Canales, Ángeles

Eller, Steffen

Karagiannis, Theodoros

Lott, Marie

Martínez-Orts, Mónica

Niemietz, Mathäus

Ott, Dimitri

Paulson, James C.

Razi, Nahid

Unverzagt, Carlo

Weishaupt, Markus

Weiss, Michael

English

EPub Bayreuth

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657Efficient Chemoenzymatic Synthesis of N-Glycans with aβ1,4-Galactosylated Bisecting GlcNAc MotifMichael Weiss,[a] Dimitri Ott,[a] Theodoros Karagiannis,[a] Markus Weishaupt,[a]Mathäus Niemietz,[a] Steffen Eller,[a] Marie Lott,[a] Mónica Martínez-Orts,[b] Ángeles Canales,[b]Nahid Razi,[c] James C. Paulson,[c] and Carlo Unverzagt*[a]Dedicated to Professor Horst Kessler on the occasion of his 80th birthday.In human serum immunoglobulin G (IgG), a rare modification ofbiantennary complex N-glycans lead to a β1,4-galactosylatedbisecting GlcNAc branch. We found that the bisecting GlcNAcon a biantennary core-fucosylated N-glycan was enzymaticallygalactosylated under stringent reaction conditions. Furtheroptimizations led to an efficient enzymatic approach to thisparticular modification for biantennary substrates. Notably, tri-and tetra-antennary complex N-glycans were not converted bybovine galactosyltransferase. An N-glycan with a galactosylatedbisecting GlcNAc was linked to a lanthanide binding tag. Thepseudo-contact shifts (PCS) obtained from the correspondingDy-complex were used to calculate the conformational prefer-ences of the rare N-glycan. Besides two extended conforma-tions only a single folded conformation was found.IntroductionImmunoglobulins are one of the best-studied classes ofglycoproteins due to their high therapeutic value. The N-glycans of immunoglobulins are known to affect the biologicalactivity of antibodies, for example, by modulating the inter-actions of the Fc part.[1] Thus, the glycosylation pattern oftherapeutic antibodies is thoroughly characterized. Originatingfrom an unexpected side reaction during the enzymaticgalactosylation of a synthetic bisected N-glycan we becameaware that galactosylated bisecting GlcNAc moieties are presentin human serum IgG. The Gal-β1,4-bisecting GlcNAc motif wasinitially discovered by Nishimura[2] and later confirmed byRudd[3] (Scheme 1). A similar motif was also found on bisectedhybrid N-glycans of the 19 A glycoprotein derived from a lectin-resistant HEK cell line.[3] In cells lacking GlcNAc-transferase II thetruncated bisected N-glycans leads to a Gal-β1,4-bisectingGlcNAc motif, which can be further modified.[4] An enzymaticgalactosylation of a single GlcNAc β1,2-linked to the central β-mannose was found for a synthetic N-glycan bearing the LEC 14antigen.[5]Results and DiscussionAs part of a collaboration with the Consortium for FunctionalGlycomics (CFG), we synthesized various bisected complex-typeN-glycans and functionalized them with a bifunctional spacerfor glycan microarray printing.[6] The bisected N-glycans[7] weresynthesized chemically following a modular approach anddeprotected to the free N-glycans.In the case of the core fucosylated bisected compound 4,precursor 1 was obtained from three building blocks[8] and wassubsequently fucosylated using thiofucoside 2[9] (Scheme 2).The low stereoselectivity (α/β ratio 5.4 : 1) required the separa-tion of the unnatural β-anomer 3b by preparative HPLC.Subsequently, the protecting groups of nonasaccharide 3 wereremoved in a multistep procedure. After treatment with ethyl-enediamine in n-butanol an acetylation/deacetylation sequenceyielded a benzylated nonasaccharide azide intermediate, which[a] M. Weiss, Dr. D. Ott, T. Karagiannis, Dr. M. Weishaupt, Dr. M. Niemietz,Dr. S. Eller, M. Lott, Prof. C. UnverzagtUniversity of Bayreuth, Bioorganic ChemistryUniversitätsstraße 30, 95447 Bayreuth (Germany)E-mail: carlo.unverzagt@uni-bayreuth.de[b] M. Martínez-Orts, Dr. Á. CanalesDpto. Química Orgánica I, Fac. Ciencias QuímicasUniversidad Complutense de MadridAvd. Complutense s/n, 28040 Madrid (Spain)[c] Dr. N. Razi, Prof. J. C. PaulsonDepts. of Molecular Medicine, and Immunology and MicrobiologyThe Scripps Research Institute10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA)Supporting information for this article is available on the WWW underhttps://doi.org/10.1002/cbic.202000268This article is part of a Special Collection on the occasion of Horst Kessler’s80th birthday. To view the complete collection, visit our homepage© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH. This isan open access article under the terms of the Creative Commons AttributionLicense, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.Scheme 1. Biantennary N-glycan with unmodified bisecting GlcNAc (A);complex N-glycans found on human serum IgG bearing a β1,4-galactosy-lated bisecting GlcNAc motif (B, C); hybrid N-glycans from 19 A glycoproteinwith a galactosylated bisecting GlcNAc motif (D, E).ChemBioChemCommunicationsdoi.org/10.1002/cbic.2020002683212ChemBioChem 2020, 21, 3212–3215 © 2020 The Authors. ChemBioChem published by Wiley-VCH GmbHWiley VCH Mittwoch, 30.12.20202022 - closed* / 171929 [S. 3212/3215] 1123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657was reduced to the anomeric amine using propanedithiol.[10]Complete removal of residual thiol was crucial prior to acidichydrolysis of the glycosylamine. The final catalytic hydro-genation gave the free N-glycan 4 in good overall yield. Theenzymatic galactosylation of 4 was carried out using UDP-Gal,bovine galactosyltransferase and calf alkaline phosphatase(CIAP).[11] Analysis of the reaction mixture after three days byMALDI-TOF-MS showed the desired galactosylated N-glycan 5but also a side product (ca. 23%) with the mass of an additionalgalactose unit and we assumed that the additional galactosemight be attached to the bisecting GlcNAc (6). We noticed thatrelative to previous galactosylations of biantennary bisected N-glycan-conjugates[12] the concentration of the N-glycan acceptor(4) was higher (4x) as well as the relative amount ofgalactosyltransferase (2x). Using even higher concentrations ofN-glycan acceptor 4 and repeated addition of galactosyltrans-ferase gave nearly complete conversion to the trigalactosylatedproduct 6. On a preparative scale 4 (3.7 mg; 19 mM) wasincubated with 800 mU of galactosyltransferase added in threeportions over 9 days, which led to complete conversionaccording to MALDI-TOF-MS. The trigalactosylated compound 6was isolated by gel filtration in a yield of 74%. Using a lowerconcentration of 4 in combination with lower amounts ofgalactosyltransferase furnished the selectively digalactosylatedcompound 5 in 78% yield.Both compounds 5 and 6 were characterized by a set of 2DNMR experiments revealing that the third galactose was indeedtransferred to O-4 of the bisecting GlcNAc moiety. This wasevident from the 1H signal of H-4B shifting from 3.06 to 3.3 ppmand the 13C signal of C-4B moving from 72.7 to 81.7 ppmindicative for a glycosylation at C4B. The glycans 5 and 6 wereequipped with a bifunctional linker and added to the repertoireof the CFG glycan array (data not shown).Next the scope of the enzymatic elongation of a bisectingGlcNAc was investigated on an analytical scale using a series offully synthetic bisected N-glycan azides (Scheme 3). The panelof potential substrates consisted of the unsubstituted bisectedN-glycan 7,[7] the corresponding core-fucosylated glycan 8[8] andbisected compounds with three or four antennae (9–11).[7]When attempting to apply the stringent conditions of galacto-sylation directly to the acceptors (19 mM 7–11), strongsubstrate inhibition was found, which only led to sluggish andincomplete galactosylation after several days (HPLC data notshown). In contrast, dilute conditions (2 mM acceptor 7–11)gave complete galactosylation of the GlcNAc termini within1 day (12–16). The galactosylated compounds were submittedScheme 2. Synthesis of N-glycan 3, deprotection to hemiacetal 4 andenzymatic galactosylation to 5 and 6. The numbering of the residues forNMR assignments is shown for 6.Scheme 3. The panel of bisected N-glycan azides (7–11) was enzymaticallygalactosylated under dilute (1.) and subsequently under stringent (2.)conditions. Transfer of an additional galactose to the bisecting GlcNAc wasobserved only in the case of the biantennary N-glycans 17 and 18. (n.d.=not detected).ChemBioChemCommunicationsdoi.org/10.1002/cbic.2020002683213ChemBioChem 2020, 21, 3212–3215 www.chembiochem.org © 2020 The Authors. ChemBioChem published by Wiley-VCH GmbHWiley VCH Mittwoch, 30.12.20202022 - closed* / 171929 [S. 3213/3215] 1123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657to stringent galactosylation conditions (19 mM acceptor 12–16).Out of the five compounds only the biantennary N-glycans 12and 13 gave the corresponding product with a galactosylatedbisecting GlcNAc (17–18) whereas no conversion according toLC–MS was found for the compounds with three or fourantennae (9–11). The galactosylation of a bisecting GlcNAcappears to be particularly sensitive to additional steric hin-drance and might not be possible in the case of complex N-glycans with more than two antennae. According to the HPLCpeak areas of the galactosylations after 3 days, the transfer ofthe third galactose occurs significantly more slowly (~1/5) inthe presence of a core fucose (13) compared with theunsubstituted bisected compound (12) (see S4 in the Support-ing Information).Because only the biantennary digalactosylated N-glycanazides were accepted by galactosyltransferase, we assumedthat the galactosylated bisecting GlcNAc residue might affectthe conformational freedom of the neighboring antennae. Wedecided to investigate the conformational preferences of the N-glycan 17 by attaching a suitable lanthanide binding tag (19)and subsequently measure the pseudo contact shifts of para-magnetic N-glycan complexes to calculate the preferredconformations.[13] On a preparative scale the bisected octasac-charide azide 7 was first converted to the digalactosylatedcompound 12 (92% yield) and subsequently to the trigalactosy-lated undecasaccharide 17 (91% after RP-HPLC). The azide of17 was reduced and coupled with the lanthanide binding tag18. Purification of the conjugate 19 by RP-HPLC was importantat this stage since a small percentage (~12%) of α-configuredproduct also formed. The ethyl esters of 19 were saponified in1 M NaOH followed by a purification of 20 by RP-HPLC andcomplexation with either LaCl3 or DyCl3 in a D2O-imidazolebuffer (Scheme 4.[13b]The conformational behavior of the trigalactosylated bi-sected N-glycan 17 was characterized by using paramagneticNMR.[14] We have previously implemented this approach todetermine the conformation of a non-bisected biantennary N-glycan and the same methodology has been employed in thiswork.[13a] The good HSQC-signal dispersion obtained for theparamagnetic dysprosium complex 20b allowed full resolutionof the anomeric signals of GlcNAc residues 5 and 5’ and thethree galactoses (G, G’ and GB), which overlapped in thediamagnetic complex 20a (Scheme 5A, B). In total 58 PCSs wereobtained and used for conformation analysis (Scheme 5C). ThePCS depends on the distance and the orientation of the NMRnuclei relative to the paramagnetic metal ion and thus providesstructural information. The experimental PCs were correlatedwith the back-calculated values from the different minimumenergy conformations of the N-glycan by using Mspinsoftware.[15] Iterative calculations were carried out with Mspin toobtain the number of conformers and the population of eachconformer that better fit the experimental PCS values. The bestcorrelation between experimental and calculated data wasobtained considering three interconverting main conformationsfor the Man4’α1,6-Man3 linkage, extended gauche–gauche (gg),extended gauche–trans (gt) and folded gg conformation, withpopulations of 40, 28, and 32%, respectively (Scheme 5D).Scheme 4. Synthesis of the trigalactosylated bisected N-glycan azide 17,conjugation with lanthanide binding tag 18, deprotection and formation ofthe diamagnetic complex 20a and the paramagnetic complex 20b.Scheme 5. A), B) HSQC sections of the anomeric region of the diamagnetic(20a) and paramagnetic complex (20b); the three galactose signals arewithin the dotted lines. C) PCS of paramagnetic complex 20b. D) Relativepercentages of the main conformations calculated from PCS.ChemBioChemCommunicationsdoi.org/10.1002/cbic.2020002683214ChemBioChem 2020, 21, 3212–3215 www.chembiochem.org © 2020 The Authors. ChemBioChem published by Wiley-VCH GmbHWiley VCH Mittwoch, 30.12.20202022 - closed* / 171929 [S. 3214/3215] 1123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657The main conformations are the extended gg and gtconformers as also found in the corresponding non-bisectedbiantennary N-glycan.[13a] However, a higher population (32%)of a single folded conformation (ψ=90°) was obtained for thegalactosylated bisected glycan compared with the non-bisectedbiantennary structure, where two folded conformations werefound (10% ψ=60° and 10% ψ=90°). Notably, the galactosy-lated bisected branch affects the preferred conformations ofthe N-glycan leading to a threefold increase of a single foldedgeometry of the α1,6 arm. The extended gt conformationserved as a starting point for a preliminary model where thebisecting GlcNAc of 12 could be placed in the acceptor site ofbovine galactosyltransferase (PDB ID: 1TW5)[16] with only minoradjustments of the 1,3 arm (see Schemes S7 and S8 in theSupporting Information). We are currently investigating theconformational preferences of complex N-glycans with thetypical bisecting GlcNAc-modification lacking the additionalgalactose unit.In summary a rare N-glycan identified in human serum IgGcould be synthesized by using stringent conditions for theenzymatic galactosylation of the bisecting GlcNAc moiety. Thisparticular conversion appears to be limited to biantennary-N-glycans. The conformational analysis of a conjugate of theglycan bearing the galactosylated bisecting GlcNAc motif usingparamagnetic NMR revealed two extended conformers but onlya single folded geometry.AcknowledgementsWe are grateful for the funding from the Deutsche Forschungsge-meinschaft and Bavaria California Technology Center (BaCaTeC).We are also grateful to the Agencia Estatal de Investigacion andMINECO of Spain for grant CTQ2016-76263-P, and support fromgrant GM62116 from the US National Institutes of Health. Wethank Prof. Javier Pérez Castells from Universidad San Pablo-CEUfor providing the protected lanthanide tag.Conflict of InterestThe authors declare no conflict of interest.Keywords: N-glycans · glycosylation · glycobiology ·immunoglobulin · paramagnetic NMR spectroscopy[1] a) T. Shinkawa, K. Nakamura, N. Yamane, E. Shoji-Hosaka, Y. Kanda, M.Sakurada, K. Uchida, H. Anazawa, M. Satoh, M. Yamasaki, N. Hanai, K.Shitara, J. Biol. Chem. 2003, 278, 3466–3473; b) P. Sondermann, A.Pincetic, J. Maamary, K. Lammens, J. V. Ravetch, Proc. Natl. Acad. Sci. USA2013, 110, 9868–9872; c) T. Li, D. J. Di Lillo, S. Bournazos, J. P. Giddens,J. V. Ravetch, L. X. Wang, Proc. Natl. Acad. Sci. USA 2017, 114, 3485–3490; d) R. M. Anthony, F. Nimmerjahn, D. J. Ashline, V. N. Reinhold, J. C.Paulson, J. V. Ravetch, Science 2008, 320, 373–376.[2] Y. Takegawa, K. Deguchi, H. Nakagawa, S. Nishimura, Anal. Chem. 2005,77, 6062–6068.[3] D. J. Harvey, M. Crispin, C. Scanlan, B. B. Singer, L. Lucka, V. T. Chang,C. M. Radcliffe, S. Thobhani, C. T. Yuen, P. M. Rudd, Rapid Commun. MassSpectrom. 2008, 22, 1047–1052.[4] Y. Wang, J. Tan, M. Sutton-Smith, D. Ditto, M. Panico, R. M. Campbell,N. M. Varki, J. M. Long, J. Jaeken, S. R. Levinson, A. Wynshaw-Boris, H. R.Morris, D. Le, A. Dell, H. Schachter, J. D. Marth, Glycobiology 2001, 11,1051–1070.[5] I. Prahl, C. Unverzagt, Angew. Chem. Int. Ed. 2002, 41, 4259–4262; Angew.Chem. 2002, 114, 4434–4437.[6] O. Blixt, S. Head, T. Mondala, C. Scanlan, M. E. Huflejt, R. Alvarez, M. C.Bryan, F. Fazio, D. Calarese, J. Stevens, N. Razi, D. J. Stevens, J. J. Skehel,I. van Die, D. R. Burton, I. A. Wilson, R. Cummings, N. Bovin, C. H. Wong,J. C. Paulson, Proc. Natl. Acad. Sci. USA 2004, 101, 17033–17038.[7] M. Mönnich, S. Eller, T. Karagiannis, L. Perkams, T. Luber, D. Ott, M.Niemietz, J. Hoffman, J. Walcher, L. Berger, M. Pischl, M. Weishaupt, C.Wirkner, R. G. Lichtenstein, C. Unverzagt, Angew. Chem. Int. Ed. 2016, 55,10487–10492; Angew. Chem. 2016, 128, 10643–10648.[8] T. Luber, M. Niemietz, T. Karagiannis, M. Mönnich, D. Ott, L. Perkams, J.Walcher, L. Berger, M. Pischl, M. Weishaupt, S. Eller, J. Hoffman, C.Unverzagt, Angew. Chem. Int. Ed. 2018, 57, 14543–14549.[9] D. Ott, J. Seifert, I. Prahl, M. Niemietz, J. Hoffman, J. Guder, M. Mönnich,C. Unverzagt, Eur. J. Org. Chem. 2012, 5054–5068.[10] C. Unverzagt, Angew. Chem. Int. Ed. Engl. 1996, 35, 2350–2353; Angew.Chem. 1996, 108, 2507–2510.[11] C. Unverzagt, H. Kunz, J. C. Paulson, J. Am. Chem. Soc. 1990, 112, 9308–9309.[12] a) S. André, C. Unverzagt, S. Kojima, M. Frank, J. Seifert, C. Fink, K.Kayser, C. W. von der Lieth, H. J. Gabius, Eur. J. Biochem. 2004, 271, 118–134; b) S. André, T. Kozar, R. Schuberth, C. Unverzagt, S. Kojima, H. J.Gabius, Biochemistry 2007, 46, 6984–6995.[13] a) A. Canales, A. Mallagaray, J. Pérez-Castells, I. Boos, C. Unverzagt, S.André, H. J. Gabius, F. J. Cañada, J. Jiménez-Barbero, Angew. Chem. Int.Ed. 2013, 52, 13789–13793; Angew. Chem. 2013, 125, 14034–14038; b) A.Canales, I. Boos, L. Perkams, L. Karst, T. Luber, T. Karagiannis, G.Domínguez, F. J. Cañada, J. Pérez-Castells, D. Häussinger, C. Unverzagt,J. Jiménez-Barbero, Angew. Chem. Int. Ed. 2017, 56, 14987–14991;Angew. Chem. 2017, 129, 15183–15187.[14] a) S. Yamamoto, T. Yamaguchi, M. Erdelyi, C. Griesinger, K. Kato, Chem.Eur. J. 2011, 17, 9280–9282; b) T. Yamaguchi, Y. Sakae, Y. Zhang, S.Yamamoto, Y. Okamoto, K. Kato, Angew. Chem. Int. Ed. 2014, 53, 10941–10944; Angew. Chem. 2014, 126, 11121–11124.[15] a) http://www.mestrelab.com Program Mspin; b) A. Navarro-Vazquez,Magn. Reson. Chem. 2012, 50, 73–9.[16] B. Ramakrishnan, E. Boeggeman, P. K. Qasba, Biochemistry 2004, 43,12513–12522.Manuscript received: April 30, 2020Revised manuscript received: June 26, 2020Accepted manuscript online: June 28, 2020Version of record online: August 19, 2020ChemBioChemCommunicationsdoi.org/10.1002/cbic.2020002683215ChemBioChem 2020, 21, 3212–3215 www.chembiochem.org © 2020 The Authors. ChemBioChem published by Wiley-VCH GmbHWiley VCH Mittwoch, 30.12.20202022 - closed* / 171929 [S. 3215/3215] 1

Efficient Chemoenzymatic Synthesis of N-Glycans with a β1,4-Galactosylated Bisecting GlcNAc Motif

https://epub.uni-bayreuth.de/5260/1/cbic.202000268.pdf

Efficient Chemoenzymatic Synthesis of N-Glycans with a β1,4-Galactosylated Bisecting GlcNAc Motif

Abstract

Similar works

Full text

Available Versions

EPub Bayreuth