Supplemental Information Array-Based Functional ...

Chemistry and Biology Chemistry & Biology, Volume 19

Supplemental Information Array-Based Functional Screening of Heparin Glycans Tania M. Puvirajesinghe, Yassir A. Ahmed, Andrew K. Powell, David G. Fernig, Scott E. Guimond, and Jeremy E. Turnbull Inventory of Supplemental Information Figure S1 (relating to Figure 1) shows the depletion of endogenous HS sulfation from culture cells and assessment of cell adhesion on aminosilane surfaces compared to standard glass slides. Figure S2 (relating to Figure 2) shows the optimisation of the immobilization of heparin saccharides for measurement of activation of ERK1/2. Figure S3 (relating to Figure 3) shows activation of ERK1/2 phosphorylation is specific for interactions between immobilized heparin saccharides, FGF2 and FGFR1. Supplemental Experimental Procedures Supplemental References

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Chemistry and Biology Figure S1 (relating to Figure 1) shows the depletion of endogenous HS sulfation from culture cells and assessment of cell adhesion on aminosilane surfaces compared to standard glass slides. (A) Chlorate treatment efficiently depletes sulfation of HS on Swiss 3T3 cells grown on aminosilane coated glass slide. (B) Chlorate-treated Swiss 3T3 cells have the same expression of β-actin and vinculin when grown on standard and aminosilane glass slides. Swiss 3T3 cells were chlorate treated, allowed to adhere on aminosilane coated glass slides and serum starved for 24 hours. Cells were fixed, permeabilized, stained with the anti-HS 10E4 antibody and DAPI nuclear stain or either β-actin (panel B, a) or the cell adhesion marker vinculin (panel B, b) (Grinnell, 1978; Pennington et al., 2007; Volberg et al., 1995) by immuno-staining with specific antibodies as described in Methods and immunofluorescence measured with a fluorescence microscope. All photographs were taken with 488 ms exposure time and 75 % exposure/time ratio for the green excitation filter and 31 ms exposure time and 75 % exposure time ratio for blue excitation filter. Images were taken using a 20 x objective (A). The inhibition of sulfation is overcome with an exogenous source of sulfate (i.e. SFM plus sodium chlorate and sodium sulfate) (Rapraeger et al., 1994), and results in normal 10E4 antibody staining (panel d) using 10 mM sodium sulfate and 30 mM NaCl. SFM contained 70 mM NaCl. Scale bar (100 microns) is labelled in red.

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Chemistry and Biology Figure S2 (relating to Figure 2) shows the optimisation of the immobilization of heparin saccharides for measurement of activation of ERK1/2. Heparin 12mer saccharides known to activate FGF signaling (Delehedde, 2002) were solubilised and spotted in different solutions: water, betaine (in water), betaine in formamide and formamide (alone). These solutions, with and without dp12, were spotted in a volume of 0.5 μl onto an aminosilane surface with 3 replicates for each condition. The slide was microwaved to reduce the reaction time (Powell et al., 2009). Chlorate treated cells were overlaid onto the slide following serum starvation (Pelech et al., 1986); FGF2 (10 ng/ml) was added to the cells for 90 min. Cells were then fixed and stained for both total and phosphorylated ERK1/2, using specific secondary antibodies conjugated to different fluorophores, corresponding to the two wavelengths detected by the microarray scanner (535/ 557-592 nm and 635/ 650-690 nm). Fluoresence intensity of spot areas was measured using Image J software. Measurements were made for phosphorylated ERK1/2 (panel A) and total ERK1/2 (panel B). Mean ± standard deviation (SD) are calculated for the of 3 spots from a single slide. Data is representative of 2 experiments.

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Figure S3 (relating to Figure 3) A: MEK1 inhibitor decreases activation of ERK1/2 phosphorylation. Swiss 3T3 cells were treated with sodium chlorate and serum starved as described below. Conditions were indicated as shown above the gels. Some cells were either treated with inhibitor (PD098059) or not and some were stimulated with FGF2 (10 ng/ml) and 12mer heparin (10 μg/ml) for 90 minutes. Protein extracts of the cells were separated using SDS-PAGE as described below. Blots were probed first for activated (phosphorylated) ERK1/2, and subsequently the PVDF membrane was stripped and re-probed with an antibody to detect total ERK1/2. ERK1/2 is activated with 12mer heparin and FGF2, but not in the presence of the inhibitor. Total ERK 1/2 protein levels remain the same in the conditions with and without inhibitor. B: DNA synthesis is inhibited by increasing concentrations of MEK1 inhibitor. DNA synthesis was measured in cells pre-treated with MEK1 inhibitor, PD098059. A DNA synthesis assay was carried out as described below. Swiss 3T3 cells were cultured for 48 hours in sulphate-free medium containing 30 mM sodium chlorate and were treated with different concentrations of PD098059, as indicated. Cells were incubated with FGF-2 (10 ng/ml) in the presence or absence of 12mer (dp12) heparin saccharides (10 μg/ml). Control cells received no stimulation and were also tested for the effect of inhibitor. Results are the mean ± S.D. of three experiments. Note that inhibitor alone was at 50 μM PD098059 and did not stimulate cells. Negative control represents no stimulation and no inhibitor added. C: Activation of ERK1/2 phosphorylation is specific for interactions between immobilized heparin saccharides, FGF2 and FGFR1. Chlorate treated cells were seeded onto an aminosilane slide already spotted with or without heparin 12mer saccharides, left to adhere overnight, and serum starved for 24 hours. Cells were treated with a MEK1 inhibitor for 15 minutes before being stimulated with FGF2 for 90 minutes. Cells were then fixed and stained for total and phosphorylated ERK1/2 as described above. The ratios of pERK/total ERK were calculated (arbitrary units). Conditions without inhibitor could not be carried out on the same slide since MEK inhibitor was added in the culture media; the level of comparable control conditions from a separate slide are shown (dashed line, positive control with 12mer and FGF2 (mean, 0.172; SD, 0.01); dotted line, negative control with FGF2 alone (mean, 0.040; SD, 0.005)). Data is representative of 2 experiments. D: Structure and preparation of heparin saccharides. Panel a: shows the predominant disaccharide repeating structure [UA2S-GlcNS(6S)] of heparin saccharides dp2 (2mer, n=1), dp12 (12mer, n=6) and dp18 (18mer, n=9) derived from porcine mucosal heparin. For clarity, the uronic acid moiety is shown as α-L-idoA but can also occasionally be its C-5 epimer, β-D-GlcA. Panel b: compositional analysis of the porcine mucosal heparin starting material (by heparinise digestion) confirmed the predominant disaccharide UA2S-GlcNS(6S) (~72%). Panel c: Oligosaccharides were fractionated using gel filtration chromatography and separated according to oligosaccharide length, with 2mer, 12mer and 18mer fractions highlighted in the chromatogram. Purified oligosaccharides were analyzed by PAGE and quantified by weighing or by measuring their absorption at 232 nm.

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E: Heparin oligosaccharides cause differential stimulation of DNA synthesis in standard cell culture assays. DNA synthesis assays were carried out as described below. Swiss 3T3 cells were cultured for 48 hours in sulphate-free medium containing 30 mM sodium chlorate and were then incubated with FGF-2 (10 ng/ml) in the presence or absence of heparin oligosaccharides; 2mer (dp2), 12mer (dp12) or 18mer (dp18), all at (10 μg/ml) as indicated. Control Swiss cells received no stimulation. Results are the mean ± S.D. of three experiments. 18mer and 12mer caused elevated DNA synthesis, whereas the 2mer did not lead to an increase of DNA synthesis above that of the negative control. F: Immunoflourescence microscopy analysis of ERK1/2 phosphorylation in HSdeficient cells responding to FGF2 on slide arrays in the presence of immobilized heparin oligosaccharides. Fluorescence images of Swiss 3T3 cells grown on aminosilane surfaces modified by surface attachment of 2mer, 12mer or 18mer heparin saccharides, or control (no saccharide). HS sulfate-deficient (chlorate-treated) cells were seeded onto an aminosilane slide spotted with the saccharides in betaine and treated as described in Fig. S2 to measure FGF2 signaling responses. The array was immunostained for phosphorylated ERK1/2. Images were taken under a 40 x objective with a 150 ms exposure time and 252 % exposure/time ratio. Scale bar corresponds to 50 μm and is shown in red. G: Dose response of FGF signaling to soluble and immobilized heparin saccharides in HS-deficient cells. a. DNA synthesis assays were carried out as described in the supplementary experimental section. Swiss 3T3 cells were chlorate treated, serum starved and incubated with FGF-2 (10 ng/ml) and different concentrations of heparin 12mer for 90 minutes as indicated. Control Swiss cells were grown without sodium chlorate. Results are the mean ± S.D. of three experiments. b. Swiss 3T3 cells were treated with sodium chlorate and serum starved as previously described, cells were overlaid onto slide array surfaces containing 2 different concentrations of spotted 12mer and 18mer oligosaccharides, or controls (no sugar), and exposed to FGF2 for response mesaurements (as described in Fig S2). Average fluorescence intensities (arbitrary units) measured by the slide scanner were: control, 8.6; 0.01mM 12mer, 11.4; 1mM 12mer, 18.9; 0.01mM 18mer, 13.6; 1mM 18mer, 23.2.

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Figure S3A

Figure S3B

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SUPPLEMENTAL EXPERIMENTAL PROCEDURES Fluorescence Microscopy: A Zeiss Axiosop 2 plus microscope was fitted with a manually-controlled stage and colour wheels, diachronic beamsplitter and an ebq 100 Netz power mercury lamp. Axiovision Rel 4.6 3.0 software was used for image analysis (Carl Zeiss). Filter sets used were 49- with excitation 265 and emission 445/50 (for DAPI). Zeiss Filter sets 20, green with excitation 546/12 and emission 575/640. Filter set 10 with excitation 450-490 and emission 515-565. Filter sets 38 with excitation 470/40 and emission 525/550. The following lenses were used: Zeiss Plan-NEOFLUAR 20 x /0.50 (440341 [01]). 40 x Plan-NEOFLUAR 40 x./ 1.3 oil ∞/0.17 and 63X Zeiss PlanAPOCHROMAT 63 x /1.4 OIL ∞/0.17. Immunocytochemistry: Immunocytochemistry was used to assess the removal of sulfated HS polysaccharides using 10E4 antibody in an indirect immunostaining protocol. Swiss 3T3 cells were grown in treatment medium A for two days with a medium change after 24 hours using ATCC cell growth recommendations. Cells were split using trypsin-EDTA (TE) (Sigma, 0.25 % (w/v)) and solubilized in either treatment medium A, or treatment medium B (all pre-treatment medium was supplemented with 10 % (v/v) dialysed FCS). Cells at 20-30 % confluence (5000 to 10,000 cells/cm2) were transferred to aminosilane coated glass slides (Corning), and left to adhere overnight. Cells were serum starved in the appropriately supplemented pretreatment medium with 0.1 % (w/v) dialyzed BSA and fixed using 4 % (w/v) paraformaldehyde, after which chamber wells were washed three times with PBS. Blocking was carried out with 10 % (v/v) normal goat serum (NGS), 0.1 % (v/v) Triton X-100 in PBS (PBSTX) for 1 h at room temperature. Anti-heparan sulfate 10E4 was used as the primary antibody (Seikagaku) using 1:100 dilution in 1 % (v/v) NGS in PBSTX and incubated for 1 hr. Wells were washed with PBS. Fluorescently labeled secondary antibody was added (1:500 dilution) and incubated in the dark for 1 h (Alexa 488-conjugated IgM goat anti-mouse antibody, Abcam). Wells were washed with PBS and slides were mounted and stored in the dark at 4 oC until ready for imaging. Antibody controls included omission of primary antibody, 10E4 and heparitinase I enzyme treatment of the fixed cells prior to 10E4 antibody incubation. Heparitinase I was diluted 5 mU enzyme/ ml buffer (100mM sodium acetate, pH 7, 0.1 mM calcium acetate, 50 μg/ml BSA) and following blocking with 10 % (w/v) NGS in PBSTX, added to fixed cells and incubated at 37 oC for 1 h. Assessment of cellular cytoplasmic microfilaments of actin and focal adhesion vinculin molecules: β-actin primary polyclonal rat antibody (Cell Signaling) was used at a dilution of 1:50 on cellular samples to assess polymerised actin fibres. Anti-rat IgM 488 was used as the secondary antibody for immunofluorescence. Immunoblots used horse radish peroxidase-linked anti-rat as a secondary antibody. The presence of vinculin was assessed using a direct immunostaining protocol, using manufacturer’s directions (Sigma Aldrich) using a 1/100 in PBSTX and 5 % (w/v) BSA followed by goat anti-mouse IgG1 secondary antibody. The presence of nuclear double stranded DNA was detected using 0.5 μg/ml DAPI stain (Invitrogen).The primary antibody phosphorylated ERK1/2 (Thr202/Tyr204) was used with Alexa Fluor ® 555 secondary antibody (Invitrogen).

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Chemistry and Biology DAPI nuclear stain: The presence of nuclear double stranded DNA was detected using DAPI stain (Invitrogen). Cells were incubated at a concentration of 0.5 μg/ml of DAPI and PBS was added into the final washing step and left for 5 to 10 minutes, in the dark Measurement of DNA synthesis using a tritiated thymidine assay: Swiss 3T3 fibroblasts were passaged with 0.25 % TE into treatment medium A supplemented with 10 % dialysed calf serum. Cells were incubated for 48 hours at 37 oC with medium replacement after 24 hours. Cells were split using 0.25 % TE and transferred into 24-well plates at a density of 30,000 cells per well in 500 μl of medium. Cells were counted using a haemocytometer. Cells were allowed to adhere overnight at 37 oC and washed with treatment medium A supplemented with 0.1 % dialysed BSA and incubated 24 hours at 37 oC in the same medium. Appropriate growth factors and saccharides were added and incubated at 37 oC at 5 % CO2 for 18 hours. Radioactive thymidine was added at 200 μl of 40 μCi/ml [3H] thymidine and incubated for 6 hours at 37 oC. The liquid from the wells was aspirated after incubation and cells were washed twice with PBS. The cells were fixed by adding 500 μl of 5 % ice-cold trichloroacetic acid (TCA) and immediately removed and left for 30 minutes at 4 oC. The solution was aspirated and washed with 500 μl of 5 % ice-cold TCA, which was removed immediately. Each well was then washed twice with 500 ul of ice-cold 70 % ethanol. The ethanol was aspirated and plates were left to dry on top of a hot oven for 15 minutes. When wells were completely dry, 500 μl of 0.2 N NaOH solution was added to each well and incubated for 30 minutes at 37 oC to solubilise cells. The solubilised cells (300 μl) were added to 1 ml of Ultima Gold™ Optiphase scintillation cocktail (Perkin-Elmer) and the radioactivity was quantified using a 10 minute count cycle on a TRICARB 2100 TR Packard (Canberra Company) liquid scintillation analyzer.

Heparin oligosaccharide preparation: Defined heparin-derived oligosaccharides (2mer, 12mer and 18mer) were prepared as described (Powell et al., 2010). Briefly, porcine mucosal heparin (PMH)) was partially digested using heparinase I (IBEX Technologies, Inc.). Partial digests were fractionated by gel filtration chromatography on a Superdex 30 preparation grade column (16 mm I.D. x 200 cm length) run at 0.5 ml/min in 0.5 M ammonium hydrogen carbonate. Pooled peak fractions were desalted and lyophilized. Purified oligosaccharides were analyzed by PAGE and quantified by weighing or by measuring their absorption at 232 nm. Compositional analysis was used to confirm the presence of the major disaccharide repeat of the trisulfated disaccharide UA(2S)-GlcNS(6S) (Powell et al, 2010). Effect of oligosaccharide concentration on cell responses: Studies adding saccharides in solution to cell cultures demonstrated that incubation of different concentrations of heparin 12mers result in dose-dependent effects on DNA synthesis (panel a), consistent with previous data in the literature (Delehedde et al., 2002). In glycobioarray format, experiments immobilizing different concentrations of heparin 12 and 18mers demonstrated a reduction in the relative fluorescence of phosphorylated ERK at 0.01mM spotting concentration compared to 1mM. In addition, differences in fluorescence intensity were detected at both low and high concentrations of the 12mer and 18mer saccharides, providing evidence that this technology is sufficiently sensitive to detect dose-response differences between different saccharide structures. This data is consistent with previous studies showing that saccharides

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SUPPLEMENTAL REFERENCES Delehedde, M., Lyon, M., Gallagher, J.T., Rudland, P.S., and Fernig, D.G. (2002). Fibroblast growth factor-2 binds to small heparin-derived oligosaccharides and stimulates a sustained phosphorylation of p42/44 mitogen-activated protein kinase and proliferation of rat mammary fibroblasts. Biochem. J. 366, 235-244. Grinnell, F. (1978). Cellular adhesiveness and extracellular substrata. Int Rev Cytol 53, 65-144. Pelech, S.L., Olwin, B.B., and Krebs, E.G. (1986). Fibroblast growth factor treatment of Swiss 3T3 cells activates a subunit S6 kinase that phosphorylates a synthetic peptide substrate. Proc Natl Acad Sci U S A 83, 5968-5972. Pennington, S.R., Foster, B.J., Hawley, S.R., Jenkins, R.E., Zolle, O., White, M.R.H., McNamee, C.J., Sheterline, P., and Simpson, A.W.M. (2007). Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts. J Biol Chem 282, 32112-32120. Powell, A.K., Zhi, Z.L., and Turnbull, J.E. (2009). Saccharide microarrays for highthroughput interrogation of glycan-protein binding interactions. Methods Mol Biol 534, 313-329. Powell, A.K., Ahmed, Y.A., Zhi, Z. L., Turnbull, J. E. (2010). Generating heparan sulfate saccharide libraries for glycomics applications. Nature Protocols 5, 829-841. Rapraeger, A.C., Guimond, S., Krufka, A., and Olwin, B.B. (1994). Regulation by heparan sulfate in fibroblast growth factor signaling. Methods Enzymol 245, 219-240. Volberg, T., Geiger, B., Kam, Z., Pankov, R., Simcha, I., Sabanay, H., Coll, J.L., Adamson, E., and Ben-Ze'ev, A. (1995). Focal adhesion formation by F9 embryonal carcinoma cells after vinculin gene disruption. J Cell Sci 108 ( Pt 6), 2253-2260. Zhi, Z.L., Laurent, N., Powell, A.K., Karamanska, R., Fais, M., Voglmeir, J., Wright, A., Blackburn, J.M., Crocker, P.R., Russell, D.A., et al. (2008). A versatile gold surface approach for fabrication and interrogation of glycoarrays. Chembiochem 9, 1568-1575.

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