LINGO-1, A TRANSMEMBRANE SIGNALING PROTEIN, INHIBITS OLIGODENDROCYTE DIFFERENTIATION AND MYELINATION THROUGH INTERCELLULAR SELF- INTERACTIONS.

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1 Supplemental Data: LINGO-1, A TRANSMEMBRANE SIGNALING PROTEIN, INHIBITS OLIGODENDROCYTE DIFFERENTIATION AND MYELINATION THROUGH INTERCELLULAR SELF- INTERACTIONS. Scott Jepson, Bryan Vought, Christian H. Gross, Lu Gan, Douglas Austen, J. Daniel Frantz, Jacque Zwahlen, Derek Lowe #, William Markland, and Raul Krauss *. Supplemental Experimental Procedures: CHO cell membranes. CHO cells were grown to confluence, washed with ice-cold phosphate buffered saline (PBS), and then washed with 5 mm HEPES ph 7.5, 5 mm EDTA and 0.1 mm PMSF. 2.3 x 10 8 parent and 2.2 x 10 8 LINGO-1 CHO cells were lysed using a polytron for two consecutive 30 second pulses. Nuclei and unlysed cells were removed by centrifuging at 1000 x g for 5 min at 4 C. The supernatants were collected and centrifuged at 53,000 x g for 30 min at 4 C. Pellets were resuspended in ml buffer (50 mm HEPES ph7.5, 130 mm NaCl, 0.5 mm MgCl 2, 5 mm KCl). Total protein concentration was determined with a Bio-Rad protein assay using BSA as a standard. AP-LINGO-1 stable cell lines. A cdna sequence encoding a 6xHis tag and residues I23-G511 of human placental alkaline phosphatase (NCBI reference sequence NP_ ) was inserted between residues A33 and T34 of human LINGO-1 ectodomain (M1-E532, NCBI reference sequence NP_ ) cdna sequence to generate an M1-A33_Gly_His6_Gly_(AP_I23-G511)_(LINGO-1_T34-E532) construct. The entire sequence was cloned into a pfuse vector (Invivogen, San Diego, CA) using EcoRI and NheI to remove the higg1 Fc tag, and the construct was sequence verified. HEK293 EBNA cells (ATCC, Manassas, VA) were cultured in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 25 µg/ml of G418 (Invitrogen, Carlsbad, CA). The cells were then transfected with pfuse- AP-LINGO-1 plasmid using FuGENE6 reagent according to the product manual (Roche Applied Science, Indianapolis, IN). Single cell stable clones from transfected cells, and media containing secreted soluble AP-LINGO-1, were obtained as described for soluble LINGO-1-Fc protein. Soluble AP-LINGO-1 binding: Parent and LINGO-1 CHO cells were plated into 96-well PDL-coated plates at 2.5x10 4 cells/well and left to attach overnight. Medium was removed from the cells and 50 µl of conditioned medium containing AP-LINGO-1 added to the wells and incubated for 90 min at 37 o C. Plates were washed 5x in ice-cold binding buffer, fixed for 10min in 4% paraformaldehyde, then washed an additional 3 times. After the final wash the substrate pnpp (Vector labs; SK-5900), containing Levamisole (Vector Labs; SP-5000) as directed by the manufacturer, was added and the plates read at 405 nm on a plate reader (Molecular Devices, Spectramax) as the color developed. 1

2 SUPPLEMENTAL FIGURE 1: LINGO-1 expression decreases during mouse OPC maturation in vitro. Mouse OPCs were grown for 3 days in PDGF/FGF as described in methods. PDGF/FGF was removed and replaced for 10 ng/ml CNTF and 15 nm T3 for the times indicated in the figure to promote oligodendrocyte differentiation. Representative western blots show an increase in MBP protein expression, that correlated with a decrease in LINGO-1 expression as cells differentiated in the presence of CNTF/T3. 2

3 SUPPLEMENTAL FIGURE 2: Overexpression of LINGO-1 prevents OPC maturation. Western blots of mouse and rat OPCs expressing FL-LINGO-1 and ECTO-LINGO-1, presented in Figure 2, were quantified by densitometry. MBP values were normalized to GAPDH and are expressed as percent of Control. A) Rat OPCs. B) Mouse OPCs. For detailed description see legend to Figure 2. C) Rat OPC cultures infected with FL-LINGO or control lentiviral constructs as indicated. Differentiation was assessed by quantitative immunofluorescence microscopy for O4 and MBP after PDGF/FGF removal and subsequent culture for an additional 4 days in the presence of 1 ng/ml CNTF and 15 nm T3. Expression of FL-LINGO decreased MBP expression and did not affect the expression of O4. Values were obtained as in figure 2; **p<0.01 t-test. 3

4 SUPPLEMENTAL FIGURE 3: Soluble LINGO-1 ectodomain prevents mouse oligodendrocyte maturation. A) Mouse OPC cultures were grown for 4 days in medium containing PDGF/FGF and subsequently treated for 6 days with recombinant soluble LINGO-1 ectodomain, after removal of PDGF/FGF and addition of 1 ng/ml CNTF and 1.5 nm T3. Representative experiment, showing a dose responsive decrease in the amount of MBP protein expression assessed by quantitative immunofluorescence microscopy. Values were obtained as in figure 2; *p<0.05 One way ANOVA with Bonferroni post-hoc. B) Manual cell counts of MBP + oligodendrocytes from rat cultures treated with CNTF/T3 (described in Figure 3D) confirm that changes in MPB + cell number correlates with changes in MBP fluorescence intensity. *p<0.05, t-test. 4

5 SUPPLEMENTAL FIGURE 4: Expression of LINGO-1 lentiviral constructs. A) Western blots from rat and mouse OPCs cultures infected with FL-LINGO-1 lentivirus, described in Figure 2, probed with anti- LINGO-1 antibody show increased expression of LINGO-1. B) Astrocyte cultures infected with Control, FL-LINGO-1 and ECTO-LINGO-1 lentivirus described in Figure 5 show successful expression of the reporter egfp present in the construct, compared to uninfected cultures. For details, see Figure 5 and Experimental Procedures. 5

6 SUPPLEMENTAL FIGURE 5: LINGO-1 self-interaction is independent of the tag. A) Membranes from stable lines of LINGO-1 CHO and control CHO cells analyzed for LINGO-1 expression by western blotting show absence of LINGO-1 in the control cell line. Values in the figure indicate amount of membrane protein loaded on the gels. B) LINGO-1 CHO and control CHO cells were incubated with either 50 µg/ml slingo-1 or 50 µg/ml IgG-AP alone and processed as described in Experimental Procedures. Only slingo-1 showed increased binding to LINGO-1 CHO cells. C) Conditioned medium containing a soluble AP-LINGO-1 construct was incubated at the dilutions indicated with LINGO-1 CHO and control CHO cells. Soluble AP-LINGO-1 ectodomain displayed specific dose responsive binding to CHO cells stably transfected with FL-LINGO-1, compared to control CHO cells. AP-LINGO binding was observed in 5 independent experiments. Values represent the mean + SEM of three replicate wells. *** p<0.001 two-way ANOVA with Bonferroni post hoc. 6