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Chemoenzymatic Assembly of Bacterial Glycoconjugates for Site-Specific Orthogonal Labeling Vinita Lukose, Garrett Whitworth, Ziqiang Guan, and Barbara Imperiali J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b07146 • Publication Date (Web): 09 Sep 2015 Downloaded from http://pubs.acs.org on September 9, 2015
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ToC Graphic – Lukose et al
OH OH O HO AcHN O
OH OH O HO
OH O
HN O
HO AcHN O OH OH O O O AcHN O OH
HO HO
PglJ
O-UDP
N3
PglA
OH O
HO AcHN
O N3
NH HO
O AcHN
O
OH O
PglC HO O-UDP
AcHN AcHN O
O AcHN
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OPP-Und
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Chemoenzymatic Assembly of Bacterial Glycoconjugates for Site-Specific Orthogonal Labeling Vinita Lukose†, Garrett Whitworth†, Ziqiang Guan‡ and Barbara Imperiali†* †
Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139 (USA)
‡
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710 (USA)
Supporting Information Placeholder ABSTRACT: The cell surfaces of bacteria are replete with diverse glycoconjugates that play pivotal roles in determining how bacteria interact with the environment and the hosts that they colonize. Studies to advance our understanding of these interactions rely on the availability of chemically defined glycoconjugates in a form that can be selectively modified under orthogonal reaction conditions to serve as discrete ligands to probe biological interactions, in displayed arrays and as imaging agents. Herein, enzymes in the N-linked protein glycosylation (Pgl) pathway of Campylobacter jejuni are evaluated for their tolerance for azide-modified UDP-sugar substrates including derivatives of 2,4-diacetamidobacillosamine and Nacetylgalactosamine. In vitro analyses reveal that chemoenzymatic approaches are useful for the preparation of undecaprenol diphosphate-linked glycans and glycopeptides with site-specific introduction of azide functionality for orthogonal labeling at three specific sites in the heptasaccharide glycan. The uniquely modified glycoconjugates that can be accessed through this methodology represent valuable tools for investigating the roles of C. jejuni cell surface glycoconjugates in host pathogen interactions.
Cell-surface glycoconjugates are common participants in the interactions amongst mammalian cells and between pathogenic microorganisms and the host cells that they infect.1,2 With respect to bacterial glycolipids and glycoproteins, the surge of information from whole genome sequencing and mass spectrometry-based analysis has accelerated the pace at which new glycans are discovered and highlights the importance of these entities in bacterial pathogenesis3 and symbiosis.4 In this context, methods for the preparation and modification of complex glycans and glycoconjugates, with defined modification sites, are critical for parsing the molecular basis for crucial biological responses. Bacterial glycans present particular challenges for study due to the prevalence of highly modified “non-standard” carbohydrates, such as 2,4-diacetamidobacillosamine (diNAcBac) and pseudaminic acid, embedded within diverse glycan architectures,5,6 which exacerbates the task of chemical synthesis requiring that tailored methods for the generation of each unique glycan must be developed to study the roles of carbohydrates in each organism. Chemoenzymatic methods provide an important complement to chemical synthesis enabling access to chemically-defined materials for biological studies including the generation of glycan arrays,7 molecular imaging probes8 and vaccines.9 Many of the challenges are exemplified in the N-linked protein glyco-
sylation system of Campylobacter jejuni,10 a widespread human enteropathogen. C. jejuni requires N- glycosylation to adhere to, invade and colonize target host cells.11-13 This prominent, bacterial protein modification is accomplished by enzymes of the protein glycosylation (Pgl) pathway through initial stepwise assembly of a heptasaccharide onto an undecaprenol diphosphate (Und-PP) carrier, followed by transfer of the glycan to an acceptor protein by the oligosaccharyl transferase, PglB. The enzymes that comprise the C. jejuni Pgl pathway use complex polyprenol-linked substrates to produce glycopeptide and glycoprotein products. To study these enzymes and characterize the functions of the glycan products, reliable preparative approaches, including opportunities for introduction of bioorthogonal chemical handles that can be used to append reporter molecules for biochemical and biophysical applications, are needed. The chemical synthesis of the C. jejuni N-linked glycan is extremely labor-intensive14 and would require significant repurposing for the assembly of variants that include uniquely modified carbohydrates, therefore we set out to establish the practicality and limitations of a general chemoenzymatic approach using native and modified nucleotide sugar donors. Herein we present a systematic approach for the production of defined glycans with bioorthogonal conjugation handles representing intermediates and products in the C. jejuni pathway. Since the bacterial gene clusters encoding enzymes in the biosynthesis and utilization of polyprenol diphosphate-linked glycans in N- and O-linked bacterial protein glycosylation can now be identified using bioinformatics approaches15,16 we anticipate that this study will provide a useful guide for the application of parallel approaches for the preparation of glycoconjugate targets from other physiologically interesting pathogenic and symbiotic bacteria. Previously, we demonstrated that the C. jejuni N-glycan can be prepared on an analytical scale, in a one-pot reaction, using enzymes in the Pgl pathway.17 In this pathway, PglC, a phosphoglycosyltransferase (PGT), catalyzes the first membrane-committed step transferring phospho-diNAcBac from UDP-diNAcBac to Und-P to afford Und-PPdiNAcBac (Fig. 1). The glycosyltransferases (GTs) PglA and PglJ then elaborate the glycan by adding a single GalNAc each, while PglH, a GT with polymerase activity,18 adds three more α-1,4-GalNAcs. The native branched heptasaccharide is completed by the glucosyl transferase, PglI. After translocation to the periplasm by the flippase PglK, the glycan is transferred to protein substrates by the oligosaccharyl transferase, PglB. There is precedent for tolerance of azide-modified sugars as substrates for glycosyltransferases when the azide is incorporated into N-acetamido sugars.19 Of the seven carbohydrates that comprise
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the C. jejuni glycan, six feature N-acetyl groups that can potentially be modified with azide chemical handles. The first three enzymes in the Pgl pathway, PglC, PglA and PglJ would deliver an azide-modified carbohydrate into a single, site-specific position. In contrast, the polymerase activity of PglH18 could potentially result in insertion of azidemodified carbohydrates in place of the three terminal GalNAc residues of the glycan. Therefore, incorporating an azide-modified carbohydrate into the glycan with any of the first three enzymes in the pathway would be advantageous due to the potential for unique positional control. OH OH O HO AcHN O
Und-PP-heptasaccharide
PglH OH O
PglH
HO AcHN O OH O O
HO HO
OH
OH O AcHN
PglH
HO
PglI
OH O
O
AcHN
PglJ
A
OH O
O
HO AcHN AcHN O
O
AcHN
UDP-2-NAc-4-NAzBac
AcHN
O -O P O
Site for azide incorporation
O
PglC
O P O O-
7
80
O R
NH HO
PglC
60 40 20
AcHN
O P O P O OO-
O
O
100
HO HN
O
O
N
O
AcHN O PP Und
OH OH
+
O
HO
R
30
PglA
25 20 15
HO
10 5
OH O
HO
0
10
HO
HO
+
O
AcHN AcHN O
AcHN
40 35
OH O
HO R
O PP Und
UDP-GalNAz UDP-GalNAc
45
O UDP
PglJ HO
OH O
30
HO
25 20
R
O
OH O
15
HO
10 5 0
AcHN O PP Und
OH O
HO
O
R AcHN O
60
time, min
C
O UDP
UDP-GalNAz UDP-GalNAc
35
R = N3 UDP-GalNAz
To study the tolerance of Pgl enzymes for unnatural azido sugars, Pgl pathway enzymes (PglC, PglA, PglJ, PglH, PglI and PglB) were expressed and purified as previously described17,20 and simple substrates were prepared using a combination of chemical and enzymatic approaches. First, polyprenols (C50-60) derived from the leaves of Rhus typhina were subject to phosphorylation with phosphoramidite (FmO)2PNiPr2, followed by oxidation and deprotection to afford the corresponding polyprenol phosphates.21,22 UDP-sugars (Fig. 1B) were prepared by chemoenzymatic approaches. Specifically, UDPdiNAcBac was generated enzymatically,23 while UDP-2-NAc-4NAzBac, a new unnatural azide-modified UDP-sugar, was prepared from UDP-4-amino-4,6-dideoxy-GlcNAc (an intermediate in UDPdiNAcBac biosynthesis) through selective chloroacetylation followed by azide substitution. The C-2 position of UDP-diNAcBac was not considered for azide modification because the presence of an N-acetyl group at this site is known to be critical for catalysis in the PglB reaction.24 The UDP-GalNAz substrate was prepared starting with Nacetylgalactosamine, as previously described.25,26 With these enzymes and substrates in hand, the tolerance of each of the glycosyltransferases was evaluated by comparing the relative turnover of the native UDPsugar and the corresponding azide-derivative. In each case the product
O PP Und
OH
HO
O
AcHN HO
R =H UDP-GalNAc
Figure 1. A. Chemical structure of the C. jejuni Und-PPheptasaccharide. Sites where the azide moiety can be incorporated are highlighted; B. UDP-sugars utilized in this study.
500
B
R = N3 UDP-2-NAc-4-NAzBac
NH
O
O P O P O OO-
AcHN
[UDP-Sugar], µM
O
O
R
N
R =H UDP-diNAcBac
OH OH
OH OH
O
25
NH
O
O
O
R HO
O
O
O UDP
UDP-diNAcBac
3
B
O
R HO
+
Und-P
PglA
product formed, µM
A
quantitation method was tailored to provide the most accurate measure of activity. The tolerance of PglC, the first enzyme in the pathway, for UDP-2-NAc-4-NAzBac relative to the native substrate was examined by comparing activity with the UDP-sugar substrates in the presence of PglC and Und-P for 60 min (Fig. 2A), followed by product isolation and hydrolysis of Und-PP-monosaccharide. For quantification, the free monosaccharide was fluorescently labeled with 8aminopyrene-1,3,6-trisulfonate (APTS) and analyzed by capillary electrophoresis (CE).27 To standardize peak area and electrophoretic mobility, maltose was used as an internal standard. In the PglC reaction, the azide derivative was well tolerated with the rate of product formation corresponding to ~60-65% of the natural substrates rate (Fig. 2A). However, it was noted that the conversion was quite low for both, UDP-diNAcBac and UDP-2-NAc-4-NAzBac at ~14% and 8% respectively. In contrast, a coupled reaction using PglC and the subsequent enzyme in the pathway, PglA, afforded much higher conversions (>60%, data not shown) suggesting that addition of PglA can be effectively used to overcome the unfavorable equilibrium of PglC to increase flux through the two enzymes.
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10
time, min
60
AcHN AcHN O
O AcHN
O PP Und
Figure 2. Kinetic analysis of PglC, PglA and PglJ comparing turnover between natural and azide-modified substrates (R = NHCOCH3 or NHCOCH2N3. A. PglC (10 nM) was evaluated at increasing concentrations of UDP-2-NAc-4-NAzBac. B. Time course for PglA (10 nM) turnover with 200 μM UDP-GalNAc or UDP-GalNAz. C. Time course for PglJ (90 nM) turnover with 100 μM UDP-GalNAc and UDPGalNAz .
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The specificity of PglA for UDP-GalNAc and UDP-GalNAz was compared using a coupled assay with PglC to make the Und-PP-diNAcBac starting material in situ. Reactions were performed with an excess of the UDP-sugars and were quenched at 10 and 60 minutes, after which the disaccharide products were isolated by liquid/liquid extraction. The products were then hydrolyzed and the resulting disaccharide was labeled with 2-aminobenzamide (2-AB) for analysis by normal-phase HPLC with fluorescence detection.28 In this case, the azide-modified UDP-sugar substrate is well tolerated by PglA with product formation comparable to the native substrate after 60 minutes (Fig. 2B). HO
A PglC PglA
OH O
HO
AcHN O OH O UDP-2-NAc-4-NAzBac UDP-GalNAc HO UDP-GalNAc AcHN O AzHN O N3 Az = OH HO O HO B PglC AcHN O OH PglJ PglA Und-P O UDP-diNAcBac UDP-GalNAc HO UDP-GalNAz AzHN AcHN O OH HO O HO AzHN C O OH PglC PglJ PglA O Und-P HO UDP-diNAcBac UDP-GalNAz AcHN UDP-GalNAc AcHN O Und-P
PglJ
O AcHN O PP-Und
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were performed with strategic quenching and isolation steps to ensure that in each case the azide-modified carbohydrate was added only at a single position. The products were confirmed using HPLC and mass spectrometry analysis (Fig. S1). Successful synthesis of Und-PPtrisaccharides with azide-modified carbohydrates in the first two sites of the glycan (Fig. 2A and B) demonstrates the ability of PglA and PglJ to elaborate azide-modified glycan precursors. Finally, to establish that Und-PP-diNAcBac-GalNAc-GalNAz (Fig. 2C) can be successfully elaborated into the native C. jejuni heptasaccharide, the synthesis was completed using PglH and PglI (Fig. 3). It has previously been reported that the terminal GalNAc of the C. jejuni glycan is a key determinant in binding to the human macrophage galactose-type lectin receptor, thereby mediating interactions between C. jejuni and host cells.29 The ability to preserve this binding partnership in the full glycan, while introducing chemical handles with bioorthogonal reactivity into carbohydrates with less dominant roles in glycan-host cell receptor interactions, highlights potential future applications of these reagents. OH OH O HO AcHN O
O AcHN
OH O
HO
O PP-Und
HO HO
AcHN O OH OH O O O AcHN O OH
OH O
HO O
O
N 3 HO
AcHN O PP-Und
Und P
Scheme 1. Chemoenzymatic synthesis of azide-modified Und-PPtrisaccharides. A. Synthesis of Und-PP-2NAc-4-NAzBac-(GalNAc)2; B. Synthesis of Und-PP-diNAcBac-GalNAz-GalNAc; C. Synthesis of Und-PP-diNAcBac-GalNAc-GalNAz. To assess the activity of PglJ, first, Und-PP-diNAcBac-[3H]-GalNAc was prepared using PglC and PglA with Und-P, UDP-diNAcBac and UDP-[6-3H]-GalNAc. The resulting Und-PP-disaccharide was incubated with PglJ, in the presence of either UDP-GalNAc or UDPGalNAz (Fig. 2C). For each substrate the reaction was quenched at either 10 or 60 minutes and purified by HPLC. Product formation was quantified by scintillation counting of each fraction. While 50% less product was formed in the presence of UDP-GalNAz relative to UDPGalNAc after 10 minutes, both substrates showed > 90% conversion after 60 min, indicating that PglJ efficiently incorporates an azido sugar into the Und-PP-trisaccharide. The site selectivity of the process was supported by MS analysis and further substantiated by activity of the product with PglH and UDP-GalNAc (vide infra). Interestingly, in contrast to the observed promiscuity of PglC, PglA and PglJ, the enzyme that transfers three additional GalNAcs to the glycan, PglH, failed to react in the presence of UDP-GalNAz. In this case, the tolerance of PglH was tested by incubating enzyme with UDP-GalNAc and UDP-GalNAz and [3H]-labeled Und-PP-diNAcBac-GalNAcGalNAc trisaccharide. Reactions were analyzed by normal-phase HPLC and no azide-containing products were observed with PglH, even in the presence of a ten-fold excess of UDP-GalNAz with extended time (4 h) (data not shown). Therefore, under these conditions, PglH does not accept UDP-GalNAz as a substrate, underscoring the need for systematic analysis of each enzyme and substrate combination to assess the tolerance for substrate analogs. These biochemical studies establish the compatibility of PglC, PglA and PglJ with azide-modified UDP-sugars. To build on these results, PglC, PglA and PglJ were then used in combination to prepare three uniquely-modified Und-PP-trisaccharides (Scheme 1). The syntheses
NH O
PglC PglA
PglJ
PglH PglI
UDP-diNAcBac UDP-GalNAc
UDP-GalNAz
UDP-GalNAc UDP-Glucose
OH O AcHN AcHN O
O AcHN
O PP Und
Figure 3. Enzymatic synthesis of Und-PP-linked heptasaccharide with incorporation of an azide-modified carbohydrate (red) in the third site of the glycan. Inset: Mass spectrum showing the doubly-deprotonated [M-2H]2- ion of the product. In order for this system to be applicable to the biosynthesis of glycopeptides and potentially glycoproteins, the tolerance of the oligosaccharyl transferase, PglB, for the azide-modified Und-PP-glycans was then investigated. The ability of PglB to transfer each of these azidemodified trisaccharides to an optimal peptide substrate30 was quantified using [3H]-labeled substrates. The efficiency of peptide glycosylation by PglB is between 75 and 90 % of native levels, when the modified carbohydrate is distal to the aglycone; however, it is somewhat more impacted, at about 60%, when the azide-modified carbohydrate is situated proximal to the diphosphate leaving group (Fig. 4, Fig. S2). It has previously been shown that synthetic substrates, including highly truncated (C20) polyprenol diphosphate derivatives of 6-azido-GlcNAc and GlcNAz (Az = COCH2N3) failed to serve as substrates in peptide glycosylation by PglB.31 However, in that case, the lack of activity was attributed to the cumulative effect of unnatural moieties in both components of the polyprenol diphosphate-sugar substrate. In this context, we demonstrate that when native polyprenols are used in conjunction with azide-modified glycans, PglB catalyzes glycosylation of peptides with conversions comparable to those of the unmodified substrate. These results combined with previous studies utilizing PglB to synthesize glycoproteins32 set the stage for synthesis of diverse glycoproteins containing unnatural carbohydrates using this system. Finally, to validate the utility of these compounds for bioorthogonal conjugation, copper-catalyzed click chemistry reactions were performed. The Und-PP-diNAcBac-GalNAc-GalNAz, was conjugated to an acetylene-545 fluorophore (Click Chemistry Tools) using copper catalyzed azide-alkyne cycloaddition in a 1:1 tert-butanol/water system and characterized by mass spectromentry after purification (See Fig. S3A) In addition, the glycopeptide, modified with GalNAz at the ter-
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minal position, was reacted with an acetylene-modified biotin (Acetylene-PEG4-Biotin, Click Chemistry Tools), also using copper catalyzed azide-alkyne cycloaddition, in aqueous conditions and characterized directly without purification (Fig. S3B). A
OH OH O HO R3 O HO
OH OH O HO R3 O
OH O R2 R1 O
PglB
HO
O AcHN
O PP Und
FITC
40
OH O R2 R1 O
Und-PP
45
B product formed, µM
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O AcHN
Ala Asp Gln
N H
[email protected] A CKNOWLEDGMENT This research was supported by NIH GM-039334 (to B.I.), GM069338 and EY023666 (to Z.G.) and an NSERC Fellowship to G.W. We also acknowledge the MIT BIF, Dr. Amy Rabideau and Prof. Brad Pentelute for assistance with MS analysis, Prof. George Peng Wang for Agx1 and NahK plasmids and Dr. Monika Musial-Siwek for PglB.
NH O Ala Thr Arg Arg-CONH2 O
Notes The authors declare no competing financial interests.
35 30 25
1: R1 = AzNH, R2 = R3 = AcNH 2: R1 = R3 = AcNH, R2 = AzNH 3: R1 = R2 = AcNH, R3 = AzNH 4: R1 = R2=R3 = AcNH
20 15 10 5 0
1
2
3
4
Figure 4. A. PglB-catalyzed peptide glycosylation with azido-glycan substrates (50 μM Und-PP-trisaccharide, 250 μM peptide). B. Production of azide-modified glycopeptide products. Together, these studies reveal that azido sugar substrates are well tolerated by several of the C. jejuni Pgl enzymes. Unfortunately, attempts to apply in vivo metabolic labeling in C. jejuni, using exogenous acetylated monosaccharide derivatives, proved unsuccessful. This was likely due to inefficient processing of the precursors to the corresponding UDPsugars, which would involve deacetylation, C-1 phosphorylation, and uridinylation. Indeed it is reported that GalNAz-1-P is not a substrate for the homologous E. coli GlmU.26 Additionally, in vitro kinetic studies show that diNAcBac-1-P is not a substrate for the C. jejuni GlmU (SI Methods and Table S1). Therefore, given the lack of promiscuity in the bacterial GlmU enzymes,26 further studies with the corresponding azide were not pursued. Together these studies suggest that further attempts to incorporate azide-modified sugars into the C. jejuni Nglycan would require engineering of the C. jejuni carbohydrate biosynthesis pathways. The studies presented herein now define that metabolic labeling in C. jejuni should be feasible from the perspective of the Pgl pathway enzymes and therefore, future focus can turn to specifically engineering C. jejuni to enable conversion of cell permeable sugar derivatives into UDP-GlcNAz and UDP-2-NAc-4-NAzBac. This general concept has precedent in mammalian cell culture systems.33,34 This report defines the tolerance of the Pgl pathway enzymes towards azide-modified carbohydrate substrates and shows that chemoenzymatic approaches are valuable for the preparation of Und-PP-glycans and glycopeptides with site-specific introduction of azide functionality for orthogonal labeling. The uniquely labeled glycoconjugates promise to be extremely useful for understanding the roles of C. jejuni cell surface glycoconjugates. As a priority, sufficient quantities can be readily prepared for study of mammalian cell adhesion and invasion, in the preparation of glycan arrays, and in biophysical studies to investigate substrate binding to the Pgl pathway enzymes using fluorescence-based approaches including fluorescence and luminescence resonance energy transfer (FRET and LRET).
ASSOCIATED CONTENT Supporting Information Supporting figures and experimental methods. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author
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OH OH O HO AcHN O
OH OH O HO
OH O
HN O
HO AcHN O OH OH O O O AcHN O OH
HO HO
PglJ
O-UDP
N3
PglA
OH O
HO AcHN
O N3
NH HO
O AcHN
O
OH O
PglC HO O-UDP
AcHN AcHN O
O AcHN
OPP-Und
ACS Paragon Plus Environment
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