We demonstrate the expected preference of an immobilised oligosaccharide Man(9)(GlcNAc)(2) upon a 96-well photochemical array, for its known receptor, the cell-surface lectin Dendritic Cell-Specific ICAM3 Grabbing Nonintegrin (DC-SIGN) when compared to immobilised competing monosaccharides

Clark, Andrew J.

Dilly, Suzanne J.

Marsh, Andrew

Mitchell, Daniel A.

Taylor, Paul C.

English

Suzanne J. Dilly

Andrew J. Clark

Daniel A. Mitchell

Andrew Marsh

Paul C. Taylor

Crossref

Molecular BioSystems

Using the Man<sub>9</sub>(GlcNAc)<sub>2</sub>–DC-SIGN pairing to probe specificity in photochemical immobilization

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 University of Warwick institutional repository: http://go.warwick.ac.uk/wrap  This paper is made available online in accordance with publisher policies. Please scroll down to view the document itself. Please refer to the repository record for this item and our policy information available from the repository home page for further information.  To see the final version of this paper please visit the publisher’s website. Access to the published version may require a subscription.  Author(s): Suzanne J. Dilly, Andrew J. Clark, Daniel A. Mitchell, Andrew Marsh, Paul C. Taylor Article Title: Using the Man9(GlcNAc)2 – DC-SIGN pairing to probe specificity in photochemical immobilization Year of publication: Not yet published Link to published article: None Publisher statement:  None   CREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 3.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS ARTICLE TYPE www.rsc.org/xxxxxx  |  XXXXXXXX This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00  |  1 Using the Man9(GlcNAc)2 – DC-SIGN pairing to probe specificity in photochemical immobilization  Suzanne J. Dilly,a Andrew J. Clark,a Daniel A. Mitchell,*b Andrew Marsh,*a Paul C. Taylor*a Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X First published on the web Xth XXXXXXXXX 200X 5 DOI: 10.1039/b000000x We demonstrate specificity of an immobilised oligosaccharide Man9(GlcNAc)2 upon a 96-well photochemical array, for its known receptor, the cell-surface lectin Dendritic Cell–Specific ICAM3 Grabbing Nonintegrin (DC-SIGN). 10  Oligosaccharide surface display is used widely in medicinal chemistry discovery programmes,1, 2 glycomic approaches to understanding function,3 and associated biophysical measurements.  Display methods often presume that a 15 particular regiochemical point of attachment will lead to a desired bioactive partnership, however this may not always be the case,4 potentially resulting in sub-optimal activity.  In particular, a limited range of surface coupling chemistries are often utilized,5, 6 which, compounded by the low availability 20 of saccharide to be immobilized leads to real synthetic and biological challenges. The design of high-throughput assays for lectins has also been noted to be complicated by their tendency to bind weakly to monovalent carbohydrate ligands, hence methods for mimicking multivalency such as that found 25 in the Man9GlcNAc2/DC-SIGN pairing continue to be developed.7, 8 We here demonstrate that an array of photoactivatable chemistries 1-5, Figure 1, in a standard 96-well format9 allows rapid immobilisation of both mono- and oligosaccharides in a fashion that retains their anticipated 30 biological activity.  Recent work has shown the applicability of related surface-mediated photoactivation strategies10 towards similar goals.11, 12   DC-SIGN is a human lectin primarily expressed on the surface of dendritic cells, and is known to interact with host 35 glycoproteins of the immune system such as ICAM-3 via binding to selected glycans.13  In addition, it also binds to carbohydrate structures on the surfaces of pathogens including HIV-1 and Mycobacterium tuberculosis. 14, 15    40  a Department of Chemistry, University of Warwick, Coventry CV4 7AL. Fax: +44 24 7652 4112; Tel: +44 24 7652 4565;  E-mail: a.marsh@warwick.ac.uk Fax: +44 24 7652 4112; Tel: +44 24 7652 4375;  45 E-mail: p.c.taylor@warwick.ac.uk b Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV2 2DX. E-mail: d.mitchell@warwick.ac.uk Tel. +44 24 7696 8596 50  † Electronic Supplementary Information (ESI) available: immobilisation of saccharides and screening versus biotin-conjugated DC-SIGN. See DOI: 10.1039/b000000x/  55   Glycan array and radioligand competition assays have shown that soluble recombinant DC-SIGN binds selectively to branched N-linked high mannose oligosaccharides, notably 60 Man9GlcNAc2, 7 (Figure 2, a component of the gp120 envelope glycoprotein of HIV-1) and also to fucosylated oligosaccharides such as Lewis-x and Blood Groups A and B.16, 17    In competition assays, monosaccharides such as mannose 65 and fucose interact with DC-SIGN with KI values between 0.6 and 1.0 mM, but in the case of Man9GlcNAc2, the KI value is significantly lower at 16 µM, indicating higher affinity.17  Crystallographic studies have shown that this enhanced affinity for selected oligomeric glycan structures is supported 70 by extended binding surfaces of the lectin domain, allowing for multiple sites of protein-oligosaccharide contact.18  However, our understanding of the extent of DC-SIGN ligand identity and mode of binding remains incomplete.  75 Figure 1 Photochemistries 1 – 5 on ethylene glycol linker derivatised polystyrene 96-well plates.   We have demonstrated that Magic Tag®, a chemical genomics tool developed in our laboratories9, 19 can be applied 80 to reveal potentially interesting interactions between small biologically active molecules and polypeptides from a phage displayed library representing a proteome.  Of particular relevance is our earlier study in which we immobilised abscisic acid.9  Extension of our easy-to-use array of 85 photochemistries including a diazirine10 1, two aryl azides20  2, 3 and two benzophenones20  4, 5 to explore binding events between saccharides and lectins is an appealing concept, but we had not previously validated Magic Tag® for this purpose. Herein, we demonstrate that the known specificities of DC-90 SIGN in solution is maintained towards three immobilized monosaccharides. NNF3CON3ON3NHSOOOO1 2 3 4 5HNOOONH2aminopropyl polystyrene plateHNOOOClOO 2  |  Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] Results Photochemical immobilization on surface tethered chemistries 1 – 5 together with Corning® photochemistry Universal-BIND™ of Man9GlcNAc2 (Figure 2), the known ligand for DC-SIGN revealed that diazirine 1 gave the strongest 5 response when challenged with biotin-conjugated DC-SIGN and subsequently exposed to fluorescein isothiocyanate labelled anti-biotin (Figure 3).  The same pattern of relative fluorescence was also seen when D-mannose, 6 (the ligand used in solid-phase to purify soluble recombinant DC-SIGN 10 from bacterial cell extracts) was similarly immobilized and again exposed to DC-SIGN and streptavidin conjugates.  Figure 2 Structures of D-mannose 6 and a known ligand for DC-SIGN, Man9GlcNAc2 7. 15   Figure 3 Response of derivatised surfaces 1 - 5, blank reference and Universal-BIND™ to biotin-conjugated DC-SIGN probed with fluorescein isothiocyanate labelled anti-20 biotin.   In order to further explore the specificity of these novel readily prepared glyco-surfaces, two further monosaccharides, D-fucose  and D-glucose (Figure 4) were immobilized using 25 the same array of photochemistries (Figure 5).  In turn, these surfaces gave lower fluorescent responses than D-mannose or the natural ligand, reflecting the known affinities of these monosaccharides (L-fucose  6.7±0.5 mM, D-mannose KI = 13.1±0.4 mM and D-glucose, KI = 23±1 mM respectively ).17  30 Note the difference in expected vs. our observed response for fucose may arise because we used D-fucose herein rather than L-fucose used in the earlier solution assay, both of which occur in nature.  35      40 Figure 4 Structures of D-glucose and D-fucose.    Figure 5 Comparison of D-glucose (front), D-fucose 45 (middle) and D-mannose (back) immobilized on the photochemical array 1-5 and Universal-BIND™.   Overall, the results demonstrate that Magic Tag® has significant potential in the lectin field. The successful rapid 50 immobilization of a complex natural ligand for DC-SIGN Man9(GlcNAc)2 that subsequently gave a positive response in our assay is very pleasing.  A second important observation is that the specific nature of the interaction between Man9GlcNAc2 and DC-SIGN (KI 55 = 16 µM)17 appears to have been maintained. Both D-glucose and D-fucose, which are known to have lower binding constants with respect to DC-SIGN, gave the expected weak responses in our assay. On the other hand, D-mannose, which does bind to DC-SIGN, gave a significant response. 60  The data contain some interesting subtleties. Firstly, diazirine 1 shows greatest response to DC-SIGN when the known ligands Man9GlcNAc2, D-mannose and D-fucose are immobilized.  3-Trifluoromethyl-3-phenyl diazirine has previously been found to be a good photocrosslinker for DNA, 65 with markedly different reactivity from both aryl azides and benzophenones.  Our observations, taken in conjunction with =  7 Man9(GlcNAc)2 =  6  D-mannoseOHHOHHOHOHHOHOHD-glucoseOHHOHHOHOHHOHD-fucoseOOHHOHHOHOHHHOHOHHOHHOOOHHHOHOHHOHHOHHHOHHOHHOHHHOHOHHOHHOOHHHOHOHHOHHOHHHOOHHOHHOHOOHHHOHOHHOHHOOHHHOHOHOHHOHHAcHNHOHOHOHOHHOHHAcHNHOH7 Man9(GlcNAc)2HHOHHOHOOHHHOHOOHHOHHOOHHHOHOH7 D-mannosemannose fucose glucose -2000 0 2000 4000 6000 8000 10000 12000 14000 16000 blank 3 2 1 5 4 universal fluorescence  This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol], 00–00  |  3 work to delineate surface vs. solution chemoselectivities21 indicate the diazirine moiety to be especially useful in saccharide C-H or O-H bond insertions. Secondly, the responses to Man9GlcNAc2 and to mannose are closer than might have been expected from literature binding competition 5 data.17 This observation, while at first surprising, is in fact consistent with literature observations of surface density and avidity effects on immobilisation of saccharide ligands.8  In our case, it may be that when the monosaccharide mannose is immobilized, clusters of the sugar result that mimic to some 10 extent the natural ligand, Man9GlcNAc2, or that receptor oligomerization may be arising at the glycosylated surface.  Indeed, the interaction between DC-SIGN and mannan, a natural oligomer of mannose, has been observed by 1D saturation transfer difference NMR22 and surface-mediated 15 DC-SIGN oligomerization itself has been postulated to play a key role in pathogen infectivity.23  Conclusions The experiments described in this communication suggest that Magic Tag® photoimmobilisation is an efficient method for 20 the capture of saccharides in order to probe their interactions with lectins. Molecular recognition of the immobilised sugars by DC-SIGN reproduced the known binding profile of this lectin, though mannose gave a surprisingly strong response, probably due to surface-mediated avidity effects. The use of a 25 range of chemical functionalities in parallel enables rapid evaluation of conditions for photocapture and recognition of synthetically challenging small molecule ligands. Acknowledgements We thank Professor Richard Napier and Dr Andrew J. 30 Thompson (Warwick HRI) for helpful discussions, BBSRC & EPSRC ExGen programme  (SJD,  ref. 88/EGM17690) and BBSRC Follow-on Fund (SJD, ref. BB/C524338/1) for funding. DAM is a Research Councils UK Academic Fellow. Notes and references 35  1. C. Y. Wu, P. H. Liang and C. H. Wong, Organic & Biomolecular Chemistry, 2009, 7, 2247-2254. 2. Y. Liu, A. S. Palma and T. Feizi, Biological Chemistry, 2009, 390, 647-656. 40 3. N. Laurent, J. Voglmeir and S. L. Flitsch, Chem. Commun., 2008, 4400-4412. 4. J. J. Tate, J. Persinger and B. Bartholomew, Nucleic Acids Research, 1998, 26, 1421-1426. 5. E. Clo, O. Blixt and K. J. Jensen, European Journal of Organic 45 Chemistry, 2010, 540-554. 6. M. Kohn, J. Pept. Sci., 2009, 15, 393-397. 7. Y. M. Chabre and R. Roy, Curr. Top. Med. Chem., 2008, 8, 1237-1285. 8. N. Jayaraman, Chem. Soc. Rev., 2009, 38, 3463-3483. 50 9. S. J. Dilly, M. J. Bell, A. J. Clark, A. Marsh, R. M. Napier, M. J. Sergeant, A. J. Thompson and P. C. Taylor, Chem. Commun., 2007, 2808-2810. 10. N. Kanoh, S. Kumashiro, S. Simizu, Y. Kondoh, S. Hatakeyama, H. Tashiro and H. Osada, Angewandte Chemie-International Edition, 55 2003, 42, 5584-5587. 11. L. H. Liu, H. Dietsch, P. Schurtenberger and M. D. Yan, Bioconjugate Chemistry, 2009, 20, 1349-1355. 12. O. Norberg, L. Q. Deng, M. D. Yan and O. Ramstrom, Bioconjugate Chemistry, 2009, 20, 2364-2370. 60 13. T. B. H. Geijtenbeek, R. Torensma, S. J. van Vliet, G. C. F. van Duijnhoven, G. J. Adema, Y. van Kooyk and C. G. Figdor, Cell, 2000, 100, 575-585. 14. T. B. H. Geijtenbeek, D. S. Kwon, R. Torensma, S. J. van Vliet, G. C. F. van Duijnhoven, J. Middel, I. Cornelissen, H. Nottet, V. N. 65 KewalRamani, D. R. Littman, C. G. Figdor and Y. van Kooyk, Cell, 2000, 100, 587-597. 15. A. Tanne, B. Ma, F. Boudou, L. Tailleux, H. Botella, E. Badell, F. Levillain, M. E. Taylor, K. Drickamer, J. Nigou, K. M. Dobos, G. Puzo, D. Vestweber, M. K. Wild, M. Marcinko, P. Sobieszczuk, L. 70 Stewart, D. Lebus, B. Gicquel and O. Neyrolles, Journal of Experimental Medicine, 2009, 206, 2205-2220. 16. Y. Guo, H. Feinberg, E. Conroy, D. A. Mitchell, R. Alvarez, O. Blixt, M. E. Taylor, W. I. Weis and K. Drickamer, Nature Structural & Molecular Biology, 2004, 11, 591-598. 75 17. D. A. Mitchell, A. J. Fadden and K. Drickamer, J. Biol. Chem., 2001, 276, 28939-28945. 18. H. Feinberg, D. A. Mitchell, K. Drickamer and W. I. Weis, Science, 2001, 294, 2163-2166. 19. S. R. Ladwa, S. J. Dilly, A. J. Clark, A. Marsh and P. C. Taylor, 80 Chemmedchem, 2008, 3, 742-744. 20. S. A. Fleming, Tetrahedron, 1995, 51, 12479-12520. 21. N. Kanoh, T. Nakamura, K. Honda, H. Yamakoshi, Y. Iwabuchi and H. Osada, Tetrahedron, 2008, 64, 5692-5698. 22. S. Mari, D. Serrano-Gomez, F. J. Canada, A. L. Corbi and J. 85 Jimenez-Barbera, Angewandte Chemie-International Edition, 2005, 44, 296-298. 23. D. Serrano-Gomez, E. Sierra-Filardi, R. T. Martinez-Nunez, E. Caparros, R. Delgado, M. A. Munoz-Fernandez, M. A. Abad, J. Jimenez-Barbero, M. Leal and A. L. Corbi, J. Biol. Chem., 2008, 283, 90 3889-3903.   4. FITC anti-biotinABCDEFGH1 2 3 4 5 6 7 8 9 10 11 12NNF3CON3O3. DC-SIGN - biotin1. Man9(GlcNAc)2 vs.    mannose2. light 254 nmFluorescesaccharideDC-SIGN - biotin

Using the Man9(GlcNAc)2 – DC-SIGN pairing to probe specificity in photochemical immobilization

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Using the Man9(GlcNAc)2 – DC-SIGN pairing to probe specificity in photochemical immobilization

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