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VP8* genes (residues 1–231) of Rotateq P[8] (GenBank: GU565044), P[8]BEL(GenBank: JN849151) were synthesized in Genewiz (Suzhou, China) and cloned into the pGEX4T-1 vector. Human P[6] (5311142) (GenBank: KT162986), human P[4] (11151099) (GenBank: KT162987), human P[8]lab (11221075) (GenBank: KT162984) were described previously (Ma et al. 2015). Recombinant VP8* proteins with N-terminal GST-tag were expressed in E. coli BL21 (DE3) cells (Tiangen) as previously described (Sun et al. 2016a). GST-fusion VP8*s were purified with Glutathione beads (GE healthcare) using established procedures (Sun et al. 2016a). VP8* core (residues 64-223) fragment of Rotateq P[8] was cloned into the pET30a vector and expressed in E.coli with C-terminal his-tag. VP8* core protein was purified with HiTrap Fast Flow (GE healthcare). Further purification was performed with gel filtration using superdex 200 column with 20 mmol/L Tris–HCl (pH 8.0), 50 mmol/L NaCl. The concentration of the purified protein was determined by the BCA Kit (BD Biosciences) with 2 mg/mL BSA as a standard.
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Oligosaccharide binding assay was carried out as previously reported (Sun et al. 2016a). GST fusion VP8* proteins were coated on the 96 well plate (Costar) at 100 μg/well at 4 ℃ overnight. After blocking with 5% non-fat milk, PAA-biotin labeled oligosaccharides (including mucin core 2, mucin core 4, mucin core 6, LNT, H type 1, H type 2, A, B, Lewis a, Lewis b) were added at two concentrations 0.2 and 1 μg/well respectively. The plate was left at 4 ℃ overnight. Horseradish peroxidase-conjugated streptavidin (Abcam) was then added at 0.1 μg/well after washing the plates 5 times with phosphate-buffered saline (PBS)-0.05% Tween 20. The reactions were conducted using a 3, 30, 5, 50- tetramethylbenzidine kit (BD Biosciences) and the absorbance of 450 nm was measured. The experiment was repeated twice and samples were performed with duplicates each time.
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The Rotateq P[8] VP8* core protein in buffer (20 mmol/L Tris-HCl, 50 mmol/L NaCl, pH 8.0) was concentrated to approximately 20 mg/mL and crystallized using the sittingdrop vapor diffusion method at 18 ℃ with 1 μL of protein mixed with 1 μL of reservoir solution. Crystals were grown under the condition of 0.1 mol/L sodium acetate trihydrate pH 4.5, 2.0 mol/L ammonium sulfate. In order to obtain the protein-glycan complex, the glycans core 2 or LNFP1 were co-crystallized with the VP8* protein. The threoninelinked core 2 trisaccharide was prepared from Fmoc-protected peracetylated core 2-Thr (Sussex Research Laboratories, Ottawa, Canada) after deprotection (Johannes et al. 2015). Core 2-Thr was purified by high-performance liquid chromatography (HPLC) and analyzed by electrospray ionization mass spectrometry (ESI-MS) before use. The pentasaccharide Lacto-N-fucopentaose 1 (LNFP1) was purchased from Dextra Laboratories (UK). VP8* and core 2/LNFP1 were co-crystallized using a protein/ligand ratio of 1:50/1:200, respectively, under the same conditions as for the native crystal described above.
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Crystals were dipped briefly in cryoprotectant solution containing 20% (v/v) glycerol and then flash-frozen in liquid nitrogen. Diffraction data were collected at the Shanghai Synchrotron Radiation Facility BL17U. Data were processed using HKL2000 software (Otwinowski and Minor 1997). The structures were solved by molecular replacement using Phaser software (Read 2001) with the structure of Rotateq P[8] VP8* (Protein Data Bank [PDB] ID: 5JDB) as the search model (Sun et al. 2016a). The initial model was refined using REFMAC5, COOT, and PHENIX software (Emsley and Cowtan 2004; Adams et al. 2010). The structural analysis was carried out with the PyMOL software package (https://pymol.org/2/). The statistical data for P[8] VP8*-core 2 and VP8*-LNFP1 are presented in Table 1. Protein structure accession numbers: the structures of P[8] VP8*-core 2 and VP8*-LNFP1 have been deposited in PDB with the access codes of 6K2N and 6K2O, respectively.
Parametera RotaTeq P[8] VP8*-Core 2 RotaTeq P[8] VP8*-LNFP1 Data collection Space group P21 P6322 Cell dimensions a, b, c (Å) 66.289 80.454 114.55 80.454 73.789 116.593 α, β, γ (°) 90, 95.959, 90 90, 90, 120 Wavelength (Å) 0.97774 0.97852 Resolution (Å) 50.00-1.80 50.00-2.30 (1.86-1.8) (2.38-2.30) Rmerge (%)b 8.4 (80.4) 15.9 (86.0) I/σI 21.597 (1.500) 22.524 (4.889) Completeness (%) 98.4 (90.3) 99.57 (96.58) Redundancy 5.4 (5.4) 25.8 (27.0) Refinement Resolution (Å) 43.24-1.80 44.71-2.30 No. reflections 86599 10481 Rwork/Rfree 0.2024/0.2288 0.2035/0.2331 No. atoms Protein 7860 1336 Ligand/ion 80 52 Water 781 80 B-factors Protein 23.5 31.1 Ligand 59.8 74.7 Water 34.4 39.6 R.m.s. deviations Bond lengths (Å) 0.005 0.003 Bond angles (°) 0.69 0.54 Ramachandran plot Favored (%) 97.47 98.14 Allowed (%) 2.53 1.86 Disallowed (%) 0.00 0.00 aValues in parentheses are given for the highest resolution shell.
b Rmerge = , where I is the intensity of unique reflection hkl and$\Sigma \mathrm{hkl}|\mathrm{I}- <\mathrm{I}>| / \Sigma \mathrm{hklI} $ is the average over symmetry-related observations of unique reflection hkl, hkl is the reflection indices.$ <\mathrm{I}>$ Table 1. Crystallographic X-ray diffraction and refinement statistics.
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Human red blood cells (RBCs) of A, B, and O types and animal blood cells (Gelaidisi, Beijing) were washed twice with PBS before use and diluted to 1% RBCs. The GSTVP8* fusion proteins were twofold-serially diluted with 50 μL per well on 96-well V-bottom plates (Costar, Corning) and the original protein concentration was at 2 mg/mL. 50 μL of RBCs was added each well subsequently. The plate was incubated at 25 ℃ and agglutination was determined 1 h later. Wells without aggregates were considered as agglutination.
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We performed fluorescent focus assays on African green monkey kidney epithelial cells (MA104 cells) in the presence and absence of glycans, with infections in the absence of glycans considered to be 100% infectivity. The MA104 cells were seeded on the 96-well plate (Costar). Laboratory-adapted Wa (G1P[8]) and SA11 (G3P[2]) rotavirus strains were investigated. Dilutions of virus yielding ~200 focus forming units per well were preincubated for 30 min with media containing human milk, LNFP1, mucin core 2, lactose, or media without glycan. Human milk was used at 1:10. The glycans were tested at a final concentration of 1 mg/mL and 5 mg/mL (for LNFP1). The MA104 cells on the 96-well plate were washed twice with ice-cold serum-free 1640 medium. After cooling the cells and virus to 4 ℃, the virus-milk or virus-glycan media were added and incubated on ice for 1 h. The inoculum was removed and cells were washed twice with ice-cold serum-free 1640 medium. The plates with serumfree 1640 medium were then put back in the incubator at 37 ℃ for 20 h. The cells were then fixed with ice cold methanol. Infected cells were detected with an anti-P[8] VP8* polyclonal rabbit serum (raised in-house, 1:400 dilution), followed by a fluorescein isothiocyanate (FITC)- conjugated goat anti-rabbit secondary antibody (Abcam, 1:1000 dilution).
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Data analysis was performed using SPSS software. Analysis of variance (ANOVA) with Dunnett's correction for multiple comparisons was used. Values of P < 0.05 were considered to indicate statistical significance.