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A Sialylated Glycan Microarray Reveals Novel Interactions ofModified Sialic Acids with Proteins and Viruses*□S

Received for publication, June 20, 2011, and in revised form, July 7, 2011 Published, JBC Papers in Press, July 12, 2011, DOI 10.1074/jbc.M111.274217

Xuezheng Song‡, Hai Yu§, Xi Chen§, Yi Lasanajak‡, Mary M. Tappert¶, Gillian M. Air¶, Vinod K. Tiwari§1,Hongzhi Cao§2, Harshal A. Chokhawala§3, Haojie Zheng§, Richard D. Cummings‡4, and David F. Smith‡5

From the ‡Department of Biochemistry and the Glycomics Center, Emory University School of Medicine, Atlanta, Georgia 30322,the §Department of Chemistry, University of California, Davis, California 95616, and the ¶Department of Biochemistry andMolecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73126

Many glycan-binding proteins in animals and pathogens rec-ognize sialic acid or its modified forms, but their molecular rec-ognition is poorly understood.Herewedescribe studies on sialicacid recognition using a novel sialylated glycanmicroarray con-tainingmodified sialic acids presented on different glycan back-bones. Glycans terminating in �-linked galactose at the non-reducing end andwith an alkylamine-containing fluorophore atthe reducing endwere sialylated by a one-pot three-enzyme sys-tem to generate �2–3- and �2–6-linked sialyl glycans with 16modified sialic acids. The resulting 77 sialyl glycans were puri-fied and quantified, characterized by mass spectrometry, cova-lently printed on activated slides, and interrogated with a num-ber of key sialic acid-binding proteins and viruses. Sialic acidrecognition by the sialic acid-binding lectins Sambucus nigraagglutinin andMaackia amurensis lectin-I, which are routinelyused for detecting �2–6- and �2–3-linked sialic acids, areaffected by sialic acid modifications, and both lectins bindglycans terminating with 2-keto-3-deoxy-D-glycero-D-galac-tonononic acid (Kdn) and Kdn derivatives stronger than thederivatives ofmore commonN-acetylneuraminic acid (Neu5Ac)andN-glycolylneuraminic acid (Neu5Gc). Three human parain-fluenza viruses bind to glycans terminating with Neu5Ac orNeu5Gc and some of their derivatives but not to Kdn and itsderivatives. Influenza A virus also does not bind glycans termi-nating in Kdn or Kdn derivatives. An especially novel aspect ofhuman influenza A virus binding is its ability to equivalently

recognize glycans terminated with either �2–6-linkedNeu5Ac9Lt or �2–6-linked Neu5Ac. Our results demonstratethe utility of this sialylated glycan microarray to investigate thebiological importance of modified sialic acids in protein-glycaninteractions.

Protein-glycan interactions play important roles in cellularadhesion, signal transduction, and host-pathogen interactionsand are mediated through specific glycan recognition by gly-can-binding proteins (1–3). Sialic acids (Sia),6 which are targetsfor glycan-binding protein recognition because they commonlyoccur at the non-reducing ends of glycans, are uniquely diverseamong monosaccharides in that many different modified sialicacids have been identified (4). Sialylation of cell surface glyco-conjugates is highly regulated by sialyltransferases and/or siali-dases (5) where the most common enzymes add or cleave sialicacid in �2–3, �2–6, or �2–8 linkages to underlying glycanstructures.Sialylated glycans play important roles in the pathogenesis of

many microorganisms. The species specificity of influenza Avirus is associated with differential binding of the virus to sialicacids linked either �2–3 or �2–6 to galactose on cell surfaceglycoconjugates (6). However, modification of sialic acids, suchas hydroxylation, that converts N-acetylneuraminic acid(Neu5Ac) toN-glycolylneuraminic acid (Neu5Gc), also plays animportant role in sialic acid ligand recognition by pathogensand is a component of the surface receptor for certain bacterialtoxins (7). Humans, unlike most other mammals includingother primates, are unable to naturally synthesize Neu5Gc (8,9), although they can incorporate it from dietary sources (10).Overall, the most commonly studied sialic acids are Neu5Ac,Neu5Gc, and their 9-O-acetylated derivatives (Neu5,9Ac2 andNeu5Gc9Ac) (11–14). The investigation of sialosides contain-ing more diverse sialic acids was limited until recent successes

* This work was supported, in whole or in part, by National Institutes of Health(NIH) Grant RO1GM085448 (to D. F. S.), a bridging grant from the Consor-tium for Functional Glycomics under NIGMS, NIH Grant GM62116 (toR. D. C.), and NIH Grant GM076360 (to X. C.). This work was also supportedby Defense Advanced Research Projects Agency Grant HR0011-10-00 (toR. D. C.) and Oklahoma Center for the Advancement of Science and Tech-nology Grant HR09-001 (to G. M. A.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1 and Tables 1– 4.

1 Present address: Dept. of Chemistry, Banaras Hindu University, Varanasi221005, India.

2 Present address: National Glycoengineering Research Center, ShandongUniversity, Jinan, Shandong 250100, China.

3 Present address: College of Chemistry and Energy Biosciences Institute, Uni-versity of California, Berkeley, Berkeley, CA 94720.

4 To whom correspondence may be addressed: Dept. of Biochemistry, EmoryUniversity School of Medicine, O. Wayne Rollins Research Center, 1510Clifton Rd., Suite 4001, Atlanta, GA 30322. Tel.: 404-727-5962; Fax: 404-727-2738; E-mail: [email protected].

5 To whom correspondence may be addressed: Dept. of Biochemistry, EmoryUniversity School of Medicine, O. Wayne Rollins Research Center, 1510Clifton Rd., Rm. 4035, Atlanta, GA 30322. Tel.: 404-727-6155; Fax: 404-727-2738; E-mail: [email protected].

6 The abbreviations used are: Sia, sialic acid(s); AEAB, 2-amino-N-(2-amino-ethyl)-benzamide; GAEAB, glycan-AEAB conjugate; LNnT, Gal�1-4GlcNAc�1–3Gal�1– 4Glc; LNT, Gal�1–3GlcNAc�1–3Gal�1– 4Glc; NA2,Gal�1-4GlcNAc�1–2Man�1– 6(Gal�1– 4GlcNAc�1–2Man�1–3)Man�1-4GlcNAc�1– 4GlcNAc; NHS, N-hydroxysuccinimide; RFU, relative fluores-cence unit(s); RCA-I, R. communis agglutinin I; SNA, S. nigra agglutinin;MAL-I, M. amurensis lectin-I; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; PGC, porous graphitized carbon; hPIV, humanparainfluenza virus; Kdn, 2-keto-3-deoxy-D-glycero-D-galactonononic ac-id; SGM, sialylated glycan microarray; CFG, Consortium of FunctionalGlycomics.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 36, pp. 31610 –31622, September 9, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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in chemoenzymatic synthesis of sialosides containing naturaland non-natural sialic acid residues using multiple promiscu-ous sialoside biosynthetic enzymes (5, 15–20).We hypothesized that the existence of multiple modified

sialic acid structures has biological importance related to theirinteraction with proteins or microorganisms. To furtherexplore the recognition and function of modified sialic acidresidues, we exploited the development of new chemoenzy-matic methods to synthesize modified sialic acids (5), alongwith recent developments of glycan microarray technology,which provide an innovative approach to studying glycan rec-ognition and function by glycan-binding proteins. We adaptedour recent development of natural glycan microarrays (21–24)to the production of a diverse array of sialylated glycans usingfluorescent derivatives of glycans that terminate with �-linkedgalactose, which are sialyltransferase acceptors, in a one-potthree-enzyme approach for the microscale chemoenzymaticsynthesis of sialosides (19, 20, 25). The resultant microarrayconsists of 77 sialylated glycans incorporating 16modified sialicacids in �2–3 and �2–6 linkages to different underlying struc-tures. This novel sialylated glycan microarray is useful for rap-idly screening glycan-binding proteins and viruses for theirinteractions with a wide variety of modified sialic acids andrepresents a new glycomic technology to explore glycan func-tion and recognition.

EXPERIMENTAL PROCEDURES

Free reducing glycans lactose, lacto-N-tetraose (Gal�1-3GlcNAc�1–3Gal�1–4Glc; LNT), and lacto-N-neotetraose(Gal�1–4GlcNAc�1–3Gal�1–4Glc; LNnT) and all chemi-cals were purchased from Sigma-Aldrich and used with-out further purification. Asialo, biantennary N-glycan(Gal�1–4GlcNAc�1–2Man�1–6(Gal�1–4GlcNAc�1–2Man�1–3)Man�1–4GlcNAc�1–4GlcNAc; NA2) was prepared from achicken egg yolk glycopeptide (26) by mild acid hydrolysis andpeptide:N-glycosidase F digestion. The bifunctional linker,2-amino(N-aminoethyl) benzamide (AEAB), was prepared asdescribed previously (27). HPLC solvents were purchased fromFisher. An Ultraflex-II TOF/TOF system from Bruker Dalton-ics was used for MALDI-TOF mass spectrometry analysis ofglycan conjugates. Biotinylated ConA, Ricinus communisagglutinin I (RCA-I), Sambucus nigra agglutinin (SNA), andMaackia amurensis lectin (MAL-I) were from Vector Labora-tories; Cy5- andAlexa488-labeled streptavidinwere from Invit-rogen; and Arthrobacter ureafaciens sialidase was from Sigma.Glycan-AEAB Conjugation, Sialylation, and Purification—

Free reducing glycans were conjugatedwith AEAB as describedpreviously (27). Briefly, 1–10 mg of glycan was mixed with50–250 �l of AEAB hydrochloride salt solution freshly pre-pared at 0.35 M in DMSO/AcOH (7:3, v/v) followed by an equalvolume of sodium cyanoborohydride solution freshly preparedin the same solvent. Themixture was vortexed briefly and incu-bated at 65 °C for 2 h. Themixturewas chilled, and the productswere precipitated upon the addition of 10 volumes of acetoni-trile. After bringing the suspension to �20 °C and maintainingthat temperature for 30 min, the products were separated bycentrifugation at 10,000 � g for 3 min. The pellet was redis-solved in 200–500 �l of water and purified by HPLC. AEAB

derivatives of LNnT, LNT, and the biantennaryN-glycan, NA2,were prepared as precursors for presentation of modified sialicacids.Sialyl glycans containing various natural modifications of

sialic acid linked �2–3 or �2–6 to the three different fluores-cent glycans described above were synthesized using a highlyefficient one-pot three-enzyme system similar to that reportedpreviously (19, 20, 25, 28). The fluorescent glycan-AEAB con-jugates (GAEABs) of LNT, LNnT, or NA2 (130–200 �g) wereincubated with three enzymes, including Escherichia coli sialicacid aldolase (28) (10–15 �g), Neisseria meningitidis CMP-sialic acid synthetase (28) (10–15 �g), and Photobacteriumdamselae �2–6-sialyltransferase (25) (10–15 �g; used for thesynthesis of �2–6-linked sialosides) or Pasteurella multocida�2–3-sialyltransferase PmST1 (19) (3–6 �g; used for the syn-thesis of �2–3-linked sialosides) in a reaction mixture of 25 �lcontaining 100 mM Tris-HCl buffer, pH 8.5. The reactionmixtures were incubated at 37 °C for 2 h (for preparing �2–3-sialylated glycans using PmST1) or 24 h (for preparing �2–6-sialylated glycans using P. damselae �2–6-sialyltransferase). ATris-HCl buffer, pH 7.5, was used for substrates containing abase sensitive functional group (e.g. acetyl or lactyl group). Thefollowing were added to this mixture: (a) sialic acid precursors(20, 25, 28, 29) (e.g. mannose, N-acetylmannosamine, or theirderivatives) at 2.0 or 4.0 eq relative to LNT or LNnT and NA2(biantennary-AEAB conjugate), respectively; (b) sodium pyru-vate (5 eq for LNT or LNnT, 10 equiv. for NA2); (c) CTP (2.0 eqfor LNT or LNnT, 4.0 eq for NA2); and (d)MgCl2 (20mM). Thereaction mixtures were incubated at 37 °C. Product formationwas monitored by TLC in EtOAc/MeOH/H2O/HOAc (4:2:1:0.1) and visualized by a UV lamp to follow modification of thefluorescent sialic acid acceptor. When the product formationreached a maximum, the reaction mixtures were frozen. Thereactionmixtureswere thenmixedwith 2 volumes of ethanol toprecipitate enzymes and cooled at �20 °C for 30 min. Aftercentrifugation at 10,000� g for 3min, the supernatant contain-ing the products and excess reactants was purified by porousgraphitized carbon (PGC)-HPLC. The collected peaks weredried in a SpeedVac, reconstituted in 100 �l of water, and puri-fied again by PGC-HPLC when necessary. The purified majorproduct of each reaction was reprofiled using ion exchangeHPLC to confirmhomogeneity and charge state and to quantifyeach glycan based on fluorescence and UV. They were alsocharacterized byMALDI-TOF to confirm the generation of theanticipated product of the sialyltransferase from startingN-acetylhexosamine, hexosamine, hexose, or appropriatelymodified derivative, pyruvate, and CTP.High Performance Liquid Chromatography—A Shimadzu

HPLCCBM-20A system, coupled with a UV detector SPD-20Aand a fluorescence detector RF-10Axl, was used forHPLC anal-ysis and separation of GAEABs. The column effluent was mon-itored by UV absorption at 330 nm and/or fluorescence at 330nm excitation and 420 nm emission. Both UV absorption andfluorescence intensity were used for the quantification of theAEAB derivatives using lacto-N-fucopentaose (LNFPIII)-AEAB as a standard. For reverse phase HPLC with a PGC col-umn (150 � 4.6 mm), the mobile phase was acetonitrile andwater with 0.1% trifluoroacetic acid (TFA). For the glycan anal-

A Novel Sialylated Glycan Microarray

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ysis and purification, the concentration of acetonitrileincreased from 15 to 45% in 30 or 15 min. For ion exchangeHPLC using a Varian IonoSpher HPLC column (250 � 4.6mm), an isocratic program with 100 mM ammonium acetate,pH 4.5, was used.Printing, Binding Assay, and Scanning—NHS-activated

slides were purchased from Schott (Louisville, KY). Non-con-tact printing was performed using a Piezorray printer fromPerkinElmer Life Sciences. The printing, binding assay, andscanning conditions were essentially the same as described pre-viously (21, 23). Briefly, all samples were printed at 100 �M inphosphate buffer (300 mM sodium phosphates, pH 8.5) in rep-licates of four. Before the assay, the slides were rehydrated for 5min in TSM buffer (20 mM Tris-HCl, 150 mM sodium chloride(NaCl), 2 mM calcium chloride (CaCl2), and 2 mM magnesiumchloride (MgCl2). Samples are applied to the rehydrated slide inTSM buffer containing 1% BSA and 0.05% Tween 20 in a finalvolume of 70–100 �l under a coverslip that covers the printedarea on the slide, and the slides are incubated in a humidifiedchamber at room temperature for 1 h. After incubation, thecoverslip is gently removed, and excess sample is washed awayby gently dipping the slides in buffer contained in Coplin jars.Biotinylated lectins binding to glycans on the array weredetected by a secondary incubation with cyanine 5- orAlexa488-streptavidin, whereas labeled viruses were assayedimmediately after washing.The slides were scanned with a PerkinElmer Life Sciences

ProScanarray microarray scanner equipped with four laserscovering an excitation range from 488 to 637 nm. For cyanine 5fluorescence, 649 nm (excitation) and 670 nm (emission) wereused. For Alexa488 fluorescence, 495 nm (excitation) and 510nm (emission) were used. All images obtained from the scannerare in grayscale and colored for easy discrimination. Thescanned imageswere analyzedwith the ScanArray Express soft-ware, which provided raw values in relative fluorescence units(RFU) from each spot in an Excel spreadsheet. The data fromthe scanner were processed in the Excel workbook to averageand determine the S.D. of the four replicates. The %CV (coeffi-cient of variation expressed inpercent) is calculated as 100�S.D./

mean. For any value with a %CV of�30, the data are evaluated todetermine the presence of anunacceptable value. The range of theacceptance for the raw RFU is the value of one S.D. below andabove average. Thus, if a raw RFU value of the four replicates isoutside therange, it isdiscarded, andonly therawRFUintherangeis used to calculate the final value of average, S.D., and %CV.Virus Preparations—Human parainfluenza and influenza A

viruses were grown in LLC-MK2 cells andMadin-Darby caninekidney cells, respectively, in infectionmediumas described pre-viously (30). Virus was purified through a sucrose gradient at10–60% (human parainfluenza virus; hPIV) or 10–40% (influ-enza), harvested by ultracentrifugation, resuspended in calci-um-magnesium saline, and labeled directly with AlexaFluor488 (Molecular Probes) as described previously (31).

RESULTS

Preparation of Sialylated GAEABs—We chose a syntheticstrategy that combined two earlier developments, the bifunc-tional fluorescent tagAEAB (27) and the one-pot three-enzymesialylation reaction (19, 20, 25) (Fig. 1). GAEABs are fluorescentand enable us to isolate and process glycans on a microscale. Inaddition, they have a primary amino group for immobilizationonto activated glass surfaces to generate glycan microarrays.Using GAEABs with terminal galactose residues as acceptorsfor sialyltransferases, we explored a one-pot three-enzyme sia-lylation system to synthesize terminally sialylated glycans withvarious sialic acid modifications. The GAEAB acceptorsincluded lactose, LNnT, LNT, and NA2, which were preparedfrom natural glycans as described previously (27) and purifiedprior to themultienzyme sialylation. Aliquots (50�g to 1mg) ofthe GAEABs were subjected to one-pot three-enzyme enzy-matic sialylation in a combinatorial fashion (16). The applica-tion of N-acetylmannosamine, N-glycolylmannosamine, Man,or their derivatives in the enzymatic reactions containing asialic acid aldolase, a CMP-sialic acid synthetase, CTP, and asialyltransferase allows formation of corresponding sialosidescontaining terminal Neu5Ac, Neu5Gc, 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn), or their derivatives,respectively, as summarized in supplemental Table 1. Because

FIGURE 1. The design and preparation of an SGM. Fluorescent glycans (GAEABs) terminating in �-linked galactose were acceptors in a one-pot three-enzymesynthetic scheme with pyruvate, CTP and mannose, N-acetylmannosamine, or various derivatives to generate fluorescent sialyl glycans containing �2–3- and�2– 6-linked sialic acid forms. The purified fluorescent sialyl glycans were structurally confirmed and printed as an SGM.




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the enzymatic sialylation efficiency varied dramatically amongdifferent reactions, the products were purified by HPLC. Pro-files of four of the 77 reaction mixtures are shown as examplesin Fig. 2, and the structures and homogeneity of each purifiedsialylated glycan were confirmed by MALDI-TOF and HPLCanalysis shown for each sialyl glycan in supplemental Fig. 1.Because the sialylated glycan products are fluorescent, evensmall amounts of the isolated products (�1 nmol) are accu-rately quantified for printing on NHS-activated glass slides. Asshown in Table 1 and supplemental Table 2, we generated 77�2–3- or �2–6-sialylated structures incorporating 16 differentterminal sialic acids (Fig. 3a) on four different underlying gly-can structures (Fig. 3b) that were tagged with a functional fluo-rescent linker (Fig. 3c). Each glycan was quantified based on itsfluorescence and printed in replicates of n � 4 on NHS-acti-vated glass slides as described previously (27) to provide a sia-lylated glycan microarray (SGM). The selection of this set ofsialic acid structures is based on structures of known impor-tance, natural occurrence, and higher abundance, along withcurrent chemical accessibility. Of the 16 sialic acid structuresselected for synthesis, four (Neu5Ac, Neu5,9Ac2, Neu5Ac9Lt,and Kdn) are known to occur in humans, and only three(Neu5Ac9Me, Kdn9Me, and Kdn7Me) are not known to occuror are yet to be discovered in nature. The large diversity of sialicacid structures on common acceptors printed as a glycanmicroarray facilitates defining the specific interactions of dif-ferent binding proteins and microorganisms.Lectin Binding Confirms Efficient Printing of Sialylated

Glycans—To testwhether the sialylated glycanswere efficientlyprinted on the SGM and to evaluate its utility, we interrogatedit with plant lectins of known specificities (Figs. 4 (lanes 1–4)and 5 (a–d)). ConA, a lectin that binds highmannose-, hybrid-,and complex-type biantennary N-glycans (32), showed strongbinding to all of the sialylated biantennary N-glycans (NA2)possessing the trimannosyl core structure (18–30 and 58–72)(glycan numbers are shown in boldface type), the acceptorbiantennary N-glycan galactoside used for the sialylation reac-tion (79), and the Man5GlcNAc2 N-glycan (80).

RCA-I, which binds to terminal Gal�1–4GlcNAc (32),showed stronger binding, as expected, to bivalent NA2 (79)compared with monovalent LNnT (78). Consistent with itsknown specificity, RCA-I bound glycans with �2–6-linkedsialic acid on Gal�1–4GlcNAc (18–30) but did not bind gly-cans terminating in �2–3-linked sialic acid, except for themonosialylated, biantennary NA2 structures (59–61, 66, 68,and 70–72). The presence of �2–6-linked sialic acid on Galresidues in Gal�1–4GlcNAc interferes with but does not pre-vent binding of RCA-I, as seen by comparing binding to NA2(79) and its disialylated (�2–6) derivative (18). The �2–6monosialylated derivatives of NA2 (28–30) show binding thatwas essentially equivalent to unsubstituted NA2 (79). Glycan20, which is an �2–6 monosialylated derivative of NA2, boundRCA-I before and after neuraminidase, although binding wasnot enhanced after neuraminidase treatment. RCA-I boundweakly to �2–6-sialylated lactose and LNnT (1–17), unlike thecorresponding �2–3-sialylated lactose and LNnT (41–57),which were not bound. Although most sialic acid derivativesmoderately decreased RCA-I binding to NA2, �2–6-sialylationwith Neu5Ac8Me (19) and Neu5Gc9Ac (25) greatly inhibitRCA-I binding.After treatment of the slides with mild acid to remove the

terminal sialic acid from the sialylated glycans on the array,RCA-I binding was generally increased over control levels insialylated type 2 glycans substituted with sialic acids. The acidtreatment slightly reduced binding to the control glycans(LNnT andNA2, 78 and 79) compared with the untreated slideprobably as a result of slight damage to the derivatized surface.Binding of RCA-I to LNnT (3–17 and 31–40) was low butdetectable on the mild acid-treated slide compared with theuntreated slide.When the SGMwas treatedwithA. ureafacienssialidase, we observed increased binding of RCA-I for thoseglycans cleavable by the enzyme as discussed below. These datademonstrate that the sialylated glycans were efficiently printedon the slides. The Neu5Ac8Me derivatives of NA2 (19 and 59)and all Kdn derivatives (67–72) showed no significant increasein binding by RCA-I after sialidase compared with theuntreated and mild acid-treated slides. Although Kdn is gener-ally more resistant than Neu5Ac and Neu5Gc derivatives, 9-O-acylation ofNeu5Ac (6, 7, and 22) andNeu5Gc (10 and 25) alsoincrease their resistance to sialidase. This finding is importantand indicates that caution is needed in studies using plant lec-tins and sialidase treatments of biological samples to exploreexpression of sialylated glycoconjugates.Binding of Sialic Acid-binding Plant Lectins to the SGM—We

analyzed binding of the sialic acid-binding lectins SNAand MAL-I to the SGM. SNA bound to the Sia�2-6Gal�1–4GlcNAc-terminated glycans (LNnT and NA2 back-bones) (Figs. 4 (lane 5) and 6a), which is consistent with itsknown specificity for this sialic acid linkage (33, 34). However,it differentiates among the sialyl derivatives and does notbind either LNnT or NA2 derivatized with �2–6-linkedNeu5Ac8Me on 4 and 19, respectively, suggesting a require-ment of a free 8-OH in sialic acid for SNA binding. Interest-ingly, SNA binds poorly to Sia�2–6 derivatives of LNT, whichcontains penultimate Gal�1–3GlcNAc, but there is significantbinding to the Kdn�2–6 derivatives of LNT. This is consistent

FIGURE 2. HPLC profiles of LNnT-AEAB and several sialylated products,includingNeu5Ac�2–6-LNnT-AEAB,Neu5Gc�2–6-LNnT-AEAB,Neu5Ac�2-3-LNnT-AEAB, and Neu5Gc�2–3-LNnT-AEAB (from bottom to top). The posi-tion of elution of the precursor, LNnT-AEAB, is indicated in the bottom profile.




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with the strong binding of SNA toKdn�2–6 derivatives of NA2(27–30) compared with the corresponding Neu5Ac andNeu5Gc derivatives of NA2 (18, 20, and 23–26).

MAL-I, which is specific for Sia�2–3Gal�1–4GlcNAc-R-terminated glycans (35), bound only sialylated glycans termi-nating with that sequence (Figs. 4 (lane 8) and 6d). MAL-I

TABLE 1The chart ID and glycan structures of the SGMStructures marked with an asterisk are monosialylated biantennary glycans. The exact branch to which the sialic acids are attached was not determined.

Chart ID Structure 1 Neu5Acα6Galβ4Glcitol-AEAB 2 Neu5Gcα6Galβ4Glcitol-AEAB 3 Neu5Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 4 Neu5Ac8Me α6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 5 Neu5Ac9Meα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 6 Neu5,9Ac2α6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 7 Neu5Ac9Ltα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 8 Neu5Gcα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 9 Neu5GcMeα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 10 Neu5Gc9Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 11 Neu5GcAcα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 12 Kdnα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 13 Kdn9Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 14 Kdn5Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 15 Kdn5,9Ac2α6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 16 Kdn9Meα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 17 Kdn7Meα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 18 Neu5Acα6Galβ4GlcNAcβ2Manα3(Neu5Acα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 19 Neu5Ac8Meα6Galβ4GlcNAcβ2Manα3(Neu5Ac8Meα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 20 *Neu5Ac9Meα6Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 21 Neu5,9Ac2α6Galβ4GlcNAcβ2Manα3(Neu5,9Ac2α6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 22 Neu5Ac9Ltα6Galβ4GlcNAcβ2Manα3(Neu5Ac9Ltα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 23 Neu5Gcα6Galβ4GlcNAcβ2Manα3(Neu5Gcα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 24 Neu5GcMeα6Galβ4GlcNAcβ2Manα3(Neu5GcMeα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 25 Neu5Gc9Acα6Galβ4GlcNAcβ2Manα3(Neu5Gc9Acα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 26 Neu5GcAcα6Galβ4GlcNAcβ2Manα3(Neu5GcAcα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 27 Kdn5Acα6Galβ4GlcNAcβ2Manα3(Kdn5Acα6Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 28 *Kdn9Meα6Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 29 *Kdn5Meα6Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 30 *Kdn7Meα6Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 31 Neu5Acα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 32 Neu5,9Ac2α6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 33 Neu5Ac9Ltα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 34 Neu5Gcα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 35 Neu5GcMeα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 36 Neu5GcAcα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 37 Kdnα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 38 Kdn9Acα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 39 Kdn5Acα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 40 Kdn5Meα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 41 Neu5Acα3Galβ4Glcitol-AEAB 42 Neu5Gcα3Galβ4Glcitol-AEAB 43 Neu5Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 44 Neu5Ac8Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 45 Neu5Ac9Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 46 Neu5,9Ac2α3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 47 Neu5Ac9Ltα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 48 Neu5Gcα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 49 Neu5GcMeα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 50 Neu5Gc9Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 51 Neu5GcAcα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 52 Kdnα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 53 Kdn9Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 54 Kdn5Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 55 Kdn5,9Ac2α3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 56 Kdn5Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 57 Kdn7Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 58 Neu5Acα3Galβ4GlcNAcβ2Manα3(Neu5Acα3Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 59 *Neu5Ac8Meα3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 60 *Neu5Ac9Meα3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 61 * Neu5,9Ac2α3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 62 Neu5Ac9Ltα3Galβ4GlcNAcβ2Manα3(Neu5Ac9Ltα3Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 63 Neu5Gcα3Galβ4GlcNAcβ2Manα3(Neu5Gcα3Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 64 Neu5GcMeα3Galβ4GlcNAcβ2Manα3(Neu5GcMeα3Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 65 Neu5Gc9Acα3Galβ4GlcNAcβ2Manα3(Neu5Gc9Acα3Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 66 *Neu5GcAcα3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 67 Kdnα3Galβ4GlcNAcβ2Manα3(Kdnα3Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 68 * Kdn9Acα3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 69 Kdn5Acα3Galβ4GlcNAcβ2Manα3(Kdn5Acα3Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 70 *Kdn9Meα3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 71 *Kdn5Meα3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 72 *Kdn7Meα3Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 73 Neu5Acα3Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 74 Neu5,9Ac2α3Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 75 Neu5Gcα3Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 76 Neu5GcMeα3Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 77 Kdn9Acα3Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 78 Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 79 Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAcitol-AEAB 80 Manα6(Manα3)Manα6(Manα3)Manβ4GlcNAcβ4GlcNAcitol-AEAB




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differentiates among the sialyl derivatives; it does not bind�2–3-sialylated LNnT or NA2 with Kdn5Me on 56 and 71,respectively. This observation and the relatively high binding ofMAL-I for glycans �2–3-sialylated with C5-substituted Kdnderivatives (Kdn5Ac (54 and 69) and Kdn5,9Ac2 (55)) indicatethat an acetyl group on the 5-OH of Kdn is needed for MAL-Ibinding. Like SNA, MAL-I bound better toO-acetylated deriv-atives of Kdn than to corresponding glycans terminated in thecommon sialic acids, Neu5Ac and Neu5Gc. The observationthat MAL-I binds poorly to �2–3-sialylated LNT, which con-tains the subterminal sequence Gal�1–3GlcNAc, is consistentwith the specificity of MAL-I for the type 2 sequenceGal�1–4GlcNAc. Removal of sialic acids from the glycan arrayby mild acid hydrolysis eliminated binding by SNA andMAL-I(Figs. 4 (lanes 7 and 10) and 6 (c and f)), and the residual bindingafter A. ureafaciens sialidase digestion (Figs. 4 (lanes 6 and 9)and 6 (b and e)) is consistent with the earlier observation of thesialidase resistance of the Kdn derivatives.To better evaluate the specificity of these sialic acid-binding

lectins for this diverse group of sialic acid derivatives, we ana-lyzed the binding of lectins in a dose-dependent manner on theSGM as described previously for SNA on a mammalian cellglycan array (34). Briefly, the binding of each lectin at differentconcentrations to each glycanwas normalized to percentages ofthe highest RFU value for each analysis, and the percentage ofmaximum binding for each glycan at the different lectin con-centrations was averaged to obtain an average ranking. The

detailed ranked analyses for SNA andMAL-I are shown in sup-plemental Tables 3 and 4.Assuming that the rankings representstrong binding at high values with rankings below 10% consid-ered weak binders or non-binders, the influence of sialic acidmodification on the SNA andMAL-I binding can be evaluated.This is, however, a complex analysis due to the number of sialicacid modifications and the fact that significant differences inranking are observed between monovalent and divalent inter-actions created by the precursor glycans (LNnT and NA2). Tosimplify the comparisons of the effects of sialic acid modifica-tion on SNA andMAL-I binding, Tables 2 and 3 only show theranking of the potential monovalent glycan ligands for SNA(Sia�2,6 derivatives of the tetrasaccharides) and MAL-I(Sia�2,3 derivatives of LNnT), respectively.Consistent with the known specificity of SNA for glycans

terminating in Sia�2–6Gal�1–4GlcNAc, ranking analysis(Table 2) showed that the highest binding glycans on the arraywere indeed those possessing sialic acids linked �2–6 to type 2precursors. Interestingly, the LNnT terminating with the mostcommon sialic acids, Neu5Ac�2–6- and Neu5Gc�2–6- (3 and8, respectively) were among the weakest of the sialylated gly-cans bound by SNA at a ranking of 8 compared with glycansterminating in modified Kdns (15, 16, 6, 14, 13, 17, and 38) atrankings of �30–100, representing a 10-fold higher level ofbinding for this lectin in certain cases. If Neu5Ac is modifiedwith amethyl group at theC8-position (4), even thisweak inter-action is lost; the 8-O-methyl derivative of Neu5Gc is not avail-

FIGURE 3. Structures of the sialic acid derivatives. a, 16 different sialic acid forms were obtained. b, four different fluorescent glycans were used assialyltransferase acceptors. c, the structure of the bifunctional fluorescent tag used as the linker to covalently couple the glycan to the NHS-derivatized glassslide.




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able (36). On the other hand, if Neu5Ac is derivatized with anO-acetyl group at the 9-position (6), SNA-binding rankincreases to 50.Modification of Neu5Acwith amethyl group atthe C9-position (5) had little effect on binding to SNA, whereasmodification at the C9-position with a larger lactyl group (7)increased binding �3-fold to a ranking of 24. Methylation oracetylation of theN-glycolyl group at C5 in Neu5Gc (9 and 11,respectively) resulted in a slight increase in SNA binding,whereas 9-O-acetylation of Neu5Gc (10), like 9-O-acetyla-tion of Neu5Ac, resulted in a significant increase in SNAbinding. The �2–6Kdn derivative of LNnT (12), which is thesimplest of the sialic acid derivatives, was bound by SNAalmost 3-fold stronger than the corresponding Neu5Ac- andNeu5Gc-terminated LNnT. This binding increased an addi-tional 4-fold when the Kdn was modified by O-acetylation atthe 5- and 9-positions (15); interestingly, this Kdn5,9Ac2

derivative (15) was still 2-fold more strongly bound than thecorresponding Neu5,9Ac2 derivative of LNnT (6). OtherO-acetylated and O-methylated derivatives of Kdn (13, 14,16, and 17) showed increased binding over glycans contain-ing Kdn alone.Consistent with the known specificity of MAL-I for glycans

terminating in Sia�2–3Gal�1–4GlcNAc, ranking analysis ofthis lectin (Table 3) showed that the strongest binding glycanson the array were those possessing sialic acids linked �2–3 totype 2 precursors. Similar to what we observed for SNA, LNnTterminating with the most common sialic acids, Neu5Ac�2–3-and Neu5Gc�2–3- (43 and 48, respectively) were among theweaker of the sialylated glycans bound byMAL-I at rankings of10 and 15, respectively, compared with glycans terminating inmodified Kdns at rankings of 37–100, representing a signifi-cantly higher binding level for this lectin in certain cases. In the

FIGURE 4. Binding of lectins and viruses to the SGM. The y axis represents 77 sialylated glycans and three controls (LNnT, NA2, and Man5), corresponding tochart ID 1– 80. All glycans are AEAB conjugates. The glycans are sorted for discrimination of �2– 6 and �2–3 linkages; the sialic acids Neu5Ac, Neu5Gc, Kdn, andderivatives; and the underlying structures lactose, LNnT, NA2, and LNT. The x axis represents lectins and viruses interrogated on the SGM, shown in individuallanes. All lectins are biotinylated and detected with cyanine 5- or Alexa488-labeled streptavidin. All viruses are labeled with Alexa488. A heat map format is usedto demonstrate the relative binding strength, with red indicating strong and yellow indicating weak. The binding signals of all lectins and viruses are normalizedindividually and shown in individual lanes, except for the sialidase treatments (lanes 3, 6, and 9) and mild acid treatments (lanes 4, 7, and 10), which arenormalized together with corresponding lectins (lanes 2, 5, and 8).

FIGURE 5. Binding of lectin ConA and RCA-I to the SGM. a, the SGM was interrogated with biotinylated Con A (10 �g/ml) followed by Alexa 488-labeledstreptavidin at 1 �g/ml. b, the SGM was interrogated with biotinylated RCA-I (10 �g/ml) followed by Alexa 488-labeled streptavidin at 1 �g/ml. c, as a control,the SGM was digested with A. ureafaciens sialidase (150 milliunits/ml, pH 5.0, 37 °C for 4 h). Then the SGM was interrogated with biotinylated RCA-I (10 �g/ml)followed by Alexa 488-labeled streptavidin at 1 �g/ml. d, as a separate control, after mild acid treatment (0.1 N HCl, 80 °C, 1 h) of the SGM to remove theacid-labile sialic acid, the SGM was interrogated with biotinylated RCA-I (10 �g/ml) followed by Alexa 488-labeled streptavidin at 1 �g/ml.




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case of MAL-I binding, modification of Neu5Ac with a methylgroup at the 8-position (44) resulted in increased binding.Othermodifications ofNeu5Ac, including 9-O-acetylation (46)and 9-O-methylation (45), increased binding by MAL-I,whereas the addition of anO-lactyl group at position 9 (47) hadlittle effect. Although the �2,3-linked Kdn derivative of LNnT(52) shared similar apparent binding levels with the corre-sponding Neu5Ac and Neu5Gc derivatives (43 and 48), MAL-I

binding was very sensitive to modifications of Kdn with signif-icant increases associated with Kdn5,9Ac2 (55), Kdn7Me (57),and Kdn5Ac (54), whereas the binding to the 9-O-acetylderivative of Kdn (53) was unchanged, and the binding to the5-O-methyl derivative of LNnT (56) was severely reduced.These results demonstrate that sialic acid modificationsaffect recognition by SNA and MAL-I in novel ways and thatthe SGM provides a new platform for studying the specific

FIGURE 6. Binding of sialic acid-binding lectins, SNA and MAL-I, to the SGM. a, the SGM was interrogated with biotinylated SNA (10 �g/ml) followed byAlexa 488-labeled streptavidin at 1 �g/ml. b, the SGM was treated with A. ureafaciens sialidase and then interrogated with biotinylated SNA (10 �g/ml) followedby Alexa 488-labeled streptavidin at 1 �g/ml. c, the SGM was treated with mild acid and then interrogated with biotinylated SNA (10 �g/ml) followed by Alexa488-labeled streptavidin at 1 �g/ml. d, the SGM was interrogated with biotinylated MAL-I (10 �g/ml) followed by Alexa 488-labeled streptavidin at 1 �g/ml.e, the SGM was treated with A. ureafaciens sialidase and then interrogated with biotinylated MAL-I (10 �g/ml) followed by Alexa 488-labeled streptavidin at 1�g/ml. f, the SGM was treated with mild acid and then interrogated with biotinylated MAL-I (10 �g/ml) followed by Alexa 488-labeled streptavidin at 1 �g/ml.




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effects of sialic acid modifications on sialic acid-recognizinglectins.The Binding of Viruses to the SGM—Influenza viruses bind

sialic acid on cell surfaces via hemagglutinin, and their specific-ities toward �6-linked sialic acid and �3-linked sialic acid areassociated with their ability to infect humans or birds, respec-tively (6). Their specificities for the various modifications ofsialic acid, however, have not been systematically studied due tothe lack of available glycans. We screened several humanviruses, including influenza A/Oklahoma/447/08 H1N1, influ-

enza A/Oklahoma/483/08 H3N2, and hPIVs hPIV1 (strainC-35), hPIV2, and hPIV3 on the SGM (Fig. 4, lanes 11–15).Although binding of the three strains of hPIV was restricted

to sialic acids in �2–3-linkages to type 2 glycan chains(Gal�1–4GlcNAc) of LNnTandNA2 (Figs. 4 (lanes 11–13) and7 (a–c)), each demonstrated significant differences in bindingto the diverse sialic acids on the SGM. These compounds werenot available on the glycan microarray of the Consortium ofFunctional Glycomics (CFG), on which hPIV1 and hPIV3 wereanalyzed and reported earlier (37). Although hPIV1 and hPIV2bound glycans with similar efficiency based on the RFU values,they demonstrated very different specificities for different sialicacid derivatives. The strictest specificity was observed withhPIV1, which bound to the monovalent Neu5Ac derivative ofLNnT (43) and had stronger interaction with the correspond-ing Neu5GcMe derivative (49), whereas no binding wasobserved with the Neu5Gc derivative (48). This observationsuggests that the virus hemagglutinin prefers the hydrophobiccenter provided by the CH3 group associated with theN-acetylgroup of the sialic acid at the 5-position of Neu5Ac and that themethoxy group of the glycolyl function onNeu5Gcmay even bemore preferred. This is consistent with the observation that thebulkier Neu5GcAc is not bound. Presentation of these sialylderivatives as divalent structures on NA2 (58, 64, and 63) didnot enhance binding by hPIV1. This is in contrast with com-monly observed stronger binding of proteins to ligands pre-sented at higher valence and may be due to interferencebetween the two branches on an N-glycan core.hPIV2 had a much broader specificity and bound to the

monovalent Neu5Ac (43), Neu5Ac9Lt (47), and Neu5GcMe(49) derivatives of LNnT that were bound by hPIV1 as well asthe corresponding Neu5Gc (48) and Neu5GcAc (51) and thecorresponding divalent (NA2) derivatives (58, 62, and 64). Nobinding of hPIV2 was observed toward either the monova-lent or divalent derivatives of Neu5Ac8Me (44 and 59),Neu5Ac9Me (45 and 60), and Neu5,9Ac2 (46 and 61), suggest-ing that binding requires a free 9-OH on the sialic acid. In lightof this observation, it is not obvious why this virus binds the9-O-lactyl derivative of Neu5Ac linked �2–3 to LNnT (47) or�2–3 to NA2 (62) to approximately the same degree (relativeRFU) as the corresponding �2–3-linked Neu5Ac derivatives(43 and 58). The hPIV2 tolerated the hydrophilic HO- on theglycolyl of Neu5Gc on Neu5Gc�2–3LNnT (48), but the corre-sponding divalent structure on NA2 was not bound. UnlikehPIV1 specificity, the bulkier Neu5GcAc derivative of LNnT(51) was bound by hPIV2, but the divalent Neu5GcAc deriva-tive of NA2 was not available for comparison (66 is a monova-lent Neu5GcAc derivative of NA2). By contrast, the binding ofhPIV3 to the SGM was approximately an order of magnitudelower in RFU than the other hPIVs (Fig. 7, a–c). The lower RFUvalues are presumably due to variations in the preparation of theviruses and/or differences in the fluorescent labeling efficiency ofvirus;however, this shouldnotaffect therelativebindingofvirus todifferent glycans. Although somewhat similar to hPIV2, the pat-tern of binding exhibited broader specificity, showing binding totheNeu5AcandNeu5Gcderivatives�2–3-linked to type1under-lying structures (LNT) (73 and 75). None of the hPIVs showedbinding to Kdn derivatives. Clearly, additional studies are needed

TABLE 2The relative ranking of the sialylated glycans that bind SNABinding of biotinylated SNA to the SGMwas carried out at three concentrations (10,1, and 0.1 �g/ml), and each glycan on the array was ranked as a percentage basedon its RFU relative to the RFU of the strongest binding glycan at each concen-tration (rank � 100 � (glycan RFU/RFUmaximum at each concentration)). Therelative rankings were averaged over at least two lectin concentrations in thelinear binding range to obtain the average rank of each glycan and then sortedfrom high to low. An average rank of 100 is assigned to the strongest bindingglycan.

Average Rank Structure Chart ID

100 Kdn5,9Ac2α6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 15 50 Kdn9Meα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 16 50 Neu5,9Ac2α6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 6 33 Kdn5Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 14 32 Kdn9Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 13 32 Kdn7Meα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 17 27 Kdn9Acα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 38 25 Neu5Gc9Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 10 24 Neu5Ac9Ltα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 7 23 Kdnα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 12 22 Kdn5Acα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 39 20 Neu5GcAcα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 11 18 Neu5GcMeα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 9 12 Kdn5Meα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 40 8 Neu5Gcα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 8 8 Neu5Acα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 3 7 Neu5Ac9Meα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 5 2 Kdnα6Galβ3GlcNAcβ3Galβ4Glcitol-AEAB 37 0 Neu5Ac8Meα6Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 4

1 Manα6(Manα3)Manα6(Manα3)Manβ4GlcNAcβ4GlcNAcitol-AEAB 80

TABLE 3The relative ranking of the sialylated glycans that bind MAL-IBinding of biotinylated MAL-I to the SGM was carried out at two concentrations(10 and 1 �g/ml), and each glycan on the array was ranked as a percentage based onits RFU relative to the RFU of the strongest binding glycan at each concentration(rank � 100 � (glycan RFU/RFU maximum at each concentration)). The relativerankings were averaged over at least two lectin concentrations in the linear bindingrange to obtain the average rank of each glycan and then sorted from high to low.

Average Rank Structure Chart ID

100 Kdn5,9Ac2α3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 55 42 Kdn7Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 57 37 Kdn5Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 54 32 Neu5Ac9Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 45 27 Neu5Ac8Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 44 23 Neu5GcAcα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 51 18 Neu5Gc9Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 50 15 Neu5Gcα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 48 14 Kdn9Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 53 13 Neu5GcMeα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 49 10 Neu5Acα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 43 10 Neu5,9Ac2α3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 46 9 Kdnα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 52 8 Neu5Ac9Ltα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 47 1 Kdn5Meα3Galβ4GlcNAcβ3Galβ4Glcitol-AEAB 56 3 Manα6(Manα3)Manα6(Manα3)Manβ4GlcNAcβ4GlcNAcitol-AEAB 80




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using structural approaches to define the interactions of suchunusual sialic derivatives with hPIV hemagglutinins. Binding ofhPIV2 toNeu5Gcwas seen on the CFG array, but the othermod-ified derivatives are not present on the CFG array.Influenza viruses A/Oklahoma/447/08 H1N1 and A/Okla-

homa/483/08H3N2were analyzed for binding to the SGM, andboth bound only glycans with �2–6-linked sialic acid. Theseviruses demonstrated a preference for only two sialic acid deriv-atives, namely the common �2–6-linked Neu5Ac (1, 3, 18, and31) and interestingly the �2–6-linked Neu5Ac9Lt (7, 22, and33), an equally strong binder (Figs. 4 (lanes 14 and 15) and 7 (dand e)). The H1N1 had a broader specificity for the underlyingglycan, binding the�2–6-sialylatedNA2 structures (18 and 22)thatwere not boundby theH3N2.The broader specificity of theH1N1 compared with H3N2 was observed on the defined gly-can array from the CFG (38), but binding to Neu5Ac9Lt, whichis not present on the CFG defined array, has not been observed

previously. In general, the hydrophobicity at 5-position andhydrophilicity at 9-position substitution groups are usuallyimportant in determining virus binding (Fig. 8).

DISCUSSION

Our results demonstrate the development of a novel sialy-lated glycan microarray that permits studies on the roles ofsialic acid modifications on molecular recognition by proteinsand viruses. Analyses of specific expression and functional rec-

FIGURE 7. Binding of viruses to the SGM. Various preparations of fluorescently labeled virus, hPIV1 (a), hPIV2 (b), hPIV3 (c), influenza A viruses A/Oklahoma/447/08 H1N1 (d), and A/Oklahoma/483/08 H3N2 (e) were suspended in binding buffer and applied to the SGM.

FIGURE 8. The general structure of a modified sialic acid with 5-positionand 9-position substitution group noted.




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ognition of modified sialic acids have been difficult due to theirunavailability. Previous development of sialoside arrays focusedon sialosides containing four common natural sialic acid forms(Neu5Ac, Neu5Gc, Neu5Ac9Ac, and Neu5Gc9Ac) presentedon diverse underlying glycans (17). An earlier chemoenzymati-cally synthesized biotinylated sialoside array with diverse sialicacid forms was presented on microplates without purification,which introduced some complexity to protein-binding studies(16). We report here the development of a comprehensive andwell defined sialylated glycan microarray printed covalently onglass slides. The diversity of the SGM includes four type 1 andtype 2 underlying structures, �2–3/6-sialyl linkages, and 16 dif-ferent modified sialic acids, 13 of which are known to exist innature (39), and the natural occurrence of Neu5Ac9Me,Kdn9Me, andKdn7Me is yet to be explored. Althoughmodifiedsialic acid-containing glycans are challenging to synthesize, weadapted the bifunctional fluorescent labeling of natural glycansand the one-pot multienzyme sialylation in a combinatorialfashion to synthesize this otherwise difficult-to-achieve largemicroscale library of sialylated glycans. The fluorescent labeledprecursor enabled the parallelmicroscale enzymatic synthesis andfacilitated the purification of products byHPLC. The well charac-terized one-pot multienzyme sialylation technology enabled thegeneration of terminal sialic acid diversity. The final products canbe directly immobilized ontoNHS- or epoxy-activated glass slideswithout further modification. Although the purification of thefinal products from the reactionmixtureswas relatively time-con-suming and yields varied, the final product library represents adurable resource for systematic study of sialic acid-binding pro-teins and organisms in the format of a microarray.Much of our understanding about the occurrence of sialic

acids in cells and tissues has come from chemical analysis orhistochemical staining of samples with the sialic acid-specificlectins SNA andMAL-I (40, 41). Detailed specificity studies onSNA using the glycan array from the CFG, which has a limitednumber of different sialic acid derivatives, indicated that SNAhas high binding activity toward Sia�2–6Gal�1,4GlcNAc-containing glycans (34). However, our results show that sialicacid recognition by SNA and MAL-I are affected by sialic acidmodifications and raise new questions as to the use of theselectins to generally define sialic acid expression in tissues.An unexpected observation was that both lectins generally

bound well to Kdn, in comparison with the more commonNeu5Ac and Neu5Gc derivatives. Kdn is a deaminated sialicacid that occurs widely in all vertebrates and in bacteria (42)and has been observed in human erythrocytes (11, 43). Impor-tantly, certain modifications of sialic acid residues interferedwith the binding of lectins. For example, SNA bound poorly toNeu5Ac8Me�2–6, andMAL-I bound poorly to Kdn5Me�2–3.Furthermore, inspection of the binding patterns of these sialicacid-specific lectins after treatment of sialylated glycans byeither mild acid hydrolysis or sialidase indicated that somemodifications of sialic acid also result in resistance to sialidase.Whereas glycans with Kdn were more resistant to A. ureafa-ciens sialidase than those with either Neu5Ac or Neu5Gc, sev-eral modifications of Neu5Ac and Neu5Gc, including 8-meth-ylation and 9-acylation, inhibited their desialylation by thissialidase. This sialidase was studied because it is routinely used

in sialylation-related studies because it is themost general siali-dase known and is capable of releasing sialic acids in �2–3,�2–6, and �2–8 linkages (44). Our studies indicate that histo-logical staining using lectins and specific sialidase treatmentshould include additional methods, such as specific antibodiesto modified sialylated glycan determinants (45), in order toexplore sialic acid expression on cells and in tissues. In addition,the information provided by this and other glycan microarrayscomposed of modified sialic acids will greatly facilitate ourunderstanding of sialidase specificities and perhaps contributeto the design of new sialidase inhibitors and identification ofnew types of sialidases.An important utility of the SGMwill be its use as a discovery

platform to detect novel binding specificities of proteins, anti-bodies, bacteria, and viruses. Here we have described screeningassays using several samples of viruses that are known or sus-pected to bind to sialylated glycans. During the initiation ofinfection,many viruses, such as influenza A, bind to cell surfacesialic acids (6, 8). Although significant data have accumulatedthat correlate virus binding specificities directed at �2–3- or�2–6-linked sialic acid with infection of avian and human pop-ulations, respectively, most studies have focused attention ondifferences in the underlying glycans.However, very little infor-mation is available on the relative binding of such infectiousagents to glycans presenting modified sialic acid residues. TheSGM, which includes the most comprehensive group of natu-rally occurring sialic acid structures on a single slide, providesan ideal discovery platform to explore the impact of modifiedsialic acids on molecular recognition.We found that three hPIVs showed very different binding

patterns on the SGM. Interestingly, these viruses bound only tomodified Neu5Ac or Neu5Gc found in mammals but not toKdn and its derivatives, which are expressedmore commonly inprokaryotes and invertebrates. Althoughmost hydroxyl groupsand the amide at position 5 of Neu5Ac and Neu5Gc are neces-sary for hPIV binding (Kdn lacks the 5-amide), the subtlechanges at the 5- and 9-positions can result in different bindingaffinities. In most cases, the hydrophobicity at the 5-positionand the hydrophilicity at the 9-position need to be retained forbinding by hPIVs (Fig. 8). We observed a similar phenomenonfor influenza viruses H1N1 and H3N2, for which �2–6-linkedNeu5Ac and Neu5Ac9Lt are the best ligands. It is interestingthat for several of the viruses interrogated on the SGM, the bestrecognized ligands were actually not directed toward Neu5Ac,the most abundant form of sialic acid. For example, the highestbound ligand for hPIV1 and -2 contained Neu5GcMe, and forboth H3N2 and H1N1 influenza A virus, the highest boundligand was Neu5Ac9Lt. This is an exciting finding because thismodified sialic acid occurs in significant amounts in humans(i.e. 20% of the sialic acid in normal human serum isNeu5Ac9Lt) (46). It is noteworthy that in previous studies, themotifs recognized by hPIV1 and -3 also include fucosylationof GlcNAc and sulfation of Gal in the Neu5Ac�2-3Gal�1–3/4GlcNAc motif (37). Furthermore, the difference inN-glycolyl and N-acetyl neuraminic acid has been a source ofenigma for antibodies recognizing sialyl Lewis X and 6-sulfosialyl Lewis X (47, 48). Although our current modified sialicacid array lacks fucosylation and sulfation, future expansion of




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this array using fucosylated and sulfated acceptors will facilitatefurther investigation into the context of relevant modificationsto glycan backbones.The general occurrence and localizationof thesemodified sialic

acid structures in human tissues are poorly understood. Ourresults indicate the need to reexaminemodified sialic acid expres-sion in human and animal tissues in regard to virus interactionsbeyond analyses of the common Neu5Ac and Neu5Gc glycanderivatives. The resulting binding information and specificityanalysis could also further the development of pharmacotherapyfor virus infections (49). The SGM could serve as a general plat-form to promote the functional studies of modified sialic acidstowardmore glycan binding proteins and viruses in the future.

Acknowledgments—We thank Shelly Gulati (University of OklahomaHealth Sciences Center) for influenza virus preparations and Dr.Jamie Heimburg-Molinaro (EmoryUniversity School ofMedicine) formanuscript editing and review.

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Cummings and David F. SmithVinod K. Tiwari, Hongzhi Cao, Harshal A. Chokhawala, Haojie Zheng, Richard D. Xuezheng Song, Hai Yu, Xi Chen, Yi Lasanajak, Mary M. Tappert, Gillian M. Air,

Acids with Proteins and VirusesA Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic

doi: 10.1074/jbc.M111.274217 originally published online July 12, 20112011, 286:31610-31622.J. Biol. Chem.

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