1

Glycan specificity of P[19] rotavirus and comparison with those of other related P 1

genotypes 2

Yang Liu1, Theresa A. Ramelot2, Pengwei Huang1, Yan Liu3, Zhen Li3, Ten Feizi3, 3

Weiming Zhong1, Fang-Tzy Wu4, Ming Tan1,5, Michael A. Kennedy2 and Xi Jiang1,5* 4

1Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, 5

Cincinnati, OH 6

2Department of Chemistry and Biochemistry, Miami University, Oxford, OH 7

3Glycosciences Laboratory, Department of Medicine, Imperial College London, London, 8

UK 9

4Center for Research, Diagnostics and Vaccine Development, Centers for Disease 10

Control, Taipei, Taiwan 11

5Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 12

13

*Corresponding author 14

Email: Jason.jiang@cchmc.org 15

16

Short title: Glycan binding specificity of P[19] rotavirus 17

JVI Accepted Manuscript Posted Online 24 August 2016J. Virol. doi:10.1128/JVI.01494-16Copyright © 2016 Liu et al.This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

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Abstract 18

The P[19] genotype belongs to the P[II] genogroup of group A rotaviruses (RVs). 19

However, unlike the other P[II] RVs that mainly infects humans, P[19] RVs commonly 20

infect animals (porcine), making P[19] unique to study RV diversity and host ranges. 21

Through in vitro binding assays and saturation transfer difference (STD) NMR, we found 22

that P[19] could bind mucin cores 2, 4, and 6, as well as type 1 histo-blood group 23

antigens (HBGAs). The common sequences of these glycans serve as minimal binding 24

units, while additional residues, such as the A, B, H, and Lewis epitopes of the type 1 25

HBGAs, can further define the binding outcomes and therefore, likely the host ranges 26

for P[19] RVs. This complex binding property of P[19] is shared with those of the other 27

three P[II] RVs (P[4], P[6] and P[8]) in that all of them recognized the type 1 HBGA 28

precursor, although P[4] and P[8], but not P[6], also bind to mucin cores. Moreover, 29

while essential for P[4] and P[8] binding, the addition of the Lewis epitope blocked P[6] 30

and P[19] binding to type 1 HBGAs. Chemical shift NMR of P[19] VP8* identified a 31

ligand binding interface that has shifted away from the known RV P-genotype binding 32

sites but is conserved among all P[II] RVs and two P[I] RVs (P[10] and P[12]), 33

suggesting an evolutionary connection among these human and animal RVs. Taken 34

together, these data are important for hypotheses on potential mechanisms for RV 35

diversity, host ranges, and cross-species transmission. 36

Importance 37

In this study, we found that this P[19] strain and other P[II] RVs recognize mucin cores 38

and the type 1 HBGA precursors as the minimal functional units and that additional 39

saccharides adjacent to these units can alter binding outcomes and thereby possibly 40

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host ranges. These data may help to explain why some P[II] RVs, such as P[6] and 41

P[19], commonly infect animals but rarely humans, while others, such as the P[4] and 42

P[8] RVs, mainly infect humans and are predominant over other P genotypes. 43

Elucidation of the molecular bases for strain-specific host ranges and cross-species 44

transmission of these human and animal RVs is important to understand RV 45

epidemiology and disease burden, which may impact development of control and 46

prevention strategies against RV gastroenteritis. 47

Introduction 48

Rotaviruses (RVs) are the principal cause of severe diarrhea in children, responsible for 49

approximately 200,000 deaths in children younger than 5 years old worldwide in 2011 50

(1-3). It has been shown that RV attachment to cell surface carbohydrates, mediated by 51

the VP8* domain of the VP4 spike protein, is the important first step for successful 52

infection (4-7). RVs are diverse in recognizing different receptors, causing infections in 53

humans and different animal species. For example, early studies showed that infection 54

with some animal RVs relies on the terminal sialic acid (SA) and are sialidase-sensitive, 55

but the majority of human and animal RVs are sialidase-insensitive (8-12). However, a 56

recent study indicated that a sialidase-insensitive human RV, Wa (P[8]), recognizes an 57

internal SA residue (13). In addition, other host cell surface molecules, such as the heat 58

shock cognate protein 70 (14-16) and integrins (17-19), have been identified as 59

potential RV receptors, although their precise roles in RV attachment, penetration, and 60

pathogenesis in host cells remain elusive. 61

The recent findings that almost all major RV genotypes in the P[II]-P[IV] genogroups 62

infect humans and recognize HBGAs (20-27) have led to a plausible hypothesis that 63

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HBGAs are important host factors or cellular receptors for human RVs. Direct evidence 64

for HBGA-RV interaction has been obtained by resolving the VP8* protein crystal 65

structures for two human RVs (P[14] and P[11]) in complex with their HBGA 66

oligosaccharide ligands (21, 22). The association between RV infection and a child’s 67

secretor status has also been observed through epidemiologic studies (28-31). 68

Together, these findings suggest that HBGAs play important roles in RV infection and 69

pathogenesis. 70

Although direct co-crystal data remain lacking, recognition of HBGAs by the most 71

prevalent human rotavirus genotypes of P[4], P[6] and P[8] has been reported by in vitro 72

binding assays. For example, the VP8*s of the human P[8] and P[4] RVs recognize the 73

Lewis b and H-type 1 HBGAs, while the P[6] RVs bind the H type 1 only (23). However, 74

a STD-NMR based study showed that while the human P[4] (strain DS-1) and P[6] 75

(strain RV-3) RV VP8* could bind the A-type HBGAs with the involvement of the α1-2 76

fucose, the VP8* of the P[8] human strain Wa did not recognize the A and the Lewis b/H 77

type 1 antigens (20). On the other hand, a binding study with a human milk glycan 78

library confirmed the binding of the P[6] (RV-3) VP8* to the terminal type 1 HBGA 79

sequences with or without the H epitope but without the Lewis epitope and an additional 80

internal Lewis x determinant (32). 81

The group A RVs are diverse and have been divided into 37 P genotypes (P[1] – P[37]) 82

in five P genogroups (P[I] – P[V]) based on sequences of the viral surface spike proteins 83

VP4 or VP8* (25, 33) (Fig 1). While the majority of RVs exclusively or nearly exclusively 84

infect either humans or particular animal species, others appear to commonly infect 85

both humans and animals (P[6], P[9], P[11], and P[14] in P[II]-P[IV]) or infect mainly 86

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animals with occasional infection of both humans and animals (P[1], P[2], P[3], P[7], 87

P[10], and P[28] in P[I]) (Fig 1). Mechanisms for cross-species transmission have been 88

suggested for some RVs, such as the P[III] RVs that can infect both humans and 89

animals probably via recognition of the A antigens shared by humans and wild and 90

domestic animals (25). In addition, the P[11] RVs recognize HBGA precursors or 91

backbones whose chain length and branching are developmentally controlled in 92

neonates and young infants (24, 26). These carbohydrates are evolutionarily conserved 93

between humans and some animals. However, the reasons behind P[1], P[2], P[3], P[7], 94

P[10], and P[28] RVs infection in animals or occasionally both human and animals 95

remain unknown, limiting our understanding of RV epidemiology and thus, the ability to 96

develop rational prevention strategies. 97

In this study, we characterized a P[19] RV that commonly infects animals (porcine), but 98

rarely humans (Fig.1). A unique binding property to the di-saccharide core structure 99

(GlcNAcβ1-6GalNAc) of mucin cores 2, 4, and 6 and the extended terminal type 1 100

HBGA tri-saccharide (Galβ1-3GlcNAcβ1-3Gal) was identified for an overlapping binding 101

interface. This P[19] RV binding interface differed from known ones but appeared to be 102

conserved among P[II] RVs and two P[I] RVs (P[10] and P[12]) (Fig.1). Additional 103

saccharides surrounding the core sequences also impact the glycan binding specificities 104

for these RVs. This may explain the observed diversity in binding specificity, host 105

ranges, and possibly pathogenicity of RVs in different human populations and animal 106

species. 107

Materials and Methods 108

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Expression and purification of VP8* proteins in E. Coli. The VP8* sequences 109

(amino acids 46-231) from a human RV P[19] (GenBank accession number: 110

DQ887060) was expressed in E. coli BL21 and purified by GST-tag affinity purification, 111

as described previously (25). Recombinant VP8* proteins of other P[II] RVs (P[4], P[6] 112

and P[8]) and a P[10] RVs were also purified using the same expression system. 15N- or 113

15N,13C-labeled P[19] VP8* proteins were also made for the NMR studies using the 114

same E. coli culture procedures with the addition of 15N-minimal growth medium (22 mM 115

KH2PO4, 50 mM Na2HPO4, 8.5 Mm NaCl, 0.1 mM CaCl2, 2 mM MgSO4, 1x BME 116

vitamins, 18 mM 15NH4Cl, and 22 mM Glucose) or 15N,13C-minimal growth medium 117

supplied with 22 mM 13C-Glucose (20). The GST tag of the labeled VP8* proteins was 118

removed for the NMR study by cutting with thrombin (Sigma-Aldrich Co., St. Louis, MO) 119

followed by FPLC gel filtration to collect the VP8* protein fractions and concentrated by 120

ultracentrifugation. The purified VP8* proteins were stored at -80 oC until needed. 121

Binding of P[19] VP8* to human saliva, human milk, and porcine mucins. A set of 122

previously characterized adult saliva samples with known ABO, secretor, and Lewis 123

phenotypes were used in binding assays with the GST-VP8* fusion proteins (23). In 124

addition, a set of saliva samples from neonates and young infants at different ages in 125

the first year of lives described in a previous study (24) was studied for age-specific host 126

ranges of P[6] RVs. A pooled human milk sample from the Cincinnati human milk bank 127

(34) and a commercial porcine mucin (Sigma) were also tested for binding to P[19] 128

VP8*. The carbohydrate profiles for the human milk and porcine mucin have been 129

determined in our previous studies (25, 34). 130

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Glycan array screening and ELISA-based oligosaccharide binding assay. Initial 131

ligand screenings for P[19] VP8* was performed by the Protein-Glycan Interaction Core 132

of CFG (Consortium for Functional Glycomics) (The glycan library information was 133

available from the website http://www.functionalglycomics.org/). The recombinant GST-134

VP8* proteins were applied to individual glycan arrays at a protein concentration of 200 135

µg/ml and the bound GST-VP8* were detected using a fluorescent-labeled anti-GST 136

monoclonal antibody. Relative fluorescent units (RFU) of each glycan were calculated to 137

rank the reactivity in the interaction with P[19] VP8*. A further microarray analysis was 138

performed at the carbohydrate microarray facility of the Glycosciences Laboratory at the 139

Imperial College, London, the United Kingdom, using a focused array of sequence-140

defined glycan probes arrayed as lipid-linked probes (35). The probes are prepared 141

from reducing oligosaccharides by reductive amination with the amino lipid, 1,2-142

dihexadecyl-sn-glycero-3-phosphoethanolamine, designated DH. The P[19] VP8* was 143

overlaid at 50 µg/ml, followed by rabbit anti-GST antibody Z-5 (Santa Cruz, Dallas, TX), 144

and biotinylated anti-rabbit IgG (Sigma). Binding was detected with Alexa Fluor-647-145

labelled streptavidin (Molecular Probes, Eugene, OR). The probes have been described 146

previously (36, 37) except for DFiLNO, a difucosylated decasaccharide received from 147

Dextra as an unknown impurity of TFiLNO (1-2,-2,-3) and characterized by ES-CID-148

MS/MS (W Chai, unpublished). 149

The ELISA-based oligosaccharide binding assays were carried out as previously 150

described (38). Oligosaccharides used in the study included biotin-labeled PAA-151

conjugates obtained from CFG and Glycotech (GlycoTech, Inc., Gaithersburg, MD), and 152

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biotin-labeled BSA conjugates of A, B, and H type 1 to type 4, kindly provided by Dr. 153

Todd L. Lowary. 154

Two-dimensional heteronuclear single quantum correlation (1H-15N-HSQC) NMR 155

experiments. Titrations of P[19] VP8* with mucin core 2 (kindly provided by Dr. James 156

Paulson) and LNFPI (V-lab) were followed by in 1H-15N-HSQC experiments and used to 157

calculate solution dissociation constants (KD values) for binding of the two ligands to 158

P[19] VP8*. The KD values were defined as KD =[VP8*][Ligand]/[VP8*-Ligand], where 159

[VP8*-Ligand] refers to the concentration of the complex of VP8* and the glycan ligand, 160

and [VP8*] and [Ligand] are the concentrations of free protein and ligand. NMR data 161

were collected at 20 oC with samples of ~300 µL of Tris NMR buffer (10 mM Tris-HCl, 162

100 mM NaCl, 1 mM EDTA, 10% D2O, pH 8.0) in 5 mm Shigemi NMR tubes on a 600 163

MHz Bruker Avance III spectrometer. Samples of 0.25 mM 15N-labeled VP8* were 164

titrated with each ligand at 0, 0.1, 0.3, 0.5, 1.0, 1.4, 1.9, 2.7, 4.3, and 6 mM, and 2-D 1H-165

15N-HSQC spectra were acquired after each titration step. Spectra were processed with 166

NMRPipe software (39) and analyzed with Sparky 167

(http://www.cgl.ucsf.edu/home/sparky) . The weighted chemical shift change upon 168

titration was determined for 10 strongly affected backbone amide peaks that were not 169

overlapped according to the following formula for chemical shift change (CSC) (40) : 170

CSC= Σ {[(ΔδHN)

2 + (ΔδN/6.5)

2]}

1/2, ΔδH

N = δH

N

free - δHN

bound and, ΔδN = δNfree - δNbound, 171

where δNfree and δNbound are the amide 15N chemical shifts in the absence and presence 172

of ligand; δHN

free and δHN

bound are the backbone amide hydrogen chemical shifts, 173

respectively. The concentration of free ligand, [L-free], was determined by [L-free] = [L-174

total] - (1/f) × [P-total], where f is the fraction of VP8* in the bound state is CSC/CSC-175

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max, and [L-total] and [P-total] are the total concentrations of ligand and protein at each 176

titration. The CSC-max and KDs were determined by fitting the CSC/CSC-max at each 177

ligand titration versus the [L-free] to the equation f = [L-free]/( KD +[L-free]), with the 178

variables CSC-max and KD. The [L-free] values were calculated with the updated CSC-179

max, and this process was repeated until there were no further changes. The error bars 180

for KD were determined based on the average and standard deviation of the 10 peaks 181

that had their CAC/CSC-max vs [L-free] plotted and fit to KD in the in the same way. 182

P[19] VP8* chemical shift assignments. In order to assign the backbone chemical 183

shifts, 2-D and 3-D NMR data were collected for ~300 µL of 1.1 mM 15N-13C-labeled 184

P[19] VP8* in Tris NMR buffer at 20 oC on 600 MHz Varian Inova, 600 MHz Bruker 185

Avance III, and 850 MHz Bruker Avance II NMR spectrometers equipped with 186

conventional 5-mm HCN probes. Backbone 1H, 13C, and 15N resonance assignments 187

were made using the PINE server and then corrected and completed manually based 188

on following 3-D spectra (41): HNCACB, CBCA(CO)NH, HNCO, HNCA, HN(CO)CA, 189

HN(CA)CO, HBHA(CO)NH, and 15N-edited NOESY-HSQC (τm = 100 ms). Backbone 190

assignments were 91% complete for residues 63-225, and only 5-10 HN cross peaks 191

could not be assigned. However, none of the unassigned peaks shifted in the titration 192

studies. 193

Ligand chemical shift assignments. Chemical shift assignments of mucin core 2 and 194

LNFPI were achieved by analysis of 2-D 1H-13C HSQC, HMBC, HSQC-TOCSY, 1H-1H 195

COSY, TOCSY, and NOESY, and 1-D 1H NMR experiments recorded at 20 oC and 196

referenced to internal TSP(20, 42). The NMR samples were 2 mM mucin core 2 in Tris 197

NMR buffer and 5 mM LNFPI in phosphate NMR buffer (20 mM sodium phosphate, 100 198

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mM NaCl, 10% D2O), both at pH 8.0. Chemical shift assignments for LNFPI were in 199

agreement with published values (43), although differences can be attributed to pH, 200

temperature, and referencing variations. 201

STD NMR experiments. All saturation transfer difference (STD) NMR spectra were 202

acquired in Shigemi Tubes (Shigemi, USA) with a Bruker 600 MHz Avance III 203

spectrometer at 10 oC using a conventional HCN probe and the TopSpin 3.1 pulse 204

sequence STDDIFFGG19.3. The NMR sample for mucin core 2 in complex with P[19] 205

VP8* was prepared with 200 uM protein and 6.0 mM ligand in a final volume of 300 μL 206

Tris NMR buffer. The LNFPI STD sample was prepared with 130 μM P[19] VP8* and 207

6.6 mM LNFPI in a final volume of 300 μL of phosphate NMR buffer. This results in total 208

protein-ligand ratios of 1:35 and 1:50, for the mucin core 2 and LNFPI samples, 209

respectively. The protein resonances were saturated at 0 ppm with a cascade of 40 210

Gaussian shaped pulses with duration of 50 ms resulting in a total saturation time of 2 211

sec. The off-resonance saturation was applied to 30 ppm and 1024 experiments were 212

acquired for each with a 5 sec recycle delay. A spin-lock filter with strength of 10 kHz 213

and duration of 100-200 ms was applied to suppress the broad protein resonance 214

signals, and Watergate 3-9-19 was used to suppress the residual water signal. The STD 215

spectra were obtained by subtracting the on-resonance spectrum from the off-216

resonance reference spectrum. STD amplification factors were determined by the 217

equation (Io - Isat) / Io, where Io and Isat are the intensity of the signals in the reference 218

and saturated spectra, respectively, and the normalized STD amplification percentage 219

was determined by dividing by the largest STD amplification factor for each ligand. The 220

protons with the largest STD amplification percentage along with data from the 2-D 221

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STD-tocsy spectrum, which was used to resolve overlap from the 1D spectra, were 222

used to assign the binding epitopes. 223

Homology modeling of P[19] VP8* and site-directed mutagenesis. A homology 224

model of the P[19] VP8* protein was built based on the X-ray crystal structure of DS1 225

strain of RVs (PDB ID: 2AEN) as the template by SWISS-MODEL automated protein 226

structure homology-modelling server (http://swissmodel.expasy.org/). Mutant VP8* 227

proteins with single amino acid substitution were constructed from template wild type 228

VP8* constructs using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La 229

Jolla, CA). 230

Virus propagation, inhibition, and infectivity assays. A P[19] RV (G9P[19]) was 231

adapted from a positive stool sample obtained from Taiwan CDC collected on Dec.17, 232

2014 by multiple blind passages on the MA104 cells. The identity of the strain was 233

confirmed by RT-PCR and sequencing following detection of viral replication in the cell 234

culture by ELISA using Rotaclone kit (Meridian Bioscience, OH, USA). The cell-culture 235

adapted P[19] RVs were then used for inhibition assays with human milk, neonate 236

saliva, porcine mucin FPLC fractions as well as the oligosaccharides of LNFPI and 237

mucin core 2 using procedures described previously (24). In brief, the P[19] viruses (at 238

300 fluorescent forming units (FFU)/10 µl) were pre-incubated with different inhibition 239

reagents for 30 min. After rinsing twice with serum-free DMEM and chilling of all 240

reagents and the 24-well plates on ice, duplicated wells of confluent MA104 monolayer 241

were inoculated with the virus-oligosaccharide or virus-saliva/milk mixtures on ice with 242

continuous rocker platform agitation for 1 h. The inocula were then removed and the 243

cells were washed twice with ice-cold serum-free DMEM. The plates were then placed 244

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back to the 37 oC incubator for 18 to 20 h prior to quantification of infected cells by 245

immunofluorescence with a rabbit anti-rotavirus antibody followed by a FITC-labelled 246

goat anti-rabbit secondary antibody. 247

248

Results 249

The P[19] VP8* binds human saliva, human milk, and porcine mucin. Our study 250

started with in vitro binding assays of P[19] VP8* to human milk, human saliva, and 251

porcine mucin for the detection of potential host receptor ligands. The P[19] VP8* 252

proteins bound the high molecular weight gel-filtration fractions of human milk and 253

porcine mucins (Fig.2a, 2b), as well as a group of human saliva samples with variable 254

binding profiles among saliva donors (Fig. 2c), indicating the existence of receptors or 255

ligands in these natural host glycan reservoirs. The binding pattern of the P[19] VP8* to 256

human saliva differed from the P[4], P[6], and P[8] RVs (23), where the P[19] VP8* 257

bound a low proportion of samples (12%) with no correlation to the ABH or Lewis types 258

of the saliva donors (Fig. 2c), suggesting that a unique, low-abundant carbohydrate may 259

be responsible for the low prevalence of P[19] in human populations. 260

P[19] VP8* recognizes mucin cores and the type 1 HBGA precursor. To define 261

specific carbohydrates recognized by P[19] VP8*, a glycan array analysis was 262

performed by the Consortium of Functional Glycomics (CFG) against a glycan library 263

containing 610 mammalian glycans. Binding signals arose for a group of glycans 264

containing the type 1 HBGAs and/or mucin cores 2, 4, and 6 (Fig. 3a). Further binding 265

assays with different HBGA- and mucin core-related glycans (Fig. 3b-f) confirmed the 266

glycan array results. For example, among the four types (types 1-4) of HBGAs tested, 267

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the P[19] VP8* bound only the type 1 antigens (Fig. 3b-e). This result was further 268

confirmed by a focused neoglycolipid-based array with pair-wise comparisons of 269

oligosaccharide sequences that differed only in linkages between residues in each of 270

the type 1 (Galβ1-3GlcNAc) and type 2 (Galβ1-4GlcNAc) glycans (Fig. 3g). These 271

results indicate that only glycans containing the terminal Galβ1-3GlcNAc sequence 272

were recognized by P[19], further supporting the specificity toward type 1 sequences for 273

P[19]. 274

Additional modifications of the type 1 disaccharides of Galβ1-3GlcNAc affected the 275

P[19] VP8* binding signals. For example, the terminal A or B residues, as well as the 276

α1,2-linked fucose, could enhance the binding signals, while the Lewis epitope (α1,4-277

linked fucose) abolished binding (Fig. 3b). In vitro binding assays of the P[19] VP8* to 278

mucin core glycans (mucin cores 1 to 8) also confirmed the glycan array results, with 279

the positive binding signals observed only in those containing the disaccharide 280

GlcNAcβ1-6GalNAc, while further additions of residues GlcNAc β1-3 (mucin core 4) or 281

Gal β1-3 (mucin core 2) linking to GalNAc enhanced the binding (Fig. 3f). 282

The ligand binding specificity of the P[19] VP8* was further characterized by STD-NMR. 283

Data for a type 1 HBGA pentasaccharide LNFPI (Fucα1-2Galβ1-3GlcNAcβ1-3Gal) 284

demonstrated that the extended type 1 chain of an internal Gal (Fucα1-2Galβ1-285

3GlcNAcβ1-3Gal) is also involved in the interaction with P[19] VP8*, in addition to the 286

GlcNAc and the terminal (H) fucose (Fig. 4a-b). Similarly, STD-NMR of P[19] VP8* with 287

mucin core 2 showed a strong interaction with the disaccharide GlcNAcβ1-6GalNAc, in 288

which the GlcNAc residue was highly involved (Fig. 4c-d). 289

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P[19] RV replication was inhibited by human saliva, human milk, porcine mucin, 290

and LNFPI glycans. To examine the biologic significance of reactive glycan ligands for 291

P[19] RVs, viral replication inhibition assays were performed on a human P[19] RV. 292

P[19] RV titers were significantly reduced following incubation of the viruses with saliva, 293

porcine mucin fractions (gel-filtration), and human milk samples that previously bound 294

P[19] VP8* (Fig. 5a-b). In contrast, the virus titers were not reduced after treatment with 295

samples that did not bind to P[19] VP8* previously (Fig. 5b). Following treatment with 296

human milk, a typical dose-dependent reduction, with up to 77.2% of infectivity 297

decrease was observed (Fig. 5b). Similar blocking effects have also been observed for 298

the BSA conjugated LNFPI (Fig. 5c), whereas the non-conjugated mucin core 2 did not 299

result in an obvious inhibitory effect (Fig. 5c), possibly due to the monovalence of the 300

core 2 oligosaccharide used in the experiment. 301

The P[19] VP8* ligand binding interface differed from previously known ones. To 302

explore the glycan binding interface for P[19] RVs, NMR titration studies of mucin core 2 303

and LNFPI were conducted to measure chemical shift changes in backbone amide 304

resonances followed by 1H-15N heteronuclear single quantum coherence (HSQC) 305

spectra. All backbone chemical shifts files were available from BioMagResBank 306

database (accession number 25441). Although LNFPI caused slightly more amino acid 307

perturbations, the residues with HSQC chemical shift perturbations for mucin core 2 and 308

LNFPI largely overlapped (Fig. 6a-d), suggesting a common interface for the two 309

ligands. Interestingly, the KD-values of the HSQC chemical shift perturbation for P[19] 310

VP8* to mucin core 2 (KD = 0.4 mM) were higher than those to LNFPI (KD = 2.6 mM) 311

(Fig. 6e), indicating the affinity of P[19] VP8* to mucin core 2 was higher than to LNFPI. 312

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More than half of the residues did not reveal significant perturbations upon sequential 313

addition of the ligands, suggesting that there were no effects due to buffer changes. 314

Besides, NMR of P[19] VP8* with the glycan of LacNAc (Galβ1-4GlcNAc) were 315

conducted as a control experiment and no clear chemical shifts on P[19] VP8* was 316

observed, which further confirmed the specificity of mucin core 2 and LNFPI to P[19] 317

VP8*. 318

More than 90% of chemical shift perturbations occurred for a group of amino acids 319

scattered in a location near the previously identified SA- and A-HBGA binding interfaces 320

(11, 21, 44) (Fig. 7a-b). Mutants with single-amino acid changes at seven of these 321

perturbed residues (W81, L168, H170, W175, T186, R211, and E214) which could 322

constitute a binding interface of P[19] resulted in completely loss of binding to mucin 323

core glycans (Fig. 7a-d). In addition, besides the above mentioned seven amino acids, 324

we noted that single-amino acid mutations at another two perturbed residues (T218 and 325

Y189) also eliminated binding of P[19] VP8* to LNFPI (Fig. 7e-f). However, mutations at 326

three other perturbed amino acids (D188, Y189, and S190) in the corresponding SA or 327

A-HBGA binding interface, revealed only minor reductions in P[19] binding to mucin 328

cores and LNFPI, while none of other amino acids that constitutes the SA/A-HBGA 329

binding interface had effect on the binding of P[19] RV to either mucin cores or LNFPI. 330

Together, these data indicate that P[19] uses a new, but related, glycan binding 331

interface that significantly shifted away from the previously known sites. 332

Superimposition comparison of this glycan binding site with those for P[3], P[14], and 333

P[11] supported our observations (Fig. 7b, data not shown). 334

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Sequence alignment showed that all P[19] strains had identical amino acid 335

compositions on the deduced ligand binding site (Fig.8). Besides, alignment of the 336

group A RV VP8* sequences showed that the amino acids of the deduced ligand 337

binding interface of the P[19] RVs are conserved among all other three P[II] RVs (P[4], 338

P[6], and P[8]) (Fig.8), suggesting a common host factor or receptor may be responsible 339

for maintenance of the P[II] genogroup. In addition, among the diverse P[I] genogroup 340

that cause diseases in different animal species, we noticed that two genotypes (P[10] 341

and P[12]) also showed a good amino acid conservation of the deduced ligand binding 342

interface. In particular, a single residue W175 was found to be conserved among the 343

two P[I] (P[10]/P[12]) and all P[II] RVs, suggesting an evolutionary connection among 344

these human and animal RVs . 345

Similar but genotype-specific glycan binding profiles among P[II] RVs. Additional 346

binding studies for other P[II] RVs revealed similar but genotype-specific binding 347

properties among the three major human RVs (P[4], P[6], and P[8]) and the P[19] RVs. 348

Like the P[19] RVs, all of the other three P[II] RVs recognized the type 1 HBGA 349

disaccharide precursor with low affinities, while VP8* of P[4] and P[8], but not of P[6], 350

also recognized mucin cores 2, 4, and 6 (Fig. 9a-b). Since all these three major human 351

RVs previously were shown binding to the type 1 HBGAs (23), we performed binding 352

assays comparing the binding results with that of P[19] on three type 1 HBGA glycans 353

(LNFPI, LNT, and LNDFHI) with or without the secretor (H) and/or Lewis fucoses. The 354

P[6] and P[19] RVs recognized LNFPI and LNT, with the former containing the H 355

fucose, while P[4] and P[8] RVs recognized the hexasaccharide LNDFHI that contains 356

both H and Lewis fucoses (Fig. 9c-e). These data plus the conserved binding interface 357

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described above suggest a potential selection pressure in the maintenance of the P[II] 358

RVs by the host mucin cores and/or type 1 HBGAs. However, observations that 359

additional saccharide residues changed the RV binding outcomes may explain the 360

diverse host ranges for P[II] in different human populations and animal species. 361

P[10] and P[19] VP8*s shared nearly identical glycan array and saliva binding 362

profiles. To further study the genetic relationship between P[10] and P[19] RVs, we 363

also performed glycan array and in vitro binding assays of P[10] as well as other three 364

randomly selected P[I] RVs (P[13], P[15], and P[23]). Nearly identical saliva binding 365

(Fig. 2c-d) and glycan array binding profiles (Fig. 10) have been observed between the 366

P[10] and P[19] RVs, while the other tested P[I] RVs revealed completely different 367

glycan binding profiles (data not shown). This result suggests strong selection 368

pressures on the P[10] and P[19] genotypes by common host factor(s), such as the 369

mucin cores and type 1 HBGAs, which may be responsible for their common host 370

range. 371

372 Discussion 373

Three major P[II] RV genotypes, P[4], P[6], and P[8], cause the vast majority (>95%) of 374

human infections worldwide (45). Interestingly, the fourth P[II] genotype P[19], unlike the 375

others, is commonly found in animals (porcine) but rarely in humans (46-50). In this 376

study, we found that the P[19] VP8* binds human milk, human saliva, and porcine 377

mucins. The P[19] VP8* revealed a significantly lower binding profiles to the human 378

saliva than the highly prevalent P[4] and P[8] RVs, consistent with the low prevalence of 379

P[19] RVs in humans observed so far. We further demonstrated that the P[19] RVs 380

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recognize the mucin cores (2, 4, and 6) and type 1 HBGA precursors as the minimal 381

functional units. However, further modifications of these glycans with additional 382

saccharides, such as the A, B, H, or Lewis epitopes, and even SA, could influence and 383

further define the binding outcomes. Interestingly, this unique binding property is shared 384

with other P[II] RVs, supported by the observation that all the other P[II] RV genotypes 385

also recognize the mucin core and type 1 HBGA related sequences while exhibiting a 386

genotype-specific difference. 387

Specifically, all of the A, B, and H epitopes appear to be involved in P[19] RV binding, 388

while the addition of the Lewis epitope (1,4-linked fucose) to these type 1 HBGA 389

glycans completely blocked P[19] binding (Fig.3). This type of epitope blocking or 390

masking effect may explain why the P[19] RVs rarely infect humans; 90% of the general 391

population are Lewis-positive, leading to Lewis-negative type 1 HBGA phenotypes in 392

only 10% of the general population (45). On the other hand, the requirement of the 393

Lewis epitope for binding also seemed to play an important role in the high prevalence 394

of the P[4] and P[8] RVs in humans, which was suggested by their common recognition 395

of Lewis b (Leb) antigens containing the Lewis epitope (23). A strong association 396

between Lewis status and P[4]/P[8] RV infection has also been reported, where P[8] 397

infection only occurred in Lewis-positive children (31). In the current study, we further 398

demonstrated the importance of Lewis epitopes for these two genotypes by their binding 399

to the hexasaccharide LNDFHI (Fig.9), that contains the Lewis epitope, but not to the 400

type 1 glycans LNFPI and LNT that lack the Lewis epitope. 401

The differences in Lewis epitope involvement for P[II] RV binding may also play a role in 402

human versus animal host ranges, as some animal species, such as mice and pigs, 403

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lack Lewis antigens ((51), our unpublished data), and could explain why P[4] and P[8] 404

RVs rarely infect animals. On the other hand, the infection of P[19] and P[6] RVs in 405

animals may be due to their lack Lewis epitopes, leaving the mucin cores and/or the 406

type 1 HBGA precursors readily available to bind these genotypes. As an alternative 407

hypothesis it has been proposed, that shared HBGAs are responsible for cross-species 408

P[11] RV transmission between humans and animals, since P[11] recognizes 409

evolutionarily conserved type 2 HBGA precursors or intermediate products (22). 410

The P[6] RV did not bind the mucin core glycans and bound poorly to the human adult 411

saliva (23), but recognized the penta-saccharide LNT, containing the type 1 HBGA 412

sequences without H and Lewis epitopes, and the hexo-saccharide LNFPI, without 413

Lewis epitopes (Fig. 9). This result is consistent with those of another P[6] RV (strain 414

RV3) that bound a group of long glycans containing the type 1 HBGA terminal 415

sequences (with or without the H epitope but without the Lewis epitope) and an extra 416

Lewis x side chain (32). In addition, a negative association between P[6] RV infection in 417

children and the presence of the Lewis gene has been reported (31). Furthermore, in 418

the current study, we also observed an age-specific binding pattern for P[6] RVs to 419

saliva samples from neonates and young infants (data not shown), which is reminiscent 420

of the age-specific host ranges in neonates and young infants found for the P[11] RVs 421

(24, 26). Since P[6] RV infection appears independent of the Lewis epitope, we 422

hypothesize that P[6] RVs may share a similar mechanism for age-specific host ranges 423

as the P[11] RVs, by recognizing the HBGA precursor and/or intermediate products that 424

may be developmentally regulated in neonates and young infants. 425

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The finding of nearly identical saliva- and glycan-binding profiles between P[10] and 426

P[19] suggests a potential evolutionary connection between P[10] and P[19] RVs, which 427

may be selected by a common host factor such as the mucin cores and/or type 1 428

HBGAs. The conserved amino acids of the deduced binding interface, particularly the 429

exclusively conserved residue W175 between P[10]/P[12] and P[19] and other P[II] RVs 430

(P[4], P[6], and P[8]), further supports this hypothesis. Interestingly, P[10] and P[19] 431

have very similar host ranges, both pigs and humans, while the majority of other P[I] 432

RVs exclusively infect animals. This observation in our view raises concern regarding 433

the strategy to develop live, attenuated animal RV reassortant vaccines for humans, 434

because these animal RVs, such as the bovine (P[5]) and sheep (P[15]) (52, 53), may 435

not replicate in the human intestine due to lack of proper host receptors. Future studies 436

to clarify this concern are urgently required. 437

Our study raises additional issues that need to be addressed in the future. For example, 438

based on the binding properties of P[19] and other P[II] RVs, we noted a potential 439

binding specificity switch from simple mucin core glycans to more complex type 1 440

HBGAs, leading to host range changes between animal (P[I]) and human (P[II]) RVs 441

and among P[II] RVs in different human populations. However, direct evidence, 442

including supporting structural data, for such host range switching and variations 443

remains lacking. While mucin core 2 is commonly found in a variety of cells and tissues, 444

mucin cores 4 and 6 sequences were only found in a very limited type of mucin, that is 445

in human intestinal mucins (54). Thus, future studies to determine the role of mucin 446

cores versus type 1 HBGAs in human RV host ranges are necessary. Finally, during our 447

characterization of P[19] and other P[II] and P[I] RVs, we noted that the RV-glycan 448

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interactions are far more complicated than previously thought. While the minimal 449

binding units of the HBGA and/or mucin core sequences are essential for binding, 450

further terminal or internal side chain modifications, the lengths of the glycan 451

backbones, and repeated sequences could strongly influence or change the binding 452

affinities and/or specificities, and therefore, the final binding outcomes. Thus, future 453

studies are needed to define the precise amino acids within the binding interface that 454

are responsible for specific interactions between each sugar residue and the individual 455

P genotypes characterized in this study. 456

Acknowledgements 457

We would like to acknowledge the Protein-Glycan Interaction Core of the Consortium for 458

Functional Glycomics for their analysis of samples by glycan microarray. We thank Dr. 459

James C. Paulson for kindly providing the mucin core 2 oligosaccharideand, Dr. Todd L. 460

Lowary for providing the ABH-BSA conjugates, and Dr. Christina Quigley for discussion 461

and editing of the manuscript. 462

Figure Legends: 463

Figure 1. Phylogeny, host ranges, and potential host glycan ligands for group A 464

rotaviruses. The group A RVs have been divided into 37 P genotypes (P[1] – P[37]) 465

based on the major surface spike protein VP4 (33). A phylogeny dividing 35 group A 466

RVs (P[1] – P[35]) into in five P genogroups (P[I] – P[IV]), reported previously (25), is 467

shown. The assigned human and animal hosts were based on the ranks of VP4/VP8* 468

sequence numbers reported for individual species with only the top (internal row) and 469

second (our row) ranks shown. It is noted that limited numbers of sequences were 470

reported for genotypes P[10], P[18], P[20] as well as the newly identified genotypes 471

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P[28]-P[35], and the true host species for these genotypes need to be verified in future 472

studies. The carbohydrate ligands recognized by genotypes reported in the literature 473

are listed, including P[1], P[2], P[3], and P[7] (9); P[4], P[6], and P[8] (13, 20, 23); P[9], 474

P[14], and P[25] (25); P[11] (24, 26); and P[19] (in this study). Comparison of the P[7] 475

(PDB ID: 2I2S (44)) and P[14] (PDB ID: 4DRV (21)) receptor binding sites with P[19] is 476

in the bottom panel. The figure is modified based on data presented in previously 477

published references (21-23, 25, 44) 478

Figure 2. Binding profiles of P[19] VP8* with human milk and porcine mucin and a 479

comparison of saliva binding profiles between P[10] and P[19]VP8*s. The human 480

milk and porcine mucin samples were fractioned through a Superdex 200 column before 481

testing for binding to P[19] VP8* following a procedure described previously (24, 25). 482

Binding signals peaked in the high molecular weight fractions for both human milk 483

(panel a) and porcine mucin (panel b) samples. The P[19] VP8* also revealed a binding 484

profile similar to that of another RV (P[10] in P[I] genogroup, panel d) among 96 adult 485

human saliva samples tested (panel c). The ABH and Lewis statuses of the saliva 486

donors are indicated. Each column represents the mean A450 absorbance value and the 487

error bars represent the standard deviation for each sample tested in triplicate. 488

489

Figure 3. P[19] VP8* recognizes mucin cores 2/4/6 and type 1 HBGAs. (a) A list of 490

top glycans recognized by P[19] VP8* in a glycan array containing 610 glycans. (b)-(e) 491

Binding to four types (1 – 4) of HBGA glycans, of which the P[19] VP8* only recognized 492

type 1 HBGA glycans. The binding signals to the H1 trisaccharide (Fucα1-2Gal β1-493

3GlcNAc-) were weak to moderate but significantly increased with the addition of the A- 494

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or B-residues, whereas further addition of a β1-3 fucose (the Lewis epitope, green 495

dashed circle) to the GlcNAc (forming Leb) completely blocked the binding. The 496

disaccharide Galβ1-3GlcNAc of the type 1 HBGA precursor (highlighted in red dashed 497

boxes) is believed to be essential for binding, while the terminal residues fucose, A, or B 498

residues added to Gal (Fucα1-2Galβ1-3GlcNAc-, GalNAcα1-3(Fucα1-2)Galβ1-499

3GlcNAc-, Galα1-3(Fucα1-2)Galβ1-3GlcNAc-) could increase the binding. (f) Among the 500

eight mucin core glycans (core 1 to core 8) tested, only mucin cores 2, 4, and 6 501

revealed binding signals. The disaccharide GlcNAcβ1-6GalNAc is believed to be 502

essential for binding, which is highlighted in red dashed boxes. The ELISA was 503

performed in triplicate and the whole experiment was repeated once. The error bars 504

represent the standard deviations from triplicate wells. (g) Analysis with a focused array 505

of lipid-linked sequence-defined probes, which was carried out to further determine the 506

HBGA binding specificity of P[19] VP8*. Six pair-wise comparisons of type 1 and type 2 507

chains with different lengths and capping moieties are shown. The P[19] VP8* 508

recognized only the type 1 containing probes (red). The type 2 chain probes are marked 509

green. DH represents the amino lipid that the oligosaccharide probes were linked to. ‘-’ 510

indicates fluorescence intensity less than 1. 511

Figure 4. NMR spectra of LNFPI and mucin core 2 in complex with P[19] VP8*. 1H 512

NMR of P[19] VP8* (130 µM) with LNFPI (6.6 mM) is shown in (a). The LNFPI epitope 513

map when bound to P[19] VP8* is shown in (b): dark orange, strong STD NMR effects; 514

orange, weaker; and yellow, the weakest STD NMR effects. The STD amplification 515

factors are shown in the box for residues that are not overlapped in the 1D STD 516

spectrum. For 1H resonances that were overlapped in the 1D STD spectrum, further 2D 517

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STD-TOCSY was applied. The inclusion of GlcNAc-III H6s and Gal-IV H3 in the binding 518

epitope was based on the 2D STD-TOCSY and colored orange (data not shown). 1H 519

NMR of mucin core 2 (6.0 mM) with P[19] VP8* (200 µM) is shown in (c) with an 520

expanded spectral view shown at the top of the panel. ‘H6d’ indicates a downfield 521

proton and ‘H6u’ indicates an upfield proton. The epitope map of mucin core 2 when 522

bound to P[19] VP8* is shown in (d). The STD-TOCSY was used to add the overlapping 523

resonances of GlcNAc-III H3/H4/H5 in the binding epitope (data not shown). 524

Figure 5. Inhibition of P[19] RV replication in cell culture by human milk, neonatal 525

saliva, porcine mucin, and LNFPI conjugates. A representative immunofluorescence 526

microscopy image of MA104 cells infected with P[19] RV is shown in (a). Each green 527

dot represents an infected cell, as shown in the enlarged panel. The plotted blocking 528

results are shown in (b) and (c). The error bars represent standard errors from triplicate 529

repeats, and the experiment was repeated once. The statistical significance was 530

calculated by ANOVA, the asterisk refers to statistical difference <0.05. (b) Blocking 531

results with neonatal saliva, human milk, and porcine mucin FPLC fractions. Human 532

saliva samples that do (saliva (+)) and do not (saliva (-)) bind to P[19] VP8* in the ELISA 533

binding assays were used in the blocking assay as positive and negative controls, 534

respectively. The saliva samples were tested at 1:10 and 1:20 dilutions in PBS with a 535

negative control containing PBS only. Serial dilutions of the human milk samples at 1:1, 536

1:10, and 1:100 were tested. The FPLC fraction of the porcine mucin samples that 537

showed VP8* binding (F11) was tested with an FPLC fraction (F81) that did not bind 538

P[19] VP8* as a negative control. (c) Blocking results with BSA-conjugated LNFPI and 539

free mucin core 2 oligosaccharide at final concentrations of 0.25, 0.5, and 1 µg/µl. 540

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Figure 6. Chemical shift changes in P[19] VP8* upon addition of mucin core 2 (a) or 541

LNFPI (b). Titrations were followed with two-dimensional 1H-15N HSQC spectra of 0.25 542

mM 15N-labeled P[19] VP8* in Tris buffer collected at 20 oC on a Bruker Avance III 600 543

MHz NMR spectrometer. NMR data shown has increasing ligand:protein ratios of 0:1 544

(red), 2:1 (orange), 8:1 (green), and 30:1 (blue). Peaks that disappeared upon titration 545

were circled. Surface representation of the HN chemical shift changes for P[19] VP8* 546

with mucin core 2 (c) and LNFPI (d) was shown. The data were plotted onto the surface 547

of a P[19] VP8* homology model with the largest changes in dark red. The chemical 548

shift differences between 1H and 15N resonances in free- and ligand-bound proteins 549

were determined as the weighted chemical shift change. For LNFPI, three HN cross-550

peaks disappeared upon ligand titration and did not reappear at higher ligand 551

concentrations. These residues, T186, E214, and C217 were set at a maximum of Δδ av 552

= 0.25. The 2-D 1H-15N HSQC with ligand:protein ratio of 30:1 was used for the bound 553

shifts. The protein structure of P[19] VP8* was based on a homology model using the 554

crystal structure of DS1 (a P[4] RV, PDB ID: 2AEN) as the template and marked by 555

using the Pymol program. (e) Plot of normalized chemical shift changes (CSC/CSC-556

max) versus free ligand concentrations (mM). 557

Figure 7. Ligand-binding interface for P[19]VP8* confirmed by site-directed 558

mutagenesis. (a) The homology model for P[19] VP8* was constructed based on the 559

DS1 crystal structure (P[4], PDB ID: 2AEN). The surface amino acid residues with 560

chemical shift changes in HSQC NMR titration are underlined. The amino acids 561

corresponding to previously reported SA- or A antigen binding sites are colored yellow, 562

while the newly identified ligand binding site is colored pink. (b) Superimposition of P[19] 563

VP8* (in cyan) with P[3] VP8* (grey, PDB ID:1KQR). The glycan binding interfaces for 564

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the two genotypes are colored in pink and yellow, respectively. (c) The binding results 565

for P[19] VP8* and its mutants, each with a signal amino acid mutation at the 566

corresponding SA-binding amino acids (V101, F144, N146, K155, D188, Y189, S190, 567

T191), to the mucin cores are shown. Mutants with significant glycan binding A450 value 568

changes are marked by a star (*). (d) The binding results for P[19] VP8* and its 569

mutants, with chemical shift changes at around the sialic acid or A antigen binding site, 570

to mucin core glycans. Single amino acid mutations of seven residues (W81, L168, 571

H170, W175, T186, R211, E214) resulted in a complete loss of binding signals. (e) The 572

binding results of the P[19] VP8* and its mutants at the corresponding SA-binding 573

amino acids to the glycan of LNFPI. (f) Binding of mutations corresponding to amino 574

acids that showed chemical shift changes to LNFPI. All seven residues that were found 575

to be involved in mucin core binding (W81, L168, H170, W175, T186, R211, E214), and 576

an additional residue T218, also resulted in a complete loss of binding to LNFPI. The 577

binding assay was tested in triplicate and the entire experiment was repeated twice. The 578

statistical significance was calculated by ANOVA analysis and the mean A450 579

absorbance values with standard deviations are shown. 580

Figure 8. Sequence alignment of VP8*s from reported P[19] strains. The amino 581

acids which were deduced to interact with mucin cores and LNFPI glycans in the current 582

study are highly conserved among all the P[19] human and porcine strains, and are 583

marked by orange boxes. Residues that interact with sialic acid or type A HBGAs in the 584

corresponding genotypes were marked in red, and those that interact with type 2 585

HBGAs precursor for P[11] genotype were marked in blue. 586

587

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Figure 9. Binding of P[II] RV VP8*s to mucin cores 2, 4, 6, and type 1 HBGA-588

related glycans. (a) Binding of the P[4], P[6], and P[8] VP8*s to mucin cores 2, 4, and 589

6. Two doses (2 or 20 µg/ml) of the mucin cores were tested. Positive binding signals 590

were observed for P[4] and P[8], but not for P[6]. (b) Binding of the P[II] RVs to the type 591

1 precursor disaccharide lacto-N-biose (Galβ1-3GlcNAc). Low binding signals were 592

detected for all four P[II] RVs. (c)-(e) Binding of the P[II] RV VP8*s with the type 1 593

HBGAs LNT, LNFPI, and LNDFPI. The P[6] and P[19] VP8*s showed positive binding to 594

the type 1 tetrasaccharide LNT (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) (c) and the LNFPI 595

pentasaccharide (Fucα1-2 Galβ1-3GlcNAcβ1-3Galβ1-4Glc) (d) with or without the 596

terminal fucose modification (secretor), respectively. Neither of the P[4] and P[8] VP8*s 597

bound the LNT and LNFPI oligosaccharides (e). Adding a Lewis fucose to LNFPI results 598

in the LNDFHI hexasaccharide (Fucα1-2 Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc), 599

which led to negative P[6] and P[19] VP8* binding, but positive P[4] and P[8] VP8* 600

binding. The mean A450 absorbance values from three replicates with standard 601

deviations are shown. 602

Figure 10. P[10] from P[I] showed a similar glycan binding pattern to P[19] VP8*. 603

A glycan array analysis was performed for P[10] in comparison with P[19]. Relative 604

Fluorescent Units (RFU) of GST-VP8* at 200 µg/ml binding to 610 glycans are shown, 605

where RFU corresponds to the strength of binding to individual glycans. The identities of 606

the 610 glycans in the array are available at 607

http://www.functionalglycomics.org/static/consortium/resources/resourcecoreh8. The 608

P[10] VP8* (panel b)revealed a similar binding profile, with a dozen identical, high 609

reactive glycans, to that of P[19] VP8*(panel a). The top fifteen ranked glycans for each 610

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of the two genotypes are shown on the right. Thirteen of the fifteen glycans (marked in 611

bold) were shared by both P[19] and P[10]. The error bars represent the standard 612

deviation from four replicate wells. 613

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17. Coulson BS, Londrigan SL, Lee DJ. 1997. Rotavirus contains integrin ligand sequences 659 and a disintegrin-like domain that are implicated in virus entry into cells. Proc Natl Acad 660 Sci U S A 94:5389-5394. 661

18. Graham KL, Halasz P, Tan Y, Hewish MJ, Takada Y, Mackow ER, Robinson MK, 662 Coulson BS. 2003. Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain 663 via VP4 DGE sequence and recognize alphaXbeta2 and alphaVbeta3 by using VP7 664 during cell entry. J Virol 77:9969-9978. 665

19. Hewish MJ, Takada Y, Coulson BS. 2000. Integrins alpha2beta1 and alpha4beta1 can 666 mediate SA11 rotavirus attachment and entry into cells. J Virol 74:228-236. 667

20. Bohm R, Fleming FE, Maggioni A, Dang VT, Holloway G, Coulson BS, von Itzstein 668 M, Haselhorst T. 2015. Revisiting the role of histo-blood group antigens in rotavirus 669 host-cell invasion. Nat Commun 6:5907. 670

21. Hu L, Crawford SE, Czako R, Cortes-Penfield NW, Smith DF, Le Pendu J, Estes 671 MK, Prasad BV. 2012. Cell attachment protein VP8* of a human rotavirus specifically 672 interacts with A-type histo-blood group antigen. Nature 485:256-259. 673

22. Hu L, Ramani S, Czako R, Sankaran B, Yu Y, Smith DF, Cummings RD, Estes MK, 674 Venkataram Prasad BV. 2015. Structural basis of glycan specificity in neonate-specific 675 bovine-human reassortant rotavirus. Nat Commun 6:8346. 676

23. Huang P, Xia M, Tan M, Zhong W, Wei C, Wang L, Morrow A, Jiang X. 2012. 677 Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-678 specific manner. J Virol 86:4833-4843. 679

24. Liu Y, Huang P, Jiang B, Tan M, Morrow AL, Jiang X. 2013. Poly-LacNAc as an 680 age-specific ligand for rotavirus P[11] in neonates and infants. PLoS One 8:e78113. 681

25. Liu Y, Huang P, Tan M, Biesiada J, Meller J, Castello AA, Jiang B, Jiang X. 2012. 682 Rotavirus VP8*: phylogeny, host range, and interaction with histo-blood group antigens. 683 J Virol 86:9899-9910. 684

26. Ramani S, Cortes-Penfield NW, Hu L, Crawford SE, Czako R, Smith DF, Kang G, 685 Ramig RF, Le Pendu J, Prasad BVV, Estes MK. 2013. The VP8* domain of neonatal 686 rotavirus strain G10P[11] binds to type II precursor glycans. J Virol 87:7255-7264. 687

27. Tan M, Jiang X. 2014. Histo-blood group antigens: a common niche for norovirus and 688 rotavirus. Expert Rev Mol Med 16:e5. 689

28. Van Trang N, Vu HT, Le NT, Huang P, Jiang X, Anh DD. 2014. Association between 690 norovirus and rotavirus infection and histo-blood group antigen types in Vietnamese 691 children. J Clin Microbiol 52:1366-1374. 692

29. Imbert-Marcille B-M, Barbe L, Dupe M, Le Moullac-Vaidye B, Besse B, Peltier C, 693 Ruvoen-Clouet N, Le Pendu J. 2014. A FUT2 gene common polymorphism determines 694 resistance to rotavirus A of the P[8] genotype. J Infect Dis 209:1227-1230. 695

30. Payne DC, Currier RL, Staat MA, Sahni LC, Selvarangan R, Halasa NB, Englund 696 JA, Weinberg GA, Boom JA, Szilagyi PG, Klein EJ, Chappell J, Harrison CJ, 697

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Davidson BS, Mijatovic-Rustempasic S, Moffatt MD, McNeal M, Wikswo M, Bowen 698 MD, Morrow AL, Parashar UD. 2015. Epidemiologic association between FUT2 699 secretor status and severe rotavirus gastroenteritis in children in the United States. JAMA 700 Pediatr 169:1040-1045. 701

31. Nordgren J, Sharma S, Bucardo F, Nasir W, Gunaydin G, Ouermi D, Nitiema LW, 702 Becker-Dreps S, Simpore J, Hammarstrom L, Larson G, Svensson L. 2014. Both 703 Lewis and secretor status mediate susceptibility to rotavirus infections in a rotavirus 704 genotype-dependent manner. Clinical infectious diseases : an official publication of the 705 Infectious Diseases Society of America 59:1567-1573. 706

32. Yu Y, Lasanajak Y, Song X, Hu L, Ramani S, Mickum ML, Ashline DJ, Prasad BV, 707 Estes MK, Reinhold VN, Cummings RD, Smith DF. 2014. Human milk contains novel 708 glycans that are potential decoy receptors for neonatal rotaviruses. Mol Cell Proteomics 709 13:2944-2960. 710

33. Trojnar E, Sachsenroder J, Twardziok S, Reetz J, Otto PH, Johne R. 2013. 711 Identification of an avian group A rotavirus containing a novel VP4 gene with a close 712 relationship to those of mammalian rotaviruses. J Gen Virol 94:136-142. 713

34. Huang P, Morrow AL, Jiang X. 2009. The carbohydrate moiety and high molecular 714 weight carrier of histo-blood group antigens are both required for norovirus-receptor 715 recognition. Glycoconj J 26:1085-1096. 716

35. Liu Y, Childs RA, Palma AS, Campanero-Rhodes MA, Stoll MS, Chai W, Feizi T. 717 2012. Neoglycolipid-based oligosaccharide microarray system: preparation of NGLs and 718 their noncovalent immobilization on nitrocellulose-coated glass slides for microarray 719 analyses. Methods Mol Biol 808:117-136. 720

36. Palma AS, Liu Y, Childs RA, Herbert C, Wang D, Chai W, Feizi T. 2011. The human 721 epithelial carcinoma antigen recognized by monoclonal antibody AE3 is expressed on a 722 sulfoglycolipid in addition to neoplastic mucins. Biochem Biophys Res Commun 723 408:548-552. 724

37. Gao C, Liu Y, Zhang H, Zhang Y, Fukuda MN, Palma AS, Kozak RP, Childs RA, 725 Nonaka M, Li Z, Siegel DL, Hanfland P, Peehl DM, Chai W, Greene MI, Feizi T. 726 2014. Carbohydrate sequence of the prostate cancer-associated antigen F77 assigned by a 727 mucin O-glycome designer array. J Biol Chem 289:16462-16477. 728

38. Zhang D, Huang P, Zou L, Lowary TL, Tan M, Jiang X. 2015. Tulane virus 729 recognizes the A type 3 and B histo-blood group antigens. J Virol 89:1419-1427. 730

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41. Bahrami A, Assadi AH, Markley JL, Eghbalnia HR. 2009. Probabilistic interaction 737 network of evidence algorithm and its application to complete labeling of peak lists from 738 protein NMR spectroscopy. PLoS Comput Biol 5:e1000307. 739

42. Meyer B, Peters T. 2003. NMR spectroscopy techniques for screening and identifying 740 ligand binding to protein receptors. Angew Chem Int Ed Engl 42:864-890. 741

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43. Hermansson K, Jansson PE, Kenne L, Widmalm G, Lindh F. 1992. A 1H and 13C 742 NMR study of oligosaccharides from human milk. Application of the computer program 743 CASPER. Carbohydr Res 235:69-81. 744

44. Blanchard H, Yu X, Coulson BS, von Itzstein M. 2007. Insight into host cell 745 carbohydrate-recognition by human and porcine rotavirus from crystal structures of the 746 virion spike associated carbohydrate-binding domain (VP8*). J Mol Biol 367:1215-1226. 747

45. Santos N, Hoshino Y. 2005. Global distribution of rotavirus serotypes/genotypes and its 748 implication for the development and implementation of an effective rotavirus vaccine. 749 Rev Med Virol 15:29-56. 750

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47. Mukherjee A, Chattopadhyay S, Bagchi P, Dutta D, Singh NB, Arora R, Parashar 755 UD, Gentsch JR, Chawla-Sarkar M. 2010. Surveillance and molecular characterization 756 of rotavirus strains circulating in Manipur, north-eastern India: increasing prevalence of 757 emerging G12 strains. Infect Genet Evol 10:311-320. 758

48. Das S, Varghese V, Chaudhuri S, Barman P, Kojima K, Dutta P, Bhattacharya SK, 759 Krishnan T, Kobayashi N, Naik TN. 2004. Genetic variability of human rotavirus 760 strains isolated from Eastern and Northern India. J Med Virol 72:156-161. 761

49. Maneekarn N, Khamrin P, Chan-it W, Peerakome S, Sukchai S, Pringprao K, 762 Ushijima H. 2006. Detection of rare G3P[19] porcine rotavirus strains in Chiang Mai, 763 Thailand, provides evidence for origin of the VP4 genes of Mc323 and Mc345 human 764 rotaviruses. J Clin Microbiol 44:4113-4119. 765

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51. Falk PG, Bry L, Holgersson J, Gordon JI. 1995. Expression of a human alpha-1,3/4-769 fucosyltransferase in the pit cell lineage of FVB/N mouse stomach results in production 770 of Leb-containing glycoconjugates: a potential transgenic mouse model for studying 771 Helicobacter pylori infection. Proc Natl Acad Sci U S A 92:1515-1519. 772

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53. Fu C, He Q, Xu J, Xie H, Ding P, Hu W, Dong Z, Liu X, Wang M. 2012. 776 Effectiveness of the Lanzhou lamb rotavirus vaccine against gastroenteritis among 777 children. Vaccine 31:154-158. 778

54. Moran AP, Gupta A, Joshi L. 2011. Sweet-talk: role of host glycosylation in bacterial 779 pathogenesis of the gastrointestinal tract. Gut 60:1412-1425. 780

781 782

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P[18]

P[1]

P[7] P[2]

P[3]

P[15] P[23]

P[24]

P[10]

P[12] P[28]

P[20]

P[16]

P[32]

P[26] P[13]

P[22]

P[21]

P[27]

P[33] P[29]

P[5]

P[34]

P[8]

P[4] P[19]

P[6]

P[9]

P[25]

P[14]

P[31]

P[11]

P[17]

P[30]

P[35]

P[I]

P[II] P[III] P[IV]

P[V]

Leb

A antigen

Sia

Type 2

precursor

Leb

0.1

Type 1

Chain

Mucin

cores

2/4/6

H type 1

P[7] P[14] P[19]

Figure 1

GM1

Mucin cores 2/4/6

Mucin cores 2/4/6

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Figure 2

F1

F11

F21

F31

F41

F51

F61

F71

F81

0.0

0.2

0.4

0.6

0.8

OD

(450n

m)

a

FPLC fractions

F1

F1

1

F2

1

F3

1

F4

1

F5

1

F6

1

F7

1

0.0

0.1

0.2

0.3

0.4

0.5

OD

(450n

m)

b

FPLC fractions

OH

2O

H4

OH

6O

H8

OH

10

OH

12

OH

14

OH

16

OH

18

OH

20

OH

22

OH

24

OH

26

OH

28

OH

30

OH

32

OH

34

OH

36

OH

38

OH

40

OH

42

OH

44

OH

46

OH

48

OH

50

OH

52

OH

54

OH

56

OH

58

OH

60

OH

62

OH

64

OH

66

OH

68

OH

70

OH

72

OH

74

OH

76

OH

78

OH

80

OH

82

OH

84

OH

86

OH

88

OH

90

OH

92

0.0

0.2

0.4

0.6

0.8

1.0

A B AB O

OD

(450n

m)

OH

2O

H4

OH

6O

H8

OH

10

OH

12

OH

14

OH

16

OH

18

OH

20

OH

22

OH

24

OH

26

OH

28

OH

30

OH

32

OH

34

OH

36

OH

38

OH

40

OH

42

OH

44

OH

46

OH

48

OH

50

OH

52

OH

54

OH

56

OH

58

OH

60

OH

62

OH

64

OH

66

OH

68

OH

70

OH

72

OH

74

OH

76

OH

78

OH

80

OH

82

OH

84

OH

86

OH

88

OH

90

OH

92

0.0

0.2

0.4

0.6

0.8

1.0

A B AB O

OD

(450n

m)

Saliva sample

P[10]

P[19]

c

d

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a

b

H1-Tri A1-Tetra B1-Tetra Leb

0.0

0.2

0.4

0.6

0.8

OD

(450n

m)

α2

β3

α3

β

α2

β3

α3

β

α4 β3

β

α2 α2

β3

β

H3-Tri A3-Tetra B3-Tetra

0.0

0.2

0.4

0.6

0.8

OD

(450n

m)

α2

β3 α

α2

β3

α3

α

α2

β3

α3

α

d c

H2-Tri A2-Tetra B2-Tetra Ley

0.0

0.2

0.4

0.6

0.8

OD

(450n

m)

α2

β4 β

α2

β4

α3

β

α2

β4

α3

β β

α2

β4

α3 α2

β3

β

α2

β3

α3

β

α2

β3

α3

β

H4-Tri A4-Tetra B4-Tetra

0.0

0.2

0.4

0.6

0.8

OD

(4

50

nm

)

e

f

1 2 3 4 5 6 7 8

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

2 ug/ml

10 ug/ml

50 ug/ml

OD

(450n

m)

β3 β3

α3 α6 α3

β3

β6

β3

β6

β6

Mucin cores

GalNAc

Gal

Fuc

GlcNAc

Figure. 3

Chart Number

Glycan Structure Average RFU

193 GlcNAcβ1-6GalNAcα-Sp14 847

177 GlcNAcβ1-6GalNAcα-Sp14

I GlcNAcβ1-3

1587

176 GlcNAcβ1-6GalNAcα-Sp80

I GlcNAcβ1 -3

400

133 GlcNAcβ1-6GalNAcα-Sp81

I Galβ1-3

3887

134 GlcNAcβ1-6GalNAcα-Sp14

I Galβ1-3

2960

604 GlcNAcβ1-6GalNAcα-Sp14

I Neu5Acα2-3Galβ1-3

5399

567 Galβ1-3GlcNAcβ1-6GalNAcα-Sp14

I Galβ1-3

3035

505 Fucα1-2Galβ1-3GlcNAcβ1-6GalNAcα-Sp14

I Fucα1-2Galβ1-3GlcNAcβ1 -3

444

525 Galα1-3Galβ1-3GlcNAcβ1-6GalNAcα-Sp14

I Fucα1-2

1633

506 GalNAcα1-3Galβ1-3GlcNAcβ1-6GalNAcα-Sp14

I Fucα1-2

807

66 Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-Sp10 1024

65 Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-Sp80 950

558

GlcNAcβ1-3Galβ1-4GlcNAcβ1-6Manα1-6Manβ1-4GlcNAcβ1-4GlcNAcα-Sp24 I | GlcNAcβ1-3Galβ1-4GlcNAcβ1-2 GlcNAcβ1-3Galβ1-4GlcNAcβ1-2Manα1-3

505

g

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a

c d

b

Figure 4

4.0 3.0 2.0 ppm

H4-IV

Fuc-I

H6-CH3

GlcNAc-III

CH3

H3-III

H2-V H5-I

H1-IV H1s-

V,II,III

1

2

3

4

6 5

3

1

2 4

5

6

3 1

2

4

5

6

3 1

2 4

5

6

3 1

2

4

5

6

Fuc-I

Gal-II GlcNAc-III Gal-IV Glc-V

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BSA 00.

25 0.5 1 0

0.25 0.

5 1

0

50

100

150

Vir

us in

fectivity t

ite

r(%

of

co

ntr

ol)

Saliva(+) Saliva(-) Human milk Porcine mucin

01:

201:

10 01:

201:

10 0

1:1

00 1

:10

1:1

F81 F11

0

50

100

150

Vir

us in

fectivity t

ite

r(%

of

co

ntr

ol)

a

b

c

Figure 5

* *

* *

*

*

*

*

LNFPI-BSA(µg/µl) Mucin core 2 (µg/µl)

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a b

c d e

Figure 6

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 2 4 6 8

Mucin core 2

LNFPI

[Ligand](mM)

No

rma

lize

d C

SC

LNFPI

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a

d

P19 W

ild

V101A

F144A

N14

6A

K15

5A

D18

8A

Y189A

S190A

T191A

P19 W

ild

V101A

F144A

N14

6A

K15

5A

D18

8A

Y189A

S190A

T191A

P19 W

ild

V101A

F144A

N14

6A

K15

5A

D18

8A

Y189A

S190A

T191A

0.0

0.5

1.0

1.5

2.0

2.5 mucin core6

mucin core4

mucin core2

OD

(450n

m)

e

b

P19 W

ild

R17

3A

T192A

Y203A

K21

6A

C21

7A

T218A

W81

A

L168A

H17

0A

W17

5A

T186A

R21

1A

E214A

P19 W

ild

R17

3A

T192A

Y203A

K21

6A

C21

7A

T218A

W81

A

L168A

H17

0A

W17

5A

T186A

R21

1A

E214A

P19 W

ild

R17

3A

T192A

Y203A

K21

6A

C21

7A

T218A

W81

A

L168A

H17

0A

W17

5A

T186A

R21

1A

E214A

0.0

0.5

1.0

1.5

2.0

2.5mucin core6

mucin core4

mucin core2

OD

(450n

m)

Figure 7

P[19]

Wild

V101A

F144A

N14

6A

K15

5A

D18

8A

Y189A

S190A

T191A

0.0

0.5

1.0

1.5

2.0

2.5

OD

(450n

m)

P[19]

Wild

R17

3A

T192A

Y203A

K21

6A

C21

7A

W81

A

L168A

H17

0A

W17

5A

T186A

R21

1A

E214A

T218A

0.0

0.5

1.0

1.5

2.0

2.5

OD

(450n

m)

c

K216

T218

R173

T192

F144 N146 K155

D188

Y189

S190

T191

W175 T186

E214

R211 H170

L168 W81

f

*

* * *

*

*

*

* * * * * * * * * * * * *

* * *

* * *

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100 110 120 130 140 150 160 170 180 190 200 210 220

|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|...

RRV|P[3] 100 DRWLATILVEPNVTSETRSYTLFGTQEQITIAYASQTQWKFIDVVKTTQNGSYS-QYGPLQSTPKLYAVMKHN-GKIYTYNGETPNVTTK--YYSTTNYDSVNMTAFCDFYIIPREEESTCTEYINNGL

NCDV|P[1] 100 NRWLATILIEPNVQQVERTYTLFGQQVQVTVSNDSQTKWKFVDLSKQTQDGNYS-QHGSLLSTPKLYGVMKHG-GKIYTYNGETPNATTG--YYSTTNFDTVNMTAYCDFYIIPLAQEAKCTEYINNGL

SA11|P[2] 100 DRWLATILIEPNVQSENRTYTIFGIQEQLTVSNTSQDQWKFIDVVKTTANGSIG-QYGSLLSSPKLYAVMKHN-EKLYTYEGQTPNARTG--HYSTTNYDSVNMTAFCDFYIIPRSEESKCTEYINNGL

CRW8|P[7] 100 DRWLATILIEPNVQTTNRIYNLFGQQVTLSVENTSQTQWKFIDVSKTTPTGNYT-QHGSLFSTPKLYAVMKFS-GRIYTYNGTTPNATTG--YYSTTNYDTVNMTSFCDFYIIPRNQEEKCTEYINHGL

L338|P[18] 100 NRWLAIILVEPDVPTEERTYTLFGQQAQITVANDSQLKWKFIVVSKQTLDGAYA-QYGPLLSATKLYAVMKHS-GRIYTYSGETPNATTA--YYSTTNYDTVNMKAYCHFYIIPRTQESKCTEYINTGL

69M|P[10] 100 NRWLATILVEPGVASTTRTYTLFGIQEQITVENSSNTKWKFIDLMKTTSSGTYT-QHSPLLSEPKLYGIMKHG-GQLWTYNGETPNAITN--GYPTTNYDSVNMTSFCNFYIIPRSQESVRTGYINNGL

H-2|P[12] 100 NRWLATVLVEPNVVTTVRTYTLFGVQEQISVENNSTTKWKFMNLIKTTLSGNFT-LYSTLLSEPKLHGIMKHG-GQLWVYNGETQTLLLQ--DYVTSNYDSLTMTSFCDFYIIPRSQESTCTEYINNGL

WC3|P[5] 100 GRWLSVILIEPGVTSETRTYTMFGSSKQVLVSNASDTKWKFVEMMKTAIDGDYA-EWGTLLSDTKLYGMMKYG-KRLFIYEGETPNATTK--RYIVTNYASVEVRPYSDFYIISRSQESACTEYINNGL

A46|P[13] 100 DMWLVIILVEPNVPQELRLYTLFGQQVNLTIENTSQTKWKFIDFRKRSQNDTYI-LENALLSETKLQAAMKYG-GKLFTFTGDTPNAAPQEWGYTTNNYSAISIKSLCDFYIVPRLPRETCRNYINQGL

Lp14|P[15] 100 NRWLATILIEPNVPETTRNYTLFGETASISVANPSQSKWRFVDVVKTTANGTYS-QYGPLLSDTKLYGVMKYN-GKLYTYNGETPNATTK--YYSTTNYDSVNMTSYCDFYIIPRAQESKCTEYVNNGL

Eb|P[16] 100 DRWIATIAIEPNVSKTKRTYTLFGITEQLTVENSYADKWKFIDFLKASTNGSYA-RYNILLSSTKLCAVAKHT-DRLYSYIRETPNAGQA--YYAFSHN-IFNLTAHCDFYIIPWAQQSLCTQYINTGL

EHP|P[20] 100 DRWLATILIEPNVASTTRTYTIFGSKEAITVENTSQNKWKFIDLAKTSLTGSYS-QYGILLSKPKLYAIMKRS-GYLYTYSGETPDAITD--YYTTTNYDSVNMTAYCDFYIIPWAQEALCTQYINNGL

Hg18|P[21] 100 NKWFATILIEQDVVAESRTYTIFGKTESIQAENTSQTEWKFIDIIKTTQDGTYS-QYGPLVLSTKLYGVMKYG-GRLYAYIGHTPNATPG--HYTIANYDTMEMSIFCEFYIMPRSQEAQCTEYINSGL

160-01|P[22] 100 DIWLVIILVEPNVPQGSRTYTIFGQQKSLMIENTSQTKWKFIDFIKESQGGTYV-PNDTLLSETKLQAAMKYG-QRLFTFIGNTPNSAPQERGYTTNNYSTINVTSLCDFYIVPRTPREVCRNYINHGL

A34|P[23] 100 DRWLATILIEPNVTSQSRTYTLFGHQEQITVENVSTTKWKFVDLAKTDINGTFT-QHGPLLSDTKLHGVMKFS-GRLYTYNGETPNATIG--YYTTTNYDTVNMVSHCDFYIIPRSEENTCTNYINNGL

TUCH|P[24] 100 DRWLATILIEPNVAAETRTYTLFGVQEQISISNTSSTKWKFVDLVKTSLTGTYS-QYGPLLSDTKLYGAMKYS-GRIYTYEGETPNAATG--YYSTTNYDSVNMTAFCDFYIIPRSQESTCTNYINNGL

134-04-15|P[26] 100 SRWLAIILVEPNVSQETRTYTIFGQQVQLQVENVSQTKWKFIDVSKTSQGGSYT-QYSTLLSNTKLDAVIKYG-GKVFTYNGETPSATSG--QYTTTNYEAIVMTLFCDFYIIPRTQEETCTNYINHGL

344-04-1|P[27] 100 DQWIAAILIEPNTNTITKNYTLFGKQEQIEVSNAS-SKWKFVDLIKKTVDGSFT-QRGPLLSDTKLYAVMKHG-SQVFTYGGETPNAATV--GYATLNYSELIIAVYCDFYIIPRAQEKQCTSFINNGL

P21-5|P[28] 100 DQWIAAILIEPNTSTTTRSYALFGKQESIEVSNPS-SRWKFVELVKHTVDGGFT-QHGPLLSNTKLYGVMKYG-QKVFTYQGETPNATTD--YYTVSNYSELIITVYCDFYIISRAQQAECTNFINNGL

AzuK-1|P[29] 100 NIWMAAIIIEPNLSAEQRTYRIYDEDVELLVDNASRTKWKIVDLYKTQQSGQYVRQNWRIYTAYKLYGIRIVD-TKLQSYGGETPNLSIIT--TTVTNYSNVEITTLSNFFIISSNNTEKCTTYMNQGL

IRL|P[32] 100 NRWLAIILVEPGVTQTTREYNVFGERIQLQVENTHQTKWKFIDISRANQSDSYA-QYGPLLSDTKLYAAMKYN-GLICTYVGETPNARTD--QYSTTNYRDVEMTIFCDFYIIPRAQENICTNYINNGL

Dai-10|P[33] 100 NRWLAVILIEPGVNQETRNYNIFGKQEQLVVSNTSSTQWKFVDAIKTSEDGSYN-QHGQLLSTNKLYAVMKSG-GRVYTYEGTTPNARTG--FYTTSNYSTVKVNVLCDFYIIPLAQESKCTEYINNGL

FGP51|P[34] 100 NKWIAAIMIEPSTISVTKNYTLFGQQEQVQVSNTSTTKWKLVELVKQTQNGVYT-QHGELLSDTKLYGVMKYG-GLVCTYKGETPNATTD--QYTTSNYSELSIAVHCDFYIISRAQEAECTRYINYGL

MC345|P[19] 100 DVWVAIISIEPNVNSESRQYSLFGVNKQITVVNTS-NKWKFMEMFRNNSNAEFQ-HKRTLTSSTKLVGILKHG-GRLWTYHGETPNATTD--YSTTSNLNEISVTTYAEFYIIPRSQESKCTEYINTGL

ST3|P[6] 100 DIWIALLLVEPNVTNQSRQYTLFGETKQITIENNT-NKWKFFEMFRSNVSSEFQ-HKRTLTSDTKLAGFLKHY-NSVWTFHGETPHATTD--YSSTSNLSEVETTIHVEFYIISRSQESKCVEYINTGL

L26|P[4] 100 DFWTAVIAVEPRVSQTNRQYILFGENKQFNIENNS-DKWKFFEMFKGSSQSNFS-NRRTLTSNNRLVGMLKYG-GRVWTFHGETPRATTD--SSNTADLNNISIVIHSEFYIIPRSQESKCNEYINNGL

Wa|P[8] 100 DFWTAVVAIEPHVNPVDRQYTIFGESKQFNVSNDS-NKWKFLEMFRSSSQNEFY-NRRTLTSDTRFVGILKYG-GRVWTFHGETPRATTD--SSSTANLNNISITIHSEFYIIPRSQESKCNEYINNGL

HAL1166|P[14] 100 DRWFACVLVEPNVQNTQREYVLDGQTVQLQVSNNSSTLWKFILFIKLEKNGAYS-QYSTLSTSNKLCAWMKRE-GRVYWYAGTTPNASES--YYLTINNDNSNVSCDAEFYLIPRSQTELCTQYINNGL

K8|P[9] 100 DRWFACVLVEPNVQNTQRQYVLDGQNVQLHVSNDSSTSWKFILFIKLTPYGTYT-QYSTLSTPHKLCAWMKRD-NRVYWYQGATPNASES--YYLTINNDNSNVSSDAEFYLIPQSQTAMCTQYINNGL

Dhaka6|P[25] 100 DRWFACIIVEPNVSLQSRDYVLDGQIVQLQVDNTSNTMWKFILFIKLTKSGNYS-QYSSLSTNHKLCAWIKRD-GRVYWFDGTVPNSSDN--YYLTINNDNSIISSDVDFYLIPRTQTDSCRRYINNGL

B233|P[11] 100 TFWMFTYLVLPNTAQTNVTVNVMNETVNISIDNSG-STYRFVDYIKTSSTQAYG-SRNYLNTAHRLQAYRRDGDGNISNYWGADTQGDLR-VGTYSNPVPNAVINLNADFYVIPDSQQETCTEYIRGGL

993-83|P[17] 100 NKKYACVMLAPNTEEGDKQYTILGRQITINLGNTDQNRYKFFDLASEN-GETYS-KIQELLTPNRLNAFMKDQ-GRLYVYHGTVPNISTG--YYTLDDIANVQTNIKCNYYIVPKSQTQQLEDFLKNGL

Ch-02V0002G3|P[30] 100 DQKYARVMLLPNTADGNKQYTILGHRVTINLGNTNSNMYKFCDLVSHN-GTTYT-KVEELVTPHGLNAFMKDQ-GKLYLYNGTVPNVQQV--YYTLANIENVQTNIKCNYYIVPKTQTGQLEDYIKNGL

Ch-06V0661|P[31] 100 NKKFACVMLPPNTPESDRQYTILGKQITIRLGNTDTVNYKFCDLLSND-GITYT-KSIELITPHRLNAFMKDA-GKLYAYSGTVPTIKKE--YHTLDNIENVQTNITCEYYIVPNSQVSTLEEYLKNGL

Tu-03V0002E10|P[35] 100 NRKYACVLMPPNTGNGDKQYTILGKQITINLGNTDQSKYKFFDLVSTD-GSTYS-KIQELTTPHRLNAFMKDQ-GRLYVYIGIVPNISTG--YYTLDDIEHVQTNIKCNYYIVSQSQTQQLEDFLKNGL

P[I]

P[II]

P[III]

P[IV]

P[V]

Figure 8

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P[4]

P[8]

P[6]

P[19]

0.0

0.5

1.0

1.5 2 ug/ml

20 ug/ml

Gal1-3GlcNAc

OD

(4

50

nm

)

Muci

n core

6

Muci

n core

4

Muci

n core

2

Muci

n core

6

Muci

n core

4

Muci

n core

2

Muci

n core

6

Muci

n core

4

Muci

n core

2

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 2 ug/ml

20 ug/ml

OD

(4

50

nm

)

P[4] P[8] P[6]

a b

P[4]

P[8]

P[6]

P[19]

0.0

0.5

1.0

1.5

LNT

OD

(4

50

nm

)

P[4]

P[8]

P[6]

P[19]

0.0

0.5

1.0

1.5

LNFP I

OD

(4

50

nm

)

P[4]

P[8]

P[6]

P[19]

0.0

0.5

1.0

1.5

LNDFH I

OD

(4

50

nm

)

c e d

Figure 9

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12

65

17

61

01

12

61

51

17

62

01

22

62

51

27

63

01

32

63

51

37

64

01

42

64

51

47

65

01

52

65

51

57

66

01

0

1000

2000

3000

4000

5000

6000

65,66

133,134

176,177

193 505,506

525

558

567

604

RFU

12

65

17

61

01

12

61

51

17

62

01

22

62

51

27

63

01

32

63

51

37

64

01

42

64

51

47

65

01

52

65

51

57

66

01

0

2000

4000

6000

8000

10000

65,66

133,134

176,177

395

558

505

567

539,546,

549,551

604

571

585

587

583

RFU

b

Glycan number

a

Figure 10

Chart

Number Glycan structure

Average

RFU

604 GlcNAcb1-6(Neu5Aca2-3Galb1-3)GalNAca-Sp14 5399

133 GlcNAcb1-6(Galb1-3)GalNAca-Sp8 3887

567 Galb1-3GlcNAcb1-6(Galb1-3)GalNAc-Sp14 3035

134 GlcNAcb1-6(Galb1-3)GalNAca-Sp14 2960

525 Gala1-3(Fuca1-2)Galb1-3GlcNAcb1-6GalNAc-Sp14 1633

177 GlcNAcb1-6(GlcNAcb1-3)GalNAca-Sp14 1587

66 Fuca1-2Galb1-3GlcNAcb1-3Galb1-4Glcb-Sp10 1024

65 Fuca1-2Galb1-3GlcNAcb1-3Galb1-4Glcb-Sp8 950

193 GlcNAcb1-6GalNAca-Sp14 847

506 GalNAca1-3(Fuca1-2)Galb1-3GlcNAcb1-6GalNAca-Sp14 807

558 GlcNAcb1-3Galb1-4GlcNAcb1-6(GlcNAcb1-3Galb1-4GlcNAcb1-2)Mana1-6(GlcNAcb1-3Galb1-

4GlcNAcb1-2Man a1-3)Manb1-4GlcNAcb1-4GlcNAc-Sp24 505

505 Fuca1-2Galb1-3GlcNAcb1-6(Fuca1-2Galb1-3GlcNAcb1-3)GalNAca-Sp14 444

176 GlcNAcb1-6(GlcNAcb1-3)GalNAca-Sp8 400

583

GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-6(GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAb1-

2)Mana1-6(GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-

6)GlcNAcb-Sp24 306

585

GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-6(GlcNAcb1-3Galb1-4GlcNAcb1-

3Galb1-4GlcNAcb1-3Galb1-4GlcNAb1-2)Mana1-6(GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-

3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp24 302

Chart

Number Glycan structure

Average

RFU

133 GlcNAcb1-6(Galb1-3)GalNAca-Sp8 7535

604 GlcNAcb1-6(Neu5Aca2-3Galb1-3)GalNAca-Sp14 6576

66 Fuca1-2Galb1-3GlcNAcb1-3Galb1-4Glcb-Sp10 6174

65 Fuca1-2Galb1-3GlcNAcb1-3Galb1-4Glcb-Sp8 6018

177 GlcNAcb1-6(GlcNAcb1-3)GalNAca-Sp14 4912

567 Galb1-3GlcNAcb1-6(Galb1-3)GalNAc-Sp14 4700

134 GlcNAcb1-6(Galb1-3)GalNAca-Sp14 3786

176 GlcNAcb1-6(GlcNAcb1-3)GalNAca-Sp8 3015

558 GlcNAcb1-3Galb1-4GlcNAcb1-6(GlcNAcb1-3Galb1-4GlcNAcb1-2)Mana1-6(GlcNAcb1-3Galb1-

4GlcNAcb1-2Man a1-3)Manb1-4GlcNAcb1-4GlcNAc-Sp24 2784

585

GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-6(GlcNAcb1-3Galb1-4GlcNAcb1-

3Galb1-4GlcNAcb1-3Galb1-4GlcNAb1-2)Mana1-6(GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-

3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp24 2531

583

GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-6(GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAb1-

2)Mana1-6(GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4(Fuca1-

6)GlcNAcb-Sp24 2372

587

GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-6(GlcNAcb1-

3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAb1-2)Mana1-6(GlcNAcb1-

3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-3)Manb1-

4GlcNAcb1-4(Fuca1-6)GlcNAcb-Sp24 1635

505 Fuca1-2Galb1-3GlcNAcb1-6(Fuca1-2Galb1-3GlcNAcb1-3)GalNAca-Sp14 1626

549 GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-6(GlcNAcb1-3Galb1-

4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-3)Manb1-4GlcNAcb1-4GlcNAcb-Sp25 1185

564

GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-

6(GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-2Mana1-

3)Manb1-4GlcNAcb1-4GlcNAcb-Sp25 1082

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