Xuezheng Song, Baoyun Xia, Sean R. Stowell, Yi Lasanajak, David F. Smith, and Richard D. Cummings

Transcription

1 Chemistry & Biology 16 Supplemental Data Novel Fluorescent Glycan Microarray Strategy Reveals Ligands for Galectins Xuezheng Song, Baoyun Xia, Sean R. Stowell, Yi Lasanajak, David F. Smith, and Richard D. Cummings SUPPLEMENTAL EXPERIMENTAL PROCEDURES Synthesis of AEAB Methyl anthranilate (MA, 20g) was added into 200mL ethylenediamine (EDA). The mixture was heated at 100 o C for 12h and evaporated to dryness. The residue was dissolved in 50mL water and cooled at 4 o C for 3 days. The precipitate was filtered and recrystallized in acetonitrile to give AEAB (yield 48%). AEAB (5g) was re-dissolved in 50mL 2N HCl and evaporated to dryness. The residue was recrystallized with acetic acid to give AEAB-HCl salt as a white crystal (yield 66%). Conjugation of free reducing oligosaccharides with DAP, AEAB, and MA 0.35M AEAB (or DAP, MA) and 1M NaCNBH 3 in DMSO/AcOH (7:3 v/v) were freshly prepared. To 1-10nmol glycan, 5µL of each above solution is added. The mixture was heated at 65 o C for 2h and dried in a Centra-vap evaporator. The dried sample was dissolved in 10µL water and applied on a glass cartridge fitted with 2 pieces of 3MM filter paper (1cm diameter). The cartridge was washed with 5 x 1mL of acetonitrile and the conjugate was eluted by 3 x 0.5mL water. Release of glycans from glycoproteins by PNGase F digestion Glycoprotein (100mg) was dissolved in 5mL denaturing buffer and boiled for 10 min. One ml of 10% NP40 and 1 ml of 10X G7 buffer were added. PNGase F (30µL, 500,000 units/ml) and 3mL water were added. The glycoprotein was digested at 37 o C for 24h. The solution was boiled for 5min and passed through a 2g C18 Sep-Pak cartridge. The flowthrough was collected and applied on a 500mg Carbograph cartridge, which was subsequently washed with water (6 column volumes). Free glycans were eluted from the Carbograph cartridge with 3 column volumes of 50% acetonitrile in 0.1% trifluoroacetic acid (TFA) and dried by Centravap prior to fluorescent derivatization. HPLC A Zorbax NH 2 column (250mm x 4.6mm) was used for NP HPLC separation. The mobile phases were acetonitrile, water, and 250 mm ammonium acetate (ph 4.5). The concentration of water increased from 16% to 40% and the concentration of ammonium acetate buffer increased from 4% to 50% over 60min. The second dimension HPLC separation was accomplished with a PGC column (150mm x 4.6mm) from Thermo. The mobile phase was acetonitrile and water with 0.1% TFA. Two time programs were used: (a) After 10min isocratic elution with 8% acetonitrile, the concentration of acetonitrile increased from 8% to 50% in 20min. (b) After 10min isocratic elution with 8% acetonitrile, the concentration of acetonitrile increased from 8% to 50% in 63min. For RP HPLC with C18 column, a Vydec C18 column was used. The mobile phase was acetonitrile and water with 0.1% TFA. The concentration of acetonitrile increased from 1% to 5% in 60min. Generation of recombinant human Gal-1 and -3 Each recombinant galectin was purified by affinity chromatography on lactosyl-sepharose and bound lectin was eluted with 100mM lactose in PBS, 14mM βme. Prior to derivatization, βme was removed from galectin samples using a PD10 gel filtration column (GE

2 Healthcare), followed by the addition of lactose (100mM final concentration) to maintain the stability of each galectin and reduce the likelihood of adduct formation at or near the carbohydrate recognition domain. Galectins were biotinylated by incubating 3-5mg/mL of each galectin with 2mM EZ-link TM Sulfo-NHS-LC-Biotin (Sulfosuccinimidyl-6-(biotinamido) Hexanoate) (Pierce) for 2h at 4ºC. Unconjugated EZ-link TM Sulfo-NHS-LC-Biotin and free lactose were separated from galectin using a PD-10 gel filtration column. Galectins were rechromatographed over lactosyl-sepharose to remove any inactive material following labeling. Bound galectin was eluted with 100mM lactose, applied to a PD-10 gel filtration column and stored at 4 C in 14mM βme in PBS until further use. Printing, binding assay, and scanning All samples were printed in phosphate buffer (300mM sodium phosphates, ph8.5). The average spot volume was within 10% variation (intra-tip) of 1/3 nanoliter. After printing, slides were placed in a high moisture chamber at 50 o C and incubated for 1h. The slides were washed and blocked with 50mM ethanolamine in 0.1M Tris buffer, ph9.0, for 1h, subsequently dried by centrifugation, and stored desiccated at -20 o C. Before assay, the slides were rehydrated for 5min in TSM buffer (20mM Tris-HCL, 150mM NaCl, 0.2mM CaCl 2 and 0.2mM MgCl 2. Biotinylated lectins were used in the binding assay and the bound lectins were detected by a secondary incubation with Cy5-SA. For multi-panel experiment on a single slide, the array layout was designed using Piezorray software according to the dimension of a standard 16-chamber adaptor. The adaptor was applied on the slide to separate a single slide to 16 chambers sealed from each other during the assay. The slides were scanned with a Perkin Elmer ProScanArray microarray scanner equipped with 4 lasers covering an excitation range from 488 to 637nm. The scanned images were analyzed with the ScanArray Express software. For Cy5 fluorescence, 649nm (Ex) and 670nm (Em) were used. All images obtained from the scanner are in grayscale and colored for easy discrimination.

3 Table S1. The Cartoon Structures of a 52 GAEAB Microarray Table S2. The List of 210 GAEABs Printed on the Glycan Microarray, Their Structure or Composition, and Their Sources Glycan # Name or ID number 1 Asialo, galactosylated, biantennary (NA2) 2 Oligomannose-8D1D3 (MAN 8-D1D3) 3 Asialo, agalacto, biantennary (NGA2) 4 Disialylated, galactosylated, fucosylated Structure/ composition

4 5 Oligomannose-7D3 (MAN 7-D3) 6 Monosialylated, galactosylated, fucosylated 7 Monosialylated, galactosylated, fucosylated, biantennary (A1F) 8 Disialylated, galactosylated, fucosylated, biantennary (A2F) 9 Monosialylated, galactosylated, biantennary (A1) 10 Oligomannose-6 (MAN 6) 11 Asialo, galactosylated, tetraantennary (NA4) 12 Oligomannose-7D1 (MAN 7-D1) 13 Disialylated, galactosylated, biantennary (A2) 14 Tetra, galacto, fuco-tetra-antennary 15 Hybrid with bisecting GlcNAc 16 Tetrasialyl LacNAc1, tetra-antennary 17 Sialyl Lewisx (Slex)

5 18 Sulpho Lewisx Lewisx tumor associated antigen LS-Tetrasaccharide a (LST a) LS-Tetrasaccharide c (LST c) Lactodifucotetraose (LDFT) 3'-Fucosyllactose (3'-FL) Sialyl Lewisa (Slea) Lacto-N-neohexaose (LNnH) 26 p-lacto-n-hexaose (plnh) '-Sialyllactose (6'-FL) 3'-Sialyllactose (3'-SL) 29 Sulpho Lewisa Lacto-N-fucopentaose I (LNFP I) Lacto-N-fucopentaose II (LNFP II) Lacto-N-fucopentaose III (LNFP III) Lacto-N-fucopentaose V (LNFP V) Lacto-N-tetraose (LNT) Lacto-Ndifucohexaose II (LNDFH II) Lacto-Ndifucohexaose I (LNDFH I) 37 Asialo, galactosylated, fucosylated 38 6'-siaLacNAc

6 39 Galb1-3GlaNAc 40 Lactose 41 Fuca1-2Gal 42 GalNAca1-3(Fuca1-2)Gal Blood Group A- tetrasaccharide Blood Group A- pentasaccharide 45 Fuca1-2Galb1-4GlcNAc 46 Gala1-3(Fuca1-2)Gal 47 Asialo, galactosylated triantennary (NA3) 48 Asialo, agalacto, triantennary (NGA3) 49 Sialylated biantennary (A2) 50 Monosialylated biantennary (A1) 51 Asialo, galactosylated, fucosylated biantennary (NA2F) 52 Monogalactosylated, biantennary (G-NGA2) Monogalactosylated, fucosylated, biantennary (G- NGA2F) Asialo, agalacto, bisected, biantennary fucosylated (NGA2FB) Oligomannose-3, 1,6- fucosyl Hybrid with bisecting GlcNAc 57 Oligomannose-2(a) 58 Oligomannose-3(a)

7 59 Oligomannose-3(b) 60 Oligomannose-4(b) 61 Oligomannose-5 62 Oligomannose-9-Glc (Man-9Glc) Oligomannose-3, xylosyl (Man3-Xyl) Oligomannose-3, xylosyl, 1,3-fucosyl (Man3-Xyl,Fuc) Monosialylated Lacto- N-fucopentose (SLNFPVI) Monosialylated Lacto- N-neohexose (MSLNnH) Monosialo, monofucosyllacto-n-hexose I (MSMFLNH I) Monosialo, monofucosyllacto-nneohexose (MSMFLNnH) Monosialo, difucosyllacto-n-neohexose (MSDFLNnH) Lacto-N-difucohexose IV (LNDFH IV) Lacto-Nneodifucohexose I (LNnD I) Monofucosyllacto- N-hexose I (MFLNH I) 73 Difucosyl-para-lacto- N-hexose II (DFpLNH II) 74 Difucosyl-para-lacto- N-neohexose (DFpLNnH)

8 75 Trifucosyl-para-lacto- N-hexose (TFpLNH) Trifucosyllacto-Nhexose (TFLNH) Monofucosyl(1-3)-iso- Lacto- N-octaose (MFiLNO(1-3)) 78 iso-lacto-n-octose (ilno) B-Pentasaccharide (B-Penta) A-Leb- Heptasaccharide (A-Hepta) 81 A-Leb-Octasaccharide (A-Octa) Trifucosyllacto-Ntetraose IV (TFLNT IV) Trifucosyllacto-Nneotetraose I (TFLNnH I) Monofucosyllacto-Nneohexose I (MFLNnH I) 85 AEAB 86 Bovine IgG-NP02-PGC01 (Hexose)3(GlcNAc)4 87 Bovine IgG-NP02-PGC02 (Hexose)5(GlcNAc)2 88 Bovine IgG-NP03-PGC01 (Hexose)3(GlcNAc)4(Fuc)1 89 Bovine IgG-NP04-PGC01 (Hexose)6(GlcNAc)2 90 Bovine IgG-NP04-PGC02 (Hexose)4(GlcNAc)4 91 Bovine IgG-NP04-PGC03 (Hexose)6(GlcNAc)2 92 Bovine IgG-NP05-PGC01 (Hexose)4(GlcNAc)4(Fuc)1 93 Bovine IgG-NP06-PGC01 (Hexose)5(GlcNAc)4 94 Bovine IgG-NP07-PGC01 (Hexose)5(GlcNAc)4(Fuc)1 95 Bovine IgG-NP08-PGC01 (Hexose)8(GlcNAc)2 96 Bovine IgG-NP09-PGC01 (Hexose)8(GlcNAc)2 97 Bovine IgG-NP10-PGC01 (Hexose)9(GlcNAc)2 98 Bovine IgG-NP12-PGC01 (Hexose)9(Glc)1(GlcNAc)2 99 Bovine IgG-NP14-PGC02 (Hexose)4(GlcNAc)4(Fuc)1(Neu5Gc)1 100 Bovine IgG-NP15-PGC02 (Hexose)5(GlcNAc)4(Neu5Gc)1 101 Bovine IgG-NP17-PGC01 (Hexose)6(GlcNAc)4(Fuc)1(Neu5Gc)1 102 Bovine IgG-NP18-PGC02 (Hexose)5(GlcNAc)4(Fuc)1(Neu5Gc)2 103 Bovine IgG-NP19-PGC01 (Hexose)5(GlcNAc)4(Neu5Gc)2 104 Bovine IgG-NP19-PGC02 (Hexose)5(GlcNAc)4(Fuc)1(Neu5Gc)2 105 Bovine IgG-NP19-PGC03 (Hexose)6(GlcNAc)5(Fuc)1(Neu5Gc)2 106 Bovine IgG-NP19-PGC04 (Hexose)6(GlcNAc)5(Fuc)1(Neu5Gc)2 107 Bovine IgG-NP20-PGC02 (Hexose)6(GlcNAc)5(Fuc)1(Neu5Gc)2

9 108 Ovalbumin-NP02-PGC01 (Hexose)1(GlcNAc)2 109 Ovalbumin-NP03-PGC01 (Hexose)2(GlcNAc)2 110 Ovalbumin-NP03-PGC02 (Hexose)2(GlcNAc)2 111 Ovalbumin-NP04-PGC01 (Hexose)3(GlcNAc)1 112 Ovalbumin-NP05-PGC01 (Hexose)2(GlcNAc)3 113 Ovalbumin-NP06-PGC01 (Hexose)3(GlcNAc)2 114 Ovalbumin-NP06-PGC02 (Hexose)2(GlcNAc)3 115 Ovalbumin-NP08-PGC01 (Hexose)3(GlcNAc)2 116 Ovalbumin-NP09-PGC01 (Hexose)3(GlcNAc)3 117 Ovalbumin-NP09-PGC02 (Hexose)4(GlcNAc)2 118 Ovalbumin-NP09-PGC03 (Hexose)3(GlcNAc)3 119 Ovalbumin-NP10-PGC01 (Hexose)3(GlcNAc)3 120 Ovalbumin-NP11-PGC01 (Hexose)3(GlcNAc)4 121 Ovalbumin-NP11-PGC02 (Hexose)4(GlcNAc)3 122 Ovalbumin-NP11-PGC04 (Hexose)5(GlcNAc)1 123 Ovalbumin-NP11-PGC05 (Hexose)3(GlcNAc)4 124 Ovalbumin-NP11-PGC06 (Hexose)4(GlcNAc)3 125 Ovalbumin-NP12-PGC01 (Hexose)3(GlcNAc)5 126 Ovalbumin-NP12-PGC02 (Hexose)3(GlcNAc)5 127 Ovalbumin-NP12-PGC03 (Hexose)3(GlcNAc)5 128 Ovalbumin-NP12-PGC04 (Hexose)5(GlcNAc)2 129 Ovalbumin-NP13-PGC01 (Hexose)5(GlcNAc)2 130 Ovalbumin-NP13-PGC02 (Hexose)4(GlcNAc)4 131 Ovalbumin-NP13-PGC03 (Hexose)3(GlcNAc)5 132 Ovalbumin-NP14-PGC01 (Hexose)3(GlcNAc)5 133 Ovalbumin-NP14-PGC02 (Hexose)3(GlcNAc)5 134 Ovalbumin-NP15-PGC01 (Hexose)6(GlcNAc)1 135 Ovalbumin-NP15-PGC02 (Hexose)4(GlcNAc)5 136 Ovalbumin-NP15-PGC03 (Hexose)5(GlcNAc)3 137 Ovalbumin-NP15-PGC04 (Hexose)4(GlcNAc)4 138 Ovalbumin-NP15-PGC05 (Hexose)3(GlcNAc)6 139 Ovalbumin-NP15-PGC06 (Hexose)5(GlcNAc)3 140 Ovalbumin-NP15-PGC07 (Hexose)4(GlcNAc)4 141 Ovalbumin-NP16-PGC01 (Hexose)3(GlcNAc)6 142 Ovalbumin-NP16-PGC02 (Hexose)3(GlcNAc)6 143 Ovalbumin-NP16-PGC03 (Hexose)6(GlcNAc)2 144 Ovalbumin-NP17-PGC01 (Hexose)5(GlcNAc)4 145 Ovalbumin-NP17-PGC02 (Hexose)4(GlcNAc)5 146 Ovalbumin-NP18-PGC01 (Hexose)3(GlcNAc)7 147 Ovalbumin-NP18-PGC02 (Hexose)5(GlcNAc)5 148 Ovalbumin-NP19-PGC01 (Hexose)3(GlcNAc)8 149 Ovalbumin-NP19-PGC02 (Hexose)5(GlcNAc)5 150 Ovalbumin-NP19-PGC03 (Hexose)4(GlcNAc)6 151 Ovalbumin-NP20-PGC01 (Hexose)4(GlcNAc)8 152 Ovalbumin-NP20-PGC02 (Hexose)4(GlcNAc)8 153 Fetuin-NP03-PGC01 [Hexose]5[GlcNAc]2 154 Fetuin-NP04-PGC01 [Hexose]5[GlcNAc]4[Neu5Ac]1 155 Fetuin-NP04-PGC02 [Hexose]4[GlcNAc]4[Fuc]3[Neu5Ac]1 156 Fetuin-NP04-PGC03 [Hexose]5[GlcNAc]5[Fuc]1[Neu5Ac]2 157 Fetuin-NP04-PGC04 [Hexose]7[GlcNAc]4[Fuc]2Neu5Ac]2 158 Fetuin-NP04-PGC05 [Hexose]7[GlcNAc]4[Fuc]2Neu5Ac]2 159 Fetuin-NP05-PGC01 [Hexose]5[GlcNAc]5[Fuc]0[Neu5Ac]1 160 Fetuin-NP06-PGC01 [Hexose][GlcNAc][Fuc][Neu5Ac] 161 Fetuin-NP07-PGC01 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]2 162 Fetuin-NP07-PGC02 [Hexose]7[GlcNAc]4[Fuc]2Neu5Ac]2 163 Fetuin-NP07-PGC03 [Hexose]7[GlcNAc]4[Fuc]2Neu5Ac]2 164 Fetuin-NP08-PGC01 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]2 165 Fetuin-NP08-PGC02 [Hexose]7[GlcNAc]4[Fuc]2Neu5Ac]2 166 Fetuin-NP09-PGC01 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]2 167 Fetuin-NP10-PGC01 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]3 168 Fetuin-NP11-PGC01 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]3 169 Fetuin-NP11-PGC02 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac4 170 Fetuin-NP11-PGC03 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]3 171 Fetuin-NP12-PGC01 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]3

10 172 Fetuin-NP12-PGC02 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac]3 173 Fetuin-NP13-PGC01 [Hexose]6[GlcNAc]5[Fuc]0[Neu5Ac4 174 Asialofetuin-RP02-PGC01 (Hexose)7(GlcNAc)4 175 Asialofetuin-RP02-PGC02 (Hexose)7(GlcNAc)4 176 Asialofetuin-RP03-PGC01 (Hexose)5(GlcNAc)4 177 Asialofetuin-RP04-PGC01 (Hexose)6(GlcNAc)5 178 Asialofetuin-RP05-PGC02 (Hexose)5(GlcNAc)4 179 Asialofetuin-RP06-PGC01 (Hexose)6(GlcNAc)5 180 Asialofetuin-RP06-PGC02 (Hexose)6(GlcNAc)5 181 Asialofetuin-RP07-PGC01 (Hexose)6(GlcNAc)5 182 Asialofetuin-RP07-PGC02 (Hexose)5(GlcNAc)4 183 Asialofetuin-RP08-PGC01 (Hexose)6(GlcNAc)5 184 Asialofetuin-RP08-PGC02 (Hexose)6(GlcNAc)5 AEAB RP02-PGC01 (Hexose)8-9(GlcNAc)2 187 RP03-PGC01 (Hexose)9(GlcNAc)2 188 RP03-PGC02 (Hexose)7(GlcNAc)2 189 RP04-PGC01 (Hexose)7(GlcNAc)2 190 RP05-PGC01 (Hexose)7(GlcNAc)2 191 RP05-PGC02 (Hexose)6(GlcNAc)2 192 RP06-PGC01 (Hexose)6-7(GlcNAc)2 193 RP07-PGC01 (Hexose)5(GlcNAc)2 194 RP08-PGC01 (Hexose)5(GlcNAc)2 195 RP10-PGC01 (Hexose)5(GlcNAc)5(Fuc)0(Neu5Ac)2 196 RP11-PGC01 (Hexose)5(GlcNAc)4(Fuc)1 197 RP11-PGC02 (Hexose)5(GlcNAc)3(Fuc)2(Neu5Ac)1 198 RP11-PGC03 (Hexose)4(GlcNAc6 199 RP12-PGC01 (Hexose)4(GlcNAc6 200 RP13-PGC01 (Hexose)6(GlcNAc)5(Fuc)1(Neu5Ac)1 201 RP13-PGC02 (Hexose)6-7(GlcNAc)4(Fuc)0(Neu5Ac)1 202 RP13-PGC03 (Hexose)5(GlcNAc)4(Fuc)1(Neu5Ac)1(Neu5Gc)1 203 RP13-PGC04 (Hexose)6(GlcNAc)3(Fuc)2(Neu5Ac)1 204 RP14-PGC02 (Hexose)5(GlcNAc)4(Fuc)1(Neu5Ac)2 205 RP15-PGC01 (Hexose)5-6(GlcNAc)4(Fuc)1 206 RP15-PGC02 (Hexose)5-6(GlcNAc)4(Fuc)1(Neu5Ac)1 207 RP15-PGC03 (Hexose)5-6(GlcNAc)4(Fuc)1(Neu5Ac)2 208 RP15-PGC04 (Hexose)5-6(GlcNAc)4(Fuc)1(Neu5Ac)2 209 RP16-PGC01 (Hexose)5-6(GlcNAc)4(Fuc)1 210 (Hexose)5(GlcNAc)4(Fuc)2(Neu5Ac)1

11 RP16-PGC02 Table S3. The Binding Data from Figure 6 in Tabular Form, Representing the Average RFU and Standard Deviation for the Varying Concentrations of Galectins Bound to Each Glycan

12

13 Figure S1. The Printing Efficiency of LNFPIII Conjugates LNFPIII-AEAB1, LNFPIII-AEAB2, and LNFPIII-DAP (the conjugate of LNFPIII and 2, 6- diaminopyridine) were printed side by side on a) NHS-activated slide and b) epoxy slide. LNFPIII, free DAP, and free AEAB were also printed as controls. Samples are printed from 1mM to 0.1µM by 2-fold serial dilution. The immobilized glycan was incubated with biotinylated AAL (1µg/ml) and further detected by Cy5-SA (5µg/ml); c) LNFPIII-DAP, 2- AA, 2-AB and AEAB conjugates are printed on different commercial slides and detected by biotinylated AAL (0.5 µg/ml) followed by Cy5-SA.

14 Figure S2. A Schematic Presentation of the Construction of a 52 GAEAB Microarray (a) Work flow of the glycan array construction. (b) Array format: each array includes 14 replicated subarrays; in each subarray, 52 GAEABs and controls were printed in 6 replicates each. (c) Theoretical fluorescent image after the array was assayed with carbohydrate binding proteins.

15 Figure S3. An Example of MALDI-TOF MS Analysis of Two GAEAB Fractions collected from HPLC (a) Neutral fraction IgG-AEAB-NP05-PGC01; MS and MS/MS data at positive mode are shown. (b) Acidic fraction IgG-AEAB-NP14-PGC02; MS and MS/MS data at positive mode and MS at negative mode are shown.