PA: Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci U S A 1998, 95:6705-6710. 21. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, Perler FB, Benner J, Kucera RB, Hirvonen CA et al.: Singlecolumn purification of free recombinant proteins using a selfcleavable affinity tag derived from a protein splicing element. Gene 1997, 192:271-281. 22. Telenti A, Southworth M, Alcaide F, Daugelat S, Jacobs WR Jr, Perler FB: The Mycobacterium xenopi GyrA protein splicing Current Opinion in Chemical Biology 2014, 22:100–107
26. Evans TC Jr, Xu MQ: Intein-mediated protein ligation: harnessing nature’s escape artists. Biopolymers 1999, 51: 333-342. 27. De Rosa L, Russomanno A, Romanelli A, D’Andrea LD: Semi synthesis of labeled proteins for spectroscopic applications. Molecules 2013, 18:440-465. This review is a nice overview of possibilities to introduce spectroscopic probes like fluorescent probes for FRET-studies and isotopic labeling in proteins by EPL explained by different protein examples. 28. Offer J: Native chemical ligation with Nalpha acyl transfer auxiliaries. Biopolymers 2010, 94:530-541. 29. Baumann L, Steinhagen M, Beck-Sickinger AG: Preparation of C terminally modified chemokines by expressed protein ligation. Methods Mol Biol 2013, 1047:103-118. A very good and detailed protocol for EPL of C-terminally modified chemokines described for CXCL12 and including hints for preparation. 30. Baggiolini M, Dewald B, Moser B: Interleukin-8 and related chemotactic cytokines — CXC and CC chemokines. Adv Immunol 1994, 55:97-179. 31. Baggiolini M, Clark-Lewis I: Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett 1992, 307:97-101. 32. Hebert CA, Luscinskas FW, Kiely JM, Luis EA, Darbonne WC, Bennett GL, Liu CC, Obin MS, Gimbrone MA Jr, Baker JB: Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils. J Immunol 1990, 145:3033-3040. 33. Lee J, Horuk R, Rice GC, Bennett GL, Camerato T, Wood WI: Characterization of two high affinity human interleukin-8 receptors. J Biol Chem 1992, 267:16283-16287. 34. Weber PJ, Bader JE, Folkers G, Beck-Sickinger AG: A fast and inexpensive method for N-terminal fluorescein-labeling of peptides. Bioorg Med Chem Lett 1998, 8:597-600. 35. David R, Machova Z, Beck-Sickinger AG: Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8. Biol Chem 2003, 384:1619-1630. 36. Richardson RM, Pridgen BC, Haribabu B, Ali H, Snyderman R: Differential cross-regulation of the human chemokine receptors CXCR1 and CXCR2. Evidence for time-dependent signal generation. J Biol Chem 1998, 273:23830-23836. 37. Frommhold D, Ludwig A, Bixel MG, Zarbock A, Babushkina I, Weissinger M, Cauwenberghs S, Ellies LG, Marth JD, BeckSickinger AG et al.: Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation. J Exp Med 2008, 205:1435-1446. 38. David R, Beck-Sickinger AG: Identification of the dimerisation interface of human interleukin-8 by IL-8-variants containing the photoactivatable amino acid benzoyl-phenylalanine. Eur Biophys J 2007, 36:385-391. 39. Jin H, Hayes GL, Darbha NS, Meyer E, LiWang PJ: Investigation of CC and CXC chemokine quaternary state mutants. Biochem Biophys Res Commun 2005, 338:987-999. www.sciencedirect.com
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40. Nordsieck K, Pichert A, Samsonov SA, Thomas L, Berger C, Pisabarro MT, Huster D, Beck-Sickinger AG: Residue 75 of interleukin-8 is crucial for its interactions with glycosaminoglycans. ChemBioChem 2012, 13:2558-2566. Successful EPL for individual and selective 15N-labeling of specific amino acids in CXCL8 and thus characterizing the role of Glu75 in more detail and detection of additional amino acids participating in GAG interaction. 41. Mobius K, Nordsieck K, Pichert A, Samsonov SA, Thomas L, Schiller J, Kalkhof S, Teresa Pisabarro M, Beck-Sickinger AG, Huster D: Investigation of lysine side chain interactions of interleukin-8 with heparin and other glycosaminoglycans studied by a methylation-NMR approach. Glycobiology 2013, 23:1260-1269. 42. Pichert A, Samsonov SA, Theisgen S, Thomas L, Baumann L, Schiller J, Beck-Sickinger AG, Huster D, Pisabarro MT: Characterization of the interaction of interleukin-8 with hyaluronan, chondroitin sulfate, dermatan sulfate and their sulfated derivatives by spectroscopy and molecular modeling. Glycobiology 2012, 22:134-145. 43. Seebach D, Gardiner J: Beta-peptidic peptidomimetics. Acc Chem Res 2008, 41:1366-1375. 44. David R, Gunther R, Baumann L, Luhmann T, Seebach D, Hofmann HJ, Beck-Sickinger AG: Artificial chemokines: combining chemistry and molecular biology for the elucidation of interleukin-8 functionality. J Am Chem Soc 2008, 130:15311-15317. 45. Juarez J, Bendall L, Bradstock K: Chemokines and their receptors as therapeutic targets: the role of the SDF-1/CXCR4 axis. Curr Pharm Des 2004, 10:1245-1259. 46. Patrussi L, Baldari CT: The CXCL12/CXCR4 axis as a therapeutic target in cancer and HIV-1 infection. Curr Med Chem 2011, 18:497-512. 47. Tiveron MC, Cremer H: CXCL12/CXCR4 signalling in neuronal cell migration. Curr Opin Neurobiol 2008, 18:237-244. 48. Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T: Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996, 382:635-638. 49. Tachibana K, Hirota S, Iizasa H, Yoshida H, Kawabata K, Kataoka Y, Kitamura Y, Matsushima K, Yoshida N, Nishikawa S
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et al.: The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 1998, 393:591-594. 50. Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, Penfold ME, Sunshine MJ, Littman DR, Kuo CJ et al.: A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med 2006, 203:2201-2213. 51. Bellmann-Sickert K, Baumann L, Beck-Sickinger AG: Selective labelling of stromal cell-derived factor 1alpha with carboxyfluorescein to study receptor internalisation. J Pept Sci 2010, 16:568-574. 52. Baumann L, Beck-Sickinger AG: Identification of a potential modification site in human stromal cell-derived factor-1. Biopolymers 2010, 94:771-778. 53. Baumann L, Beck-Sickinger AG: Photoactivatable chemokines — controlling protein activity by light. Angew Chem Int Ed Engl 2013, 52:9550-9553. The first study demonstrating the controlled photoactivation of specific stabilized CXCL12 by introduction of Nvoc and L-homoserine and Lserine, respectively. 54. Baumann L, Prokoph S, Gabriel C, Freudenberg U, Werner C, Beck-Sickinger AG: A novel, biased-like SDF-1 derivative acts synergistically with starPEG-based heparin hydrogels and improves eEPC migration in vitro. J Control Release 2012, 162:68-75. 55. Steinhagen M, Hoffmeister PG, Nordsieck K, Hotzel R, Baumann L, Hacker MC, Schulz-Siegmund M, Beck-Sickinger AG: Matrix metalloproteinase 9 (MMP-9) mediated release of MMP-9 resistant stromal cell-derived factor 1alpha (SDF-1alpha) from surface modified polymer films. ACS Appl Mater Interfaces 2014, 6:5891-5899. A well described use of EPL for immobilization of a CXCL12 containing a peptidic linker with MMP-9 cleavage site on polymer films and demonstrating the advantage for controlled release by MMP-9. The cleaved CXCL12 remained active in solution. 56. Steinhagen M, Holland-Nell K, Meldal M, Beck-Sickinger AG: Simultaneous ‘‘one pot’’ expressed protein ligation and CuIcatalyzed azide/alkyne cycloaddition for protein immobilization. Chembiochem 2011, 12:2426-2430.
Current Opinion in Chemical Biology 2014, 22:100–107
ScienceDirect Semi-synthesis of chemokines Annette G Beck-Sickinger and Nydia Panitz Protein ligation allows the introduction of a wide range of modifications into proteins that are not accessible by mutagenesis. This includes non-proteinogenic amino acids and even backbone modification. This review summarizes recent reports on modified chemokine variants by ligation technologies and includes the development of the first protein with a full secondary structure motif exchanged by a helix that exclusively consists of b-amino acids. Furthermore the first protein activatable by light by rearrangement of a depsipeptide bond is described. Combining different ligation methods, immobilization and specific release of chemokines were achieved, which is of major importance for the gradient forming activity of chemokines. Examples are shown for CXCL8 (interleukin 8, IL-8) and CXCL12 (stromal derived factor 1, SDF 1) including their chemical and structural characterization as well as the most frequently used assays. Addresses Institute of Biochemistry, Faculty of Bioscience, Pharmacy and Psychology, Leipzig University, Bruederstrasse 34, 04103 Leipzig, Germany Corresponding author: Beck-Sickinger, Annette G ([email protected])
Current Opinion in Chemical Biology 2014, 22:100–107 This review comes from a themed issue on Synthetic biomolecules Edited by Paul F Alewood and Stephen BH Kent
acid except C [2,5]. CXC-class and CC-class form the two major chemokine classes, while C-chemokines and CX3C-chemokines contain only lymphotactin (Lptn) C-chemokine, and fractalkine and neuroalkine for the CX3C-subgroup [5]. The activity of chemokines is mediated by at least 20 different heptahelix receptors, of which most are coupled to G-proteins [2]. It has been demonstrated that the chemokine–chemokine receptor system is a multiligand/multi-receptor system, for which individual chemokines can activate different receptors and one receptor can be activated by different chemokines (Figure 1c) [6]. Because of various signaling pathways chemokines exhibit multifaceted functions and play an important role in the regulation of immune surveillance, wound healing, tissue repair and inflammation [1,7]. Accordingly, the chemokine network is a highly complex system that influences various biological functions and is far from being fully understood. Most interesting are the chemotactic properties of chemokines. Understanding how cells are guided in the body to different sites of action and accordingly via different pathways, would allow the modulation of major functions including embryogenesis and immune responses, but also tumor metastasis and spreading of tumor cells [7]. Such knowledge at a molecular level could lead to the development of new therapeutic molecules with significant importance in immunology, regenerative medicine as well as oncology.
http://dx.doi.org/10.1016/j.cbpa.2014.09.024 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction Chemokines are chemotactic cytokines with a molecular weight between 8 and 12 kDa [1,2]. Up to now more than 50 proteins belong to this class, which show a highly conserved tertiary structure that consists of a free Nterminus followed by a three stranded b-sheet with an embedded C-terminal a-helix (Figure 1a,b) [3]. Chemokines can be devided into inflammatory and homeostatic cytokines according to their role in the immune system [4]. Structural classification is based on the pattern of cysteine residues (C) within the N-terminal segment of the proteins, which are termed as the CC-class, CXC-class, C-class and CX3C-class [2] where C refers to conserved cysteine residues and X may be any other amino Current Opinion in Chemical Biology 2014, 22:100–107
Synthesis of chemokines by native chemical ligation Native chemical ligation (NCL) is a chemical method that allows the covalent linkage of two unprotected peptide segments under mild conditions. One peptide includes a C-terminal a-thioester and the second peptide contains an N-terminal Cys residue. Mechanistically the first step is a trans-thioesterification leading to a thioester intermediate followed by the rapid second step, the spontaneous intramolecular S ! N acyl shift, which results in a stable amide bond between the original segments (Figure 2a) [8]. Although this reaction was originally described by T Wieland on the level of amino acids in 1953 [9], it was only with the development of NCL by Dawson et al. in the 1990s that it was applied to chemical protein synthesis [10,11]. This opened a completely new field as chemical modifications easily achievable for peptides could now be introduced into larger peptides and small proteins. The reaction is selective for the N-terminal Cys and does not influence internal Cys residues, which allows the application also for peptides www.sciencedirect.com
Semi-synthesis of chemokines Beck-Sickinger and Panitz 101
Figure 1
(a)
(c) extra
NH2
intra HOOC
CCL1 XCL1 CXCL2 (b)
XCL2 CXCL5 CXCL6 CXCL8 CXCL11 CXCL12 CXCL14 CCL18
CXCR1 XCR1 CXCR2 CXCR4 CXCR7 CCR8 native receptor? Current Opinion in Chemical Biology
Secondary and tertiary structure of chemokines with a short overview demonstrating multi-ligand–multi-receptor system. (a) and (b) Shows an overview of the chemokine CCL1 (red; PDB: 1EL0) from CC-class and fractalkine (green; PDB: 1B2T) from CX3C-class and the structure of CXCL14 (yellow; PDB: 2HDL) from CXC-class and lymphotactin (blue; PDB: 2HDM) from C-class, which confirms the similar structure of all chemokines independent of the chemokine class. All proteins are comprised of a free N-terminus followed by a three-strand b-sheet with an overlaid C-terminal ahelix. (c) Demonstrates the interaction of chemokines and their receptors as a multi-ligand–multi-receptor system.
containing multiple Cys residues. Thus no side-products are obtained and protecting groups are unnecessary. Already in the first years after the introduction NCL was used to obtain chemokines, like interleukin-8 or vMIP [10,12]. In more recent studies NCL is used to obtain chemically modified chemokines, for example, glycosylated or lipidated variants. Marcaurelle et al. synthesized the eightfold glycosylated Lptn in a one-step NCL and compared it to the non-glycosylated Lptn (Figure 2a) [13]. A similar method was used by Okamoto et al. who were able to chemically synthesize glycosylated and non-glycosylated CCL1 as well as Ser-CCL1 by using two-step NCL (Figure 2b) [14]. Characterization of the variants by chemotaxis assays demonstrated that the glycosylation at position Asn29 and the addition of N-terminal Ser (Ser0) reduces receptor binding of CCL1. Furthermore lipidated CXCL12 was produced by two chemically synthesized segments. This variant showed a significantly longer half-life compared to native CXCL12 [15]. A new variant of NCL was published by Tsuji et al. who synthesized analogs of CXCL14 in N–C-directed sequential NCL and not in the usual C–N-directed way [16]. The authors could demonstrate that the C-terminus facilitated the introduction of modifications without influencing the chemoattractant activity of CXCL14 [16]. www.sciencedirect.com
Semisynthesis of chemokines by expressed protein ligation Long proteins are often obtained by either multistep NCL [17,18] or expressed protein ligation (EPL) [19,20] a technique first described by Muir et al. in 1998 that combines NCL with intein-mediated protein splicing [20]. One or more peptide or protein segments are generated as fusion proteins to a specifically modified intein that is linked to the chitin binding domain (CBD) to form the C-terminal thioester by using the IMPACTTM-system (intein-mediated purification with an affinity chitin binding tag) (Figure 3a) [21]. Different inteins like Mxe GyrA, Sce VMA and Mth RIR1 are often used [22], whereas new intein variants are consistently engineered [23,24,25]. The CBD binds to chitin beads to remove impurities from protein expression by simple washing steps. The cleavage is induced by an excess of thiol and is most frequently performed at the N-terminus of the intein, however other approaches like thermosensitive inteins or the TWIN systems exist as well [26]. Finally the target protein thioester can be eluted from the intein-CBD tag still bound to the chitin beads (Figure 3a). For cleavage the thiol 2-mercaptoethanesulfonate (MESNa) is often applied, because of its good stability and high reactivity in NCL [27], though other thioesters have been used as well [28]. The segment with the Nterminal Cys can be synthesized by SPPS or expressed [8]. After purification of the segment containing the Current Opinion in Chemical Biology 2014, 22:100–107
102 Synthetic biomolecules
Figure 2
(a)
(b)
HS H2N
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glycosylated Lptn 49-93
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spontaneous intramolecular S→N acyl shift O HS Lptn 1-47
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Lptn 49-93 O Current Opinion in Chemical Biology
Reaction mechanism of native chemical ligation. On the left side the reaction mechanism for the native chemical ligation is demonstrated. The first step is a reversible trans-thioesterification, whereas the thiol-sulfur of the Cys attacks the carboxyl-carbon of the Ca-thioester. The formed intermediate undergoes a spontaneous intramolecular S ! N acyl shift and forms a native peptide bond. The mechanism on the left side is demonstrated for the Lptn [13]. The right side of the figure features a short version for the two-step NCL for CCL1 [14].
Ca-thioester and the fragment with the N-terminal Cys, both segments are ligated by NCL to a native peptide bond (Figure 3b) [29]. EPL has been applied to chemokines with remarkable success. In contrast to NCL where usually the third Cys is used and two segments with 30–40 residues are synthesized, EPL allows using the fourth Cys for ligation and subsequently a shorter C-terminal synthetic segment. Recent examples include CXCL8 (IL-8, interleukin 8) and CXCL12 (SDF-1a, stromal derived factor 1 alpha) for which the most advanced analogs have been achieved. Both, CXCL8 and CXCL12 belong to the CXC-chemokines. CXCL8 has been described as 72 and 77 aa variants [30–32]. It is able to activate CXCR1 and CXCR2, two rhodopsin-like GPCRs and mainly plays an important role in inflammation [33]. In order to perform structure– activity studies two segments of the chemokine were designed. CXCL8(1–54) was expressed as a fusion protein with a C-terminal intein-CBD and purified by Current Opinion in Chemical Biology 2014, 22:100–107
using the IMPACTTM-system. The second segment was synthesized, whereas Lys69 was coupled to carboxyfluorescein (CF) according to Weber et al. and ligation was performed (Figures 3 and 4b) [34]. The biological activity of CXCL8(1–77) and [K69(CF)]CXCL8(1–77) was analyzed and the CF-labeled CXCL8 revealed the same agonistic activity as CXCL8 [35]. The labeled chemokine was used for time-dependent internalization experiments and confirmed earlier studies [36]. In addition, the role of CXCL8 in sialylation of CXCR2 biology could be identified by the CF labeled analog of CXCL8 [37]. A similar approach was used to generate photo-activatable analogs of CXCL8 to map the dimerization interface of CXCL8 by introducing the non-proteinogenic amino acid benzoyl-phenylalanine at Arg65 and Ala74, respectively (Table 1) [38]. It could be demonstrated that Arg65 plays no important role for dimerization, which is in agreement with previous studies from Jin et al. [39] but that Ala74 was identified as a key residue within the dimerization interface (Table 1). www.sciencedirect.com
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Figure 3
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Mxe GyrA CBD intein
N H
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O
CXCL8(1-54)
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S CXCL8(1-54)
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Mxe GyrA CBD intein
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O N → S acyl shift
CXCL8(1-54)
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Mxe GyrA CBD intein
CXCL8(1-54)
HS N H
CXCL8(56-77) or 69 [K (CF)]CXCL8 (56-77) O
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O O + S Na O–
CXCL8(56-77) or [K69(CF)]CXCL8 (56-77)
S S
O–
H 2N
Mxe GyrA CBD intein Current Opinion in Chemical Biology
Overview of the mechanism for the expressed protein ligation. The mechanism is demonstrated for the CXCL8, which was used for many subsequent investigations. On the left side the IMPACTTM-system is illustrated. One part of CXCL8 was expressed as a fusion protein together with the Mxe GyrA intein and the CBD, whereas the target protein could be purified from impurities through binding to chitin beads. The cleavage of the CXCL8 fragment was induced by MESNa, whereby the CXCL8(1–54)-thioester was formed. After generation of the second segment by SPPS both fragments were coupled by NCL, shown on the right side, leading to a native peptide bond.
Table 1 Summary of modified chemokines formed by EPL Modification
Chemokine Human CXCL8
Carboxyfluorescein
Human CXCL8
CP18(105–121) (GGLRKRLRKFRNKIKEK) b-Peptide 1 (SDYIKQLASFP) b-Peptide 2 (WRQIKEFRAVKEAN) Benzoyl-phenylalanine
Human CXCL8 Human CXCL8 Human CXCL12 Human CXCL12 Human CXCL12 Human CXCL12
15
Selective N-labeling 6-Nitroveratryl (Nvoc) L-Serine and L-homoserine with and without Nvoc-protection group Carboxyfluorescein Introduction of MMP-9 cleavage site (CG(PLSLRS)3-Ahx-GK-Ahx-Pra-NH2)
Results Same agonistic activity like CXCL8 New hypothesis of internalization process [35] Side chains and their orientation important for activity of CXCL8 [36]
Identification of A74 as one residue for dimerization interface Influence of CXCL8 a-helix for receptor binding [38] Importance of E75 for selection of GAG binding pose [40] Nvoc-group and K56 are opportunities to study effects in vivo and in vitro [52] Improved stability through L-homoserine of at K56 and I58 Controlled activation of CXCL12 including Nvoc by UV radiation [53] Lower migration potency of CF-CXCL12 Specific binding of CF-CXCL12 to CXCR4 [51] Immobilization on azided PEG/macromer films Digestion only on cleavage site Biologically active like CXCL12 [55]
EPL offers the possibility to introduce unnatural amino acids, fluorophores, protecting groups and labels into the sequence of CXCL8 and CXCL12, and even allows the replacement of full segments. Furthermore specific immobilization of CXCL12 on macromer films was possible.
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104 Synthetic biomolecules
Specific structural investigation is often undertaken by NMR. EPL provides the unique possibility to produce large parts of the protein by recombinant expression of the 15N, 13C-modified version of a protein, whereas peptide synthesis can only introduce labeled amino acids one by one. Using EPL CXCL8 was obtained, in which residues 1–54 and 75 were labeled with 15N, 13C, residues 55–74, 76 and 77 contained no isotopes (Table 1) [40]. This unique variant allowed the clarification of the role of specific amino acids in the glycosaminoglycan (GAG) interaction [41,42]. One of the most striking analogs of CXCL8 was the first protein that replaced a full secondary structure element by a helix that solely consisted of b-amino acids. b-Amino acids have been reported to be able to
form stable structural elements with different properties compared to those consisting of a-amino acids [43]. This includes dipole moment, handedness of the helix and orientation of the side chains. The C-terminal helix of CXCL8 was fully replaced by a specially designed segment that solely consists of b-amino acids (Figure 4a and Table 1). Amino acids were selected to resemble the position of the native sequence. The protein was shown to be correctly folded and its activity maintained. Furthermore due to the different properties of helices consisting of a-amino acid or bamino acid it could be concluded that the orientation of the side chains is more important for the activity of CXCL8 than the dipole orientation and the handedness [44].
Figure 4
(a)
(b)
(c)
MMP-9
CG
(PL
SR
S)
3 -A
hx
-EK
-Pr
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biomaterial (e)
OMe MeO
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CXCL12 (1-49)
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CXCL12 (57-68)
hv
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- Nvoc Nvoc protected depsipeptide form
H2N CXCL12 (1-49)
CXCL12 (50-55)
O
O CH2
CXCL12 (57-68)
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pH ≥ 7 CXCL12 (1-49)
H N
CXCL12 (50-55)
CH2
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uncaged depsipeptide form
O CXCL12 (57-68)
HO
all-amide form, active Current Opinion in Chemical Biology
Overview of modified chemokines prepared by EPL. Protein parts, colored in green, represents fragments obtained by protein expression, whereas the segments which are stained in blue, are formed by peptide synthesis. (a) Shows CXCL8 with a modified C-terminal b-helix formed by the introduction of b-amino acid sequence SDYIKQLASFP. (b) Represents a modified CXCL8, which carries the fluorophore carboxyfluorescein at position Lys69, whereby interaction-trafficking studies with its receptors CXCR1 and CXCR2 can be investigated. (c) Demonstrates the controlled release of modified CXCL12 by MMP-9 using an additional linker peptide with specific MMP-9 cleavage site, in which the CXCL12 position Ser4 was mutated to a Val to prevent MMP-9 cleavage within the protein. Thus [S4V]-CXCL12 remains active and can be immobilized on specific biomaterials or polymer films. (d) Shows the possibility to control the activity of CXCL12 by UV-cleavable Nvoc-group. Therefore Lys56 was mutated to Ser and the amino group of Ser was coupled with Nvoc leading to an O-acyl bond (depsipeptide) between the side chain and the followed amino acid. The removal of Nvoc by irradiation with UV light promotes the formation of a native peptide bond (all-amide form) by spontaneous O ! N acyl rearrangement. Only the allamide form was active, thus activity was controlled by light. Current Opinion in Chemical Biology 2014, 22:100–107
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One of the most important chemokines is CXCL12. It consists of 68 amino acids and displays various essential functions including angiogenesis, organogenesis and immune surveillance [45–47]. Knockout mice lacking CXCL12 or its receptor CXCR4 die in utero suggesting an important role in embryogenesis [48,49]. Furthermore its role in tumor metastasis is considered to be essential. CXCL12 interacts with two heptahelix receptors, CXCR4 and CXCR7. Whereas CXCR4 transmits classical Gprotein signaling pathways, the role of CXCR7 is not fully understood [50]. EPL has been applied to produce various analogs of CXCL12 for in vitro and in vivo investigation. These, include fluorescent analogs [51] or analogs labeled with nitroveratryl (Nvoc) [52]. The N-terminal segment M[A49]-CXCL12(1–49) was recombinantly expressed and purified with the IMPACTTM-system and the C-terminal fragments based on CXCL12(50–68) were synthesized and contained chemical modifications like CF. A detailed protocol for the preparation of CXCL12 by EPL was published recently [29]. How do we control protein activity in a time-dependent and space dependent manner by light? As the simple addition of Nvoc-groups did not lead to an inactive protein and therefore did not fulfill these criteria a more significant change had to be introduced into the protein. We hypothesized that with a depsi-peptide segment, in which the peptide chain is continued via the side chain of a Ser residue, the C-terminal helix should be destroyed. The Nterminus was protected with the photolabile protecting group Nvoc and after removal the free N-terminus should attack the ester bond and lead to a rearrangement, and hopefully to an active peptide. In this case, the removal of the Nvoc-group would trigger the activity of the protein. EPL was used to produce the first protein that contains such a depsi-peptide bond, which was converted into the amide-form after photolysis (Figure 4d). Either Lys56 or Ile58 was replaced by L-serine or L-homoserine that contain the following N-terminal segment via depsi-peptide bonding, and both were coupled with Nvoc leading to an inactive chemokine [53]. The exposure to UV light led to the removal of Nvoc, the in situ generated free a-amino group reacted indeed with the ester carbonyl carbon atom and finally rearranged by an intramolecular O ! N acyl shift to the desired peptide bond (Figure 4d). The CXCL12 variants were tested for their chemotactic potential before and after UV radiation. Analogs bearing the Nvoc group showed no activity, but after UV exposure high activity was restored. This fascinating example demonstrates how EPL can open the door for new protein variants with inducible activities that are not achievable by any other methods. Chemokines usually form gradients to attract cells. CXCL12 is an important mediator in wound-healing as www.sciencedirect.com
it can guide progenitor cells that follow the CXCL12 gradient. Accordingly, immobilization on surfaces followed by an induced and controlled release to form a gradient would be a great advantage in biomaterials. This however, is still challenging. Again, EPL provides the best answer. Whereas the proof of the relevance of gradient formation could be achieved with unspecific interaction with heparin [54] a more advanced approach was reported recently. Steinhagen et al. generated CXCL12 which was C-terminally extended by a peptide that contains a matrix metalloprotease cleavage site and propargylglycine for a click reaction to polylactid acid films (Figure 4c and Table 1) [55]. Immobilization was performed in a one-pot reaction as described for enzymes combining EPL and copper(I)-catalyzed azide/alkyne cycloaddition [56]. CXCL12 was released from the film in a controlled, metalloprotease-dependent manner, active and able to fully control migration of Jurkat cells.
Conclusion The semisynthesis of chemokines by EPL opens unique possibilities for a deeper insight into the different biological processes and structural analyses of this important class of proteins. Modified chemokines carrying unnatural amino acids, fluorescent dyes or other specific probes have been generated in high yield, sufficient amount and great purity. Clearly EPL offers a new approach to insert a wide range of modifications into chemokines, which can be used as a repertoire for future studies and give new directions in biological, biophysical and medical research.
Acknowledgements Parts of the work discussed in this review were kindly supported by the Deutsche Forschungsgemeinschaft (Be1264-5/1-2, TRR67). The financial support of the graduate school ‘Building with Molecules and Nano-objects’ (BuildMoNa), the EU and the Free State of Saxony is kindly acknowledged.
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