The Y-P30 induced axonal growth and branching in young primary cortical neurons requires SDC-3.

<p>(a–c) 24 hours after plating primary cortical neurons were supplemented with 20 µM Y-P30 and finally fixed at 12 h, 24 h and 36 h after treatment. The Y-P30 induced enhancement of axonal growth and partially branching in primary cortical neurons, fixed 12 h and 24 h after supplementation, is depicted as a reconstruction (a). The quantitative analysis is shown in (b) and (c). For SDC-3 knockdown primary cortical cultures were infected immediately after plating with Lentivirus-based shSDC3-FUGW constructs or empty FUGW as a control. After 24 h the infected cultures were supplemented with Y-P30 as described previously and finally fixed 36 h later. An example for a primary cortical neuron expressing the shSDC3-FUGW construct is shown in (d). Scale bar is 20 µm. Quantitative analysis of axon length 36 h after supplementation (e) revealed that the Y-P30 induced axonal growth is significantly abolished in neurons expressing the knockdown construct, but not in neurons expressing FUGW alone. For axonal branching no differences could be detected under these conditions (f). Black boxes indicate treatment with Y-P30. N: 30–40 in each group; * p<0.05.</p

Quantitative immunoblotting of GluN2B levels in cortical primary cultures following Y-P30 application.

<p>(a+b) Representative blots probed with a GluN2B or actin antibody are depicted. (a) Protein levels were altered at DIV6 after 6 and 12 h incubation of cortical primary cultures with Y-P30. (b) No significant changes were seen at DIV18. n.s.: not significant. N: 5–7 in each group. ** p<0.01, * p<0.05.</p

Knock down of SDC-2 and -3 abolishes the Y-P30 induced CASK translocation in primary cortical neurons.

<p>(a+b) shRNA knockdown was tested in co-transfected, SDC-2-myc or SDC-3-myc over-expressing Cos7 cells. The knockdown effects of the two most efficient shRNA sequences for each SDC (a) are shown in representative Western-blots (b). Note the efficient knockdown of the respective SDC. Syndecans are glycoproteins and the different bands detected by the myc antibody probably represent differentially glycosylated protein isoforms. (c) In order to establish a Lentivirus-based knockdown system, the appropriate shRNA cassettes of the pRS vectors, containing the U6-polymerase III-promoter, were subcloned into the PacI restriction site of the FUGW vector. (d, e) In mature primary cortical cultures (DIV18), expressing the knockdown construct against SDC-2, the Y-P30 induced CASK translocation is completely abolished. Similar effects after Y-P30 supplementation were obtained in young primary cortical cultures (DIV8) expressing the SDC-3 knockdown construct, indicating different cellular effects of both SDC (n = 4–6).</p

Y-P30 induces the proteolytic cleavage of the extracellular domain of SDC-2.

<p>(a) COS7 cells, expressing SDC-2intmyc-GFP, were supplemented with 20 µM Y-P30 or a mock control. For the inhibition of matrix metalloproteinases either 50 nM GM6001 or 20 nM of MMP9/13 inhibitor I were used. After 3 h the culture media were collected, the containing proteins precipitated with ethanol and subsequently analysed with quantitative immunoblotting. In order to analyse the successful over expression and membrane-incorporation of the tagged fusion proteins, the respective cells were harvested, fractionated and the membrane proteins evaluated on western blots. A representative quantitative immunoblot analysis of the SDC-2 ecto-domain from the culture medium is shown in (a). Note that supplementation with Y-P30 increases the amount of the myc-tagged SDC-2 ecto-domain, whereas GM6001 as well as MMP9/13I abolished the Y-P30 dependent cleavage. The total expression and incorporation of the SDC-2 construct was analysed in membranes after subcellular fractionation using western-blot analysis (b). The relative amounts of the detected SDC-2 ecto-domains from the culture media are depicted in (c) as % to the control. N: 3–6; *** p<0.0001. (d) Illustrates an immunofluorescence image of the SDC-2intmyc-GFP expression in COS7 cells, showing a clear merge of the GFP-fluorescence from the C-terminus of the fusion protein and the myc-tag, incorporated into the ecto-domain of SDC-2. Scale bar is 20 µm.</p

Y-P30 regulates the nuclear distribution of CASK in primary cortical neurons.

<p>(a) CASK is a multidomain scaffolding protein. While its GUK-domain interacts with the transcription factor Tbr1, the PDZ domain is responsible for binding to the C-terminus of SDC. (b) Essential for this binding are the final four amino acids that are conserved within all SDC. The binding of Y-P30 is mediated via heparan sulfate side chains (HSSC). (c) Supplementation of primary cortical neurons with Y-P30 at DIV 18 leads to the accumulation of CASK in the nucleus as visualized in confocal images following CASK antibody staining. Scale bar is 20 µm. (d) For quantitative analysis fractions of nuclei (P1) and remaining cellular components (P2) were prepared and analysed with quantitative immunoblotting. In order to normalize the relative amounts of CASK, the P1 fraction was analysed in relation to the NeuN signal, the P2 fraction to the Actin signal. (e) In mature primary cortical cultures (DIV 18) supplementation with Y-P30 leads to a significant increase of the CASK concentration in P1 fractions after 3 and 6 h whereas the CASK concentration in P2 declines albeit not significantly at the same time points. (g) Interestingly, a decrease of the CASK concentration in P1 fractions of young neurons (DIV 8) 3 and 6 h after supplementation with Y-P30 was observed. (h, i) The effect of Y-P30 on the nuclear localization of CASK was abolished in mature neurons (DIV18) treated with Heparitinase I and Chondroitinase A, B, C, cleaving the HSSCs. Note that all fractionation assays were done in the presence of 7.5 µM anisomycin. For Western-blots 20 µg of protein were loaded per lane. Relative concentrations of CASK were analysed by measuring the optical density of the respective signal and normalized as described above. Black boxes indicate treatment with Y-P30. N = 4–11 in each group. ** p<0.001, * p<0.05.</p

Expression anaylsis of Caldendrin, L- and S-CaBP1.

<p>(A) Immunoblot analysis reveals that bacterially expressed untagged Caldendrin migrates at 33 kDa like the smaller Caldendrin isoform in cortex and hippocampus of rat brain. Bacterially produced myristoylated L- and S-CaBP1 migrate at 25 kDa and 18 kDa respectively. Immunoreactivity is detected by anti-Caldendrin/CaBP1 rabbit antibody, directed against the common C-terminus of all three isoforms. 20 µg of brain samples are compared to ≈ 10 ng of purified proteins. The western blot shows Caldendin expression in different regions of rat brain (B) and in different rat organs (C). Caldendrin is detected by anti-Caldendrin/CaBP1 rabbit antibody (rb). Equal loading in all lanes was ensured by measuring the total protein concentraion (20 µg/lane) and verified with an anti-actin mouse antibody (ms). Note that consistant with previous reports the actin band is amost absent in heart tissue due to differnential expression of this marker.</p

N – Number of binding sites, ΔH – Change in enthalpy, ΔS – Change in entropy, K_a – Association constant, K_d – Dissociation constant, K_D – Global dissociation constant.

<p>N – Number of binding sites, ΔH – Change in enthalpy, ΔS – Change in entropy, K_a – Association constant, K_d – Dissociation constant, K_D – Global dissociation constant.</p

Far and Near UV- CD spectroscopy.

<p>(A) Far UV- CD spectra of the apo, Mg²⁺-bound and Mg²⁺+Ca²⁺ bound common C-terminus of Caldendrin/CaBP1 shows no conformational change in secondary structure. (B) Near UV- CD spectra of the apo, Mg²⁺-bound and Mg²⁺+Ca²⁺ bound common C-terminus of Caldendrin/CaBP1 exhibit significant change in tertiary structure. (C) Far UV- CD spectra of the apo-, Mg²⁺-bound and Mg²⁺+Ca²⁺-bound full length Caldendrin. Mg²⁺ binding to apo-Caldendrin causes a mild change in the secondary structure of the protein. Ca²⁺ binding to Mg²⁺- bound Caldendrin has no detectable effect on the secondary structure of the protein. (D) Near UV- CD spectra of apo-, Mg²⁺-bound and Ca²⁺-bound Mg²⁺+Ca²⁺-bound full length Caldendrin. Mg²⁺ binding to apo-CDD causes a significant change in the tertiary structure of the protein, whereas Ca²⁺ binding to Mg²⁺- bound Caldendrin has no effect. (E) Ca²⁺-titration of the apo-protein had a clear effect on the structure. Representative spectra for each condition are shown.</p

Surface plasmon resonance analysis.

<p>(A) Untagged full length Caldendrin was immobilized on a CM5 surface and the full-length Caldendrin was also injected as analyte. (B) The N-terminus of Caldendrin was immobilized on a CM5 surface and also injected as analyte in running buffer. (C) The common C-terminus (CDD-Ct) was immobilized on the sensor chip and the N-terminus injected as analyte. (D) The common C-terminus (CDD-Ct) was immobilized on the sensor chip and also injected as the analyte. (A–D) The running buffer always contained 50 mM Tris-Cl and 100 mM KCl with 1 mM Mg²⁺/1 mM EGTA (red) or 1 mM Mg²⁺/500 µM Ca²⁺ (green). Amount of protein in the running buffer was 5, 10, 20, 40, and 80 µg (increasing protein levels correspond to increasing amplitudes). RU: response units.</p

Equilibrium chemical unfolding.

<p>Equilibrium chemical unfolding of full length Caldendrin with guanidinium hydrochloride (GdmCl) in the absence of any ligand (Apo-CDDfl), in presence of 5 mM MgCl₂ and 1 mM EGTA (Mg²⁺-CDDfl); and 5 mM MgCl₂ and 1 mM CaCl₂ (Mg²⁺Ca²⁺- CDDfl). The upper lane shows (A) the ellipticity values of plotted against [GdmCl] (black squares) and the fitting obtained (black curve) using a two-state unfolding model. The middle lane (B) shows the quality of model fitting in terms of residuals and the bottom lane (C) shows the normalized fits for fraction folded (FN; red curve) and fraction unfolded (FU; black curve) along with the standard free energy change of unfolding (ΔGU) obtained.</p