www.sciencemag.org/content/342/6163/1230/suppl/DC1

Supplementary Materials for

Preferential Recognition of Avian-Like Receptors in Human Influenza A H7N9 Viruses

Rui Xu, Robert P. de Vries, Xueyong Zhu, Corwin M. Nycholat, Ryan McBride, Wenli Yu, James C. Paulson,* Ian A. Wilson*

*Corresponding author. E-mail: wilson@scripps.edu (I.A.W.); jpaulson@scripps.edu (J.C.P.)

Published 6 December 2013, Science 342, 1230 (2013) DOI: 10.1126/science.1243761

This PDF file includes:

Materials and Methods Figs. S1 to S5 Tables S1 to S3 References

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Preferential recognition of avian-like receptors in human influenza A H7N9 viruses Rui Xu1, Robert P. de Vries2,3, Xueyong Zhu1, Corwin M. Nycholat2,3, Ryan McBride2,3, Wenli Yu1, James C. Paulson2,3†, Ian A. Wilson1,4† 1Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 2Department of Cell and Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 3Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. 4Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA † To whom correspondence should be addressed. E-mail: wilson@scripps.edu, jpaulson@scripps.edu Supporting Online Material Materials and Methods Expression and purification of H7 HA in a baculovirus system

H7 HA cDNAs of H7N9 A/Shanghai/1/13 (Sh1) [Global Initiative on Sharing All Influenza Data (GISAID) isolate ID: EPI_ISL_138737] and H7N9 A/Shanghai/2/13 (Sh2) (GISAID isolate ID: EPI_ISL_138738) were synthesized by Genscript (USA) without codon optimization. H7 HA ectodomains were expressed using a baculovirus expression system as described previously (21). The constructs contain an N-terminal signal sequence for secretion, the H7 HA ectodomain, a thrombin cleavage site, a trimerization foldon sequence, and a His6-tag for purification. Expressed HA was purified through a His-tag affinity purification step and subsequent column chromatography steps.

Crystallization and structural determination of H7 HA

The purified H7 HA was digested with thrombin to remove the expression tag, foldon and His tag. The product was purified by gel filtration in 20 mM Tris pH 8.0, 50 mM NaCl, and concentrated to 10 mg/mL for crystallization. H7 HA crystals were grown at 22.5°C in sitting drops by vapor diffusion against a reservoir containing 17-20% PEG3350, 0.2 M ammonium acetate. The crystals were cryoprotected by adding ethylene glycol to the mother liquor to a final concentration of 20%, and then flash cooling the crystals in liquid nitrogen. HA-ligand complexes were obtained by soaking HA crystals in cryo-solutions containing glycan ligands. Final ligand concentrations and soaking times are included in tables S1 and S2. Diffraction data were collected on synchrotron radiation sources specified in the data processing and refinement statistics in tables S1 and S2. Data were processed with HKL2000 (40). Initial phases were determined by molecular replacement using Phaser (41). Refinement was carried out in Phenix (42), and alternated with manual rebuilding and adjustment in COOT (43). Two HA protomers are present per asymmetric unit, one of which is much better defined in the electron density maps with lower B-values (fig. S4, tables S1-2). Final refinement statistics are summarized in tables

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S1-2.

Expression and purification of H7 HA in a mammalian expression system Codon-optimized H7 and H1 HA-encoding cDNAs (Genscript, USA) of A/Shanghai/2/13 (H7N9) and A/Kentucky/UR06-0258/2007 (H1N1) (GenBank accession number: CY028163) were cloned into the pCD5 expression plasmid as described previously (44). The pCD5 expression vector is adapted, such that the HA-encoding cDNAs are cloned in frame with DNA sequences coding for a signal sequence, a GCN4 trimerization motif (RMKQIEDKIEEIESKQKKIENEIARIKK) (45), and a Strep-tag II (WSHPQFEK; IBA, Germany). The HA proteins were expressed in HEK293S GnTI- cells and purified from the cell culture supernatants as described previously (46). pCD5 expression vectors were transfected into HEK293S GnTI- cells using polyethyleneimine I (PEI). At 6 h post transfection, the transfection mixture was replaced by 293 SFM II expression medium (Gibco), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter), penicillin (100 units/ml), Streptomycin (100 µg/ml), glutaMAX (Gibco), and 1.5% DMSO. Tissue culture supernatants were harvested 5–6 days post transfection. HA proteins were purified using Strep-Tactin Sepharose beads according to the manufacturer’s instructions (IBA, Germany). Plate-based glycan binding of HA from mammalian expression 96-well nunc maxisorp plates were coated with 1 µg/ml SLNLN that contained either α2-3 or α2-6 linked sialic acids. Purified, soluble trimeric HA was pre-complexed with horseradish peroxidase (HRP)-linked anti-Strep-tag mouse antibody and with HRP-linked anti-mouse IgG (4:2:1 molar ratio) prior to incubation for 15 minutes on ice. The pre-formed HA complexes were incubated for 90 minutes on the SLNLN-coated plates and washed three times with PBST, and HA binding was detected using the TMB substrate kit (Thermo Scientific), similar to our previously published protocols (44). Glycan microarray binding of mammalian-expressed HA HA samples were eluted from the strep tag beads with 100mM Tris, 150mM NaCl, 1mM EDTA, 2.5mM D-Biotin, pH 7.2. Purified soluble trimeric HA was used at 50µg/ml pre-complexed with horseradish peroxidase (HRP)-linked anti-Strep-tag mouse antibody and with Alexa488-linked anti-mouse IgG (4:2:1 molar ratio) prior to incubation for 15 min on ice in 100 µl PBS-T, and incubated on the array surface in a humidified chamber for 90 minutes. Slides were subsequently washed by successive rinses with PBS-T, PBS, and deionized H2O. Washed arrays were dried by centrifugation and immediately scanned for FITC signal on a Perkin-Elmer ProScanArray Express confocal microarray scanner. Fluorescent signal intensity was measured using Imagene (Biodiscovery) and mean intensity minus mean background was calculated and graphed using MS Excel. For each glycan, the mean signal intensity is calculated from 6 replicates. The highest and lowest signals of the 6 replicates were removed and the remaining 4 replicates were used to calculate the mean signal, standard deviation (SD), and standard error measurement (SEM). Bar graphs represent the averaged mean signal minus background for each glycan sample and error bars are the SD value. A list of glycans on the microarray is included in table S3.

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Glycan microarray binding using baculovirus-expressed HAs HA samples were dialyzed against 20 mM Tris-HCl, 50 mM NaCl, pH 8.0 overnight at

4oC after His-tag affinity purification. Proteins were concentrated to 1 mg/ml before binding assay. Procedures for the HA glycan microarray binding assay were similar to those described above with small alterations. Briefly, 15 µg/ml HA was pre-complexed with an Alexa488-linked anti-penta-His mouse antibody and with Alexa488-linked anti-mouse IgG (4:2:1 molar ratio) and applied directly to the array for 1 hour.

Glycan binding of baculovirus-expressed HAs by bio-layer interferometry Recombinant HA expression from the baculovirus expression system was buffer- exchanged into PBS buffer. Association of HA was measured on an Octet Red (ForteBio) against immobilized glycans on the sensors. 3′-SLNLN and 6′-SLNLN are biotinylated glycans and were immobilized using Super Streptavidin Biosensors (ForteBio). Association was measured for 300 seconds at 30°C by exposing the sensors to protein solution in 1x kinetic buffer (1x PBS, pH 7.4, 0.01% BSA, and 0.002% Tween 20) after which the sensors were exposed to 1x kinetic buffer for dissociation for 300 seconds at 30°C.

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Fig. S1. Glycan binding specificity of the HAs was analyzed by Bio-Layer Interferometry (BLI) on an Octet Red system (ForteBio) using recombinant HAs expressed in a baculovirus system. HA binding at 0.7 mg/ml was measured against immobilized biotinylated glycans 6′-SLNLN (A), and 3′-SLNLN (B). (C) The binding curve for 3′-SLNLN was best fitted using the 2:1 heterogeneous ligand binding model, with apparent KD values of 0.24 and 0.88 µM. Avian H7N7 HA (A/Netherlands/219/2003, Neth219) shows specific recognition for 3′-SLNLN, but not 6′-SLNLN. Sh1 and Sh2 HAs from the recent H7N9 human outbreak also display detectable binding but to 3′-SLNLN only, albeit less efficiently than Neth219 HA. The L226I mutation on the Sh2 background has little impact on receptor binding specificity or avidity. The L226Q mutant, however, shows much more efficient association with glycan 3′-SLNLN (apparent KD values of 0.024 and 0.3µM).

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Fig. S2. Binding results on the glycan microarray using recombinant HA proteins from baculovirus expression. The mean signal and standard error were calculated from six independent replicates on the array. α2-3 sialosides are shown in white bars (glycans #3-35), α2-6 in black bars (glycans #36-56, very little signal), and stripped bars denoted mixed bi-antennary glycans (glycans #57-58) that contain both α2-3 and α2-6 linked sialylated glycans. List of glycans imprinted on the array is included in table S3.

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Fig. S3. Electron density maps (2Fo-Fc) of glycan ligands in the Sh2 H7 HA crystal structures. (A) LSTa, with 10 min soak (B) LSTc with 10 min soak (C) LSTa with 2 hour soak, (D) glycan #3, (E) glycan #21, (F) glycan #23. D-F are also 2 hour ligand soaks. The maps are contoured at a 1σ level (colored in grey) in panels B, D E and F. In (A) and (C), maps are shown at the 0.8σ level. Only density for the glycan receptors is shown for clarity.

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  Fig. S4. Analysis of B-value distribution in the Sh2 H7 HA crystal structure. The two HA protomers within the asymmetric unit are colored by B-value. The warmest colors (i.e., red and orange) indicate high B-values, while cool colors (i.e., blue) indicate low B-values. The two protomers are shown in similar orientations for clarity. One protomer (A) has low B-values and clear and unambiguous electron density, while the other protomer (B) is less well-defined and has much higher B-values.

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Fig. S5. Sequence alignment of full-length H7N9 HA sequences deposited in Global Initiative on Sharing All Influenza Data (GISAID).

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Table S1. Data collection and refinement statistics for Sh2 H7 HA and glycan complexes H7 HA H7 HA H7 HA H7 HA Ligand apo LSTa LSTa LSTc Soaking condition 10mM, 10min 16mM, 2 hours 10mM, 10min Data collection Beamline

SSRL 12-2

SSRL 12-2

APS 23ID-B

SSRL 12-2

Wavelength (Å) 1.0000 1.0000 1.0331 1.0000 Space group P213 P213 P213 P213 Unit cell parameters (Å, ˚)

a = b = c = 154.5 α = β = γ = 90

a = b = c = 154.7 α = β = γ = 90

a = b = c = 153.9 α = β = γ = 90

a = b = c = 154.8 α = β = γ = 90

Resolution (Å) 50-2.70 (2.80-2.70) a

50-2.70 (2.80-2.70)

50-2.60 (2.69-2.60)

50-2.90 (3.00-2.90)

Observations 223,195 187,164 234,550 183,548 Unique reflections 33,899 (3,363) 33,887 (3,362) 37,501 (3,705) 27,559 (2,712)

Completeness (%) 99.8 (100.0) 99.8 (99.9) 99.9 (100.0) 99.9 (100.0) <I/σI> 19.2 (2.6) 19.6 (2.2) 16.9 (2.1) 18.0 (2.8) Rsym (%) b 8.9 (75.7) 7.6 (86.3) 9.8 (94.0) 10.6 (93.5) Rpim (%) c 3.8 (30.8) 3.4 (38.4) 4.0 (37.1) 4.5 (34.2) Za

d

2 2 2 2

Refinement Resolution (Å) 46.6 -2.70 48.9 -2.70 48.7 -2.60 46.7 -2.90 Reflections (total) 33,870 33,863 37,471 27,534 Reflections (test) 1,718 1,718 1,873 1,385 Rcryst (%) e 22.9 22.7 21.3 22.6 Rfree (%) f 27.7 26.7 25.9 26.2 Average B-value (Å2) 100 122 105 119 Protein: protomer 1 59 80 72 73 Protein: protomer 2 141 163 139 166 Wilson B-value (Å2) 63 68 67 72 Protein atoms 7,600 7,600 7,600 7,600 Carbohydrate atoms 129 132 132 143 Waters 105 35 67 56 RMSD from ideal geometry Bond length (Å) 0.002 0.004 0.005 0.003 Bond angles (°) 0.67 0.82 0.93 0.76 Ramachandran statistics (%) g Favored 94.0 94.0 93.0 93.0 Outliers 1.0 1.3 1.3 1.0 PDB code 4N5J 4N5K 4N61 4N60

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Table S2. Data collection and refinement statistics for Sh2 HA glycan complexes H7 HA H7 HA H7 HA Ligand glycan #3 glycan #21 glycan #23 Soaking condition 10mM, 2 hours 10mM, 2 hours 10mM, 2 hours Data collection Beamline

APS 23ID-B

APS 23ID-B

APS 23ID-B

Wavelength (Å) 1.0331 1.0331 1.0331 Space group P213 P213 P213 Unit cell parameters (Å, ˚)

a = b = c = 153.8 α = β = γ = 90

a = b = c = 153.6 α = β = γ = 90

a = b = c = 153.9 α = β = γ = 90

Resolution (Å) 50-2.50 (2.59-2.50) a 50-2.75 (2.85-2.75) 50-2.70 (2.80-2.70) Observations 392,387 137,382 209,938 Unique reflections 42,019 (4,158) 31,335 (3,134) 33,536 (3,330)

Completeness (%) 100.0 (100.0) 99.1 (100.0) 100.0 (100.0) <I/σI> 25.1 (3.3) 14.7 (1.9) 16.3 (2.5) Rsym (%) b 8.9 (77.2) 9.5 (79.4) 11.2 (77.9) Rpim (%) c 2.8 (25.3) 4.8 (36.8) 4.6 (33.9) Za d

2 2 2

Refinement Resolution (Å) 48.6 -2.50 42.6 -2.75 48.7 -2.70 Reflections (total) 41,990 31,323 33,505 Reflections (test) 2,121 1,584 1,695 Rcryst (%) e 23.0 21.6 22.5 Rfree (%) f 26.7 26.5 26.5 Average B-value (Å2) 101 98 95 Protein: protomer 1 67 68 52.   Protein: protomer 2 133 126 136  Wilson B-value (Å2) 53 64 54. Protein atoms 7,600 7,600 7,600 Carbohydrate atoms 181 215 172 Waters 100 79 178 RMSD from ideal geometry

Bond length (Å) 0.004 0.003 0.003 Bond angles (°) 0.79 0.75 0.79 Ramachandran statistics (%) g Favored 93.0 93.0 93.0 Outliers 1.3 0.6 1.3 PDB code 4N62 4N63 4N64

aNumbers in parentheses refer to the highest resolution shell. bRsym = Σhkl Σi | Ihkl,i - <Ihkl> | / Σhkl Σi Ihkl,i, where Ihkl,i is the scaled intensity of the ith measurement of reflection h, k, l, and < Ihkl> is the average intensity for that reflection. cRpim = Σhkl (1/(n-1))1/2 Σi | Ihkl,i - <Ihkl> | / Σhkl Σi Ihkl,i, where n is the redundancy. dZa is the number of HA protomers per crystallographic asymmetric unit. eRcryst = Σhkl | Fo - Fc | / Σhkl | Fo | x 100

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fRfree was calculated as for Rcryst, but on a test set of 5% of the data excluded from refinement. gCalculated using Molprobity (47).

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Table S3. Glycans imprinted on the microarray.

Glycan #

Name Structure

1 Galβ(1-4)-GlcNAcβ-ethyl-NH2

2 Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)[Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

3 NeuAcα(2-3)-Galβ(1-4)-6-O-sulfo-GlcNAcβ-propyl-NH2

4 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-6-O-sulfo-GlcNAcβ-propyl-NH2

5 NeuAcα(2-3)-6-O-sulfo-Galβ(1-4)-

GlcNAcβ-ethyl-NH2 6 NeuAcα(2-3)-6-O-sulfo-Galβ(1-4)-

[Fucα(1-3)]-GlcNAcβ-propyl-NH2

7 NeuAcα(2-3)-Galβ(1-3)-6-O-sulfo-

GlcNAcβ-propyl-NH2 8 NeuAcα(2-3)-Galβ(1-4)-Glcβ-ethyl-NH2

9 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ-ethyl-

NH2 10 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-

Galβ(1-4)-GlcNAcβ-ethyl-NH2

11 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl-NH2

12 NeuAcα(2-3)-GalNAcβ(1-4)-GlcNAcβ-ethyl-NH2

13 NeuAcα(2-3)-Galβ(1-3)-GlcNAcβ-ethyl-NH2

14 NeuAcα(2-3)-Galβ(1-3)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl-NH2

15 NeuAcα(2-3)-Galβ(1-3)-GlcNAcβ(1-3)-Galβ(1-3)-GlcNAcβ-ethyl-NH2

16 NeuAcα(2-3)-Galβ(1-3)-GalNAcβ(1-3)-Galα(1-4)-Galβ(1-4)-Glcβ-ethyl-NH2

17 NeuAcα(2-3)-Galβ(1-3)-GalNAcα-Thr-NH2

18 NeuAcα(2-3)-Galβ(1-3)-[GlcNAcβ(1-6)]-GalNAcα-Thr-NH2

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Glycan #

Name Structure

19 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-6)-[Galβ(1-3)]-GalNAcα-Thr-NH2

20 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-

Galβ(1-4)-GlcNAcβ(1-6)-[Galβ(1-3)]-GalNAcα-Thr-NH2

21 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr-NH2

22 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr-NH2

23 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα-Thr-NH2

24 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα-Thr-NH2

25 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

26 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

27 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

28 NeuAcα(2-3)-[GalNAcβ(1-4)]-Galβ(1-4)-GlcNAcβ-ethyl-NH2

29 NeuAcα(2-3)-[GalNAcβ(1-4)]-Galβ(1-4)-

Glcβ-ethyl-NH2

30 Galβ(1-3)-GalNAcβ(1-4)-[NeuAcα(2-3)]-Galβ(1-4)-Glcβ-ethyl-NH2

31 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-

GlcNAcβ-propyl-NH2

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Glycan #

Name Structure

32 NeuAcα(2-3)-Galβ(1-3)-[Fucα(1-4)]-GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-ethyl-NH2

33 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-

GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-ethyl-NH2

34 NeuAcα(2-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ(1-3)-Galβ(1-4)-[Fucα(1-3)]-GlcNAcβ-ethyl-NH2

35 NeuGcα(2-3)-Galβ(1-4)-GlcNAcβ-ethyl-NH2

36 NeuAcα(2-6)-Galβ(1-4)-6-O-sulfo-GlcNAcβ-propyl-NH2

37 NeuAcα(2-6)-Galβ(1-4)-Glcβ-ethyl-NH2

38 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ-ethyl-NH2

39 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl-NH2

40 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ-ethyl-NH2

41 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-6)]-Galβ(1-4)-GlcNAcβ-ethyl-NH2

42 NeuAcα(2-6)-GalNAcβ(1-4)-GlcNAcβ-ethyl-NH2

43 NeuAcα(2-6)-[Galβ(1-3)]-GalNAcα-Thr-NH2

44 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-6)-

[Galβ(1-3)]-GalNAcα-Thr-NH2

45 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-6)-[Galβ(1-3)]-GalNAcα-Thr-NH2

46 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr-NH2

47 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-GalNAcα-Thr-NH2

48 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα-Thr-NH2

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Glycan #

Name Structure

49 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-6)]-GalNAcα-Thr-NH2

50 Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

51 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

52 GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

53 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

54 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

55 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

56 NeuGcα(2-6)-Galβ(1-4)-GlcNAcβ-ethyl-NH2

57 NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

58 NeuAcα(2-6)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-3)-[NeuAcα(2-3)-Galβ(1-4)-GlcNAcβ(1-2)-Manα(1-6)]-Manβ(1-4)-GlcNAcβ(1-4)-GlcNAcβ-Asn-NH2

References and Notes

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