Semaphorin 3A binds to the perineuronal nets via chondroitin sulfate type E motifs in rodent brains - PubMed (original) (raw)

. 2013 Sep 20;288(38):27384-27395.

doi: 10.1074/jbc.M111.310029. Epub 2013 Aug 12.

Chin Lik Tan 2, Joao Nuno Alves 2, Erich M E Ehlert 3, Gregory M Miller 4, Linda C Hsieh-Wilson 4, Kazuyuki Sugahara 5, Arie Oosterhof 6, Toin H van Kuppevelt 6, Joost Verhaagen 7, James W Fawcett 2, Jessica C F Kwok 8

Affiliations

Semaphorin 3A binds to the perineuronal nets via chondroitin sulfate type E motifs in rodent brains

Gunnar Dick et al. J Biol Chem. 2013.

Abstract

Chondroitin sulfate (CS) and the CS-rich extracellular matrix structures called perineuronal nets (PNNs) restrict plasticity and regeneration in the CNS. Plasticity is enhanced by chondroitinase ABC treatment that removes CS from its core protein in the chondroitin sulfate proteoglycans or by preventing the formation of PNNs, suggesting that chondroitin sulfate proteoglycans in the PNNs control plasticity. Recently, we have shown that semaphorin3A (Sema3A), a repulsive axon guidance molecule, localizes to the PNNs and is removed by chondroitinase ABC treatment (Vo, T., Carulli, D., Ehlert, E. M., Kwok, J. C., Dick, G., Mecollari, V., Moloney, E. B., Neufeld, G., de Winter, F., Fawcett, J. W., and Verhaagen, J. (2013) Mol. Cell. Neurosci. 56C, 186-200). Sema3A is therefore a candidate for a PNN effector in controlling plasticity. Here, we characterize the interaction of Sema3A with CS of the PNNs. Recombinant Sema3A interacts with CS type E (CS-E), and this interaction is involved in the binding of Sema3A to rat brain-derived PNN glycosaminoglycans, as demonstrated by the use of CS-E blocking antibody GD3G7. In addition, we investigate the release of endogenous Sema3A from rat brain by biochemical and enzymatic extractions. Our results confirm the interaction of Sema3A with CS-E containing glycosaminoglycans in the dense extracellular matrix of rat brain. We also demonstrate that the combination of Sema3A and PNN GAGs is a potent inhibitor of axon growth, and this inhibition is reduced by the CS-E blocking antibody. In conclusion, Sema3A binding to CS-E in the PNNs may be a mechanism whereby PNNs restrict growth and plasticity and may represent a possible point of intervention to facilitate neuronal plasticity.

Keywords: Chondroitin Sulfate; Glycosaminoglycan; Neuronal Plasticity; Perineuronal nets; Proteoglycan; Regeneration; Semaphorin; Semaphorin3A.

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Figures

FIGURE 1.

FIGURE 1.

A, schematic diagram of the differently tagged Sema3A fusion proteins used in this study. For chicken AP-collapsin 1, the AP was fused to the N-terminal part of chicken collapsin 1 lacking a signal sequence (SS) (29). For rat GFP-semaphorin 3A, the GFP was inserted after the N-terminal signal sequence in the rat Sema3A sequence (24). Sema3A-Fc is procured from the R&D Systems and contains a His tag at the N terminus and a C-terminal Ig sequence. B, chemical structures of the various GAGs used in this study.

FIGURE 2.

FIGURE 2.

Sema3A demonstrates preferential binding to CS-E and CS-B. Recombinant Sema3A-AP was used in direct (A) and competitive (B and C) ELISA toward various CS GAGs. A, although Sema3A-AP interacts very specifically with heparin and CS-E, it does not bind to other CSs. The binding affinity was determined by AP activity measured at absorbance at 405 nm, n = 4, mean ± S.D. B, Sema3A-AP interaction with heparin in competition with various CS GAGs, HS, and heparin. The binding of Sema3A to heparin was inhibited after incubation with CS-B, CS-E, and heparin. The binding was determined by AP activity measured at 405 nm. Competitive inhibition presented as percentage of binding in relation to reading in Sema3A-AP only sample (equals 100% binding), n = 4, mean ± S.D. C, measurements of dose-dependent inhibition of various concentrations of CS-B, CS-C, and CS-E in blocking Sema3A-AP interaction toward heparin in a competitive ELISA. Both CS-B and CS-E inhibit the binding of Sema3A to heparin, whereas CS-C does not. Determination of Sema3A-AP binding and presentation as in B, n = 3, mean ± S.D. D, control experiment measuring binding to heparin using lysate from Sema3A-GFP-transfected cells, lysate from non-transfected (Cntrl lysate), and no lysate. Note that Sema3A-GFP cell lysate interacted very strongly to the immobilized heparin, whereas the control lysate gave similar signal level as the negative control “_no lysate._” This suggests that the binding of Sema3A-GFP to heparin is due to the expressed Sema3A in the lysate but not other protein contaminants. Binding affinity was measured and presented as absorbance at 405 nm, n = 3, mean ± S.D. E, Sema3A-Fc binds selectively to CS-E-enriched polysaccharides on GAG microarrays. Microarrays containing different concentrations (0.5, 1, and 5 μ

m

) of the indicated GAG polysaccharides were incubated with Sema3A-Fc, followed by a Cy3-conjugated anti-mouse IgG secondary antibody, and analyzed using a Gene-Pix 5000A scanner. Data represent the mean ± S.E. from three experiments (n = 10 per condition). CS-A is 4-sulfated, CS-B is 2,4-disulfated, CS-C is 6-sulfated, CS-D is 2,6-disulfated, and CS-E is 4,6-disulfated.

FIGURE 3.

FIGURE 3.

Sema3A binding to GAGs extracted from adult rat brain demonstrates high binding affinity to PNN GAGs. Recombinant Sema3A-AP binding to brain GAGs in direct (A) and competitive ELISA (B). A, Sema3A-AP binds with high affinity to PNN-GAGs but not to the soluble GAGs or detergent GAGs in a direct ELISA. Binding affinity was determined by AP activity measured and presented as absorbance at 405 nm, n = 3, mean ± S.D. B, PNN-GAGs (with and without ChABC or Hep I and III treatment) was preincubated with the Sema3A-AP before adding onto heparin-coated wells in an indirect ELISA. The binding of Sema3A alone is defined as 100% (1st bar). Incubation with PNN GAGs decreases the binding to ∼20% (2nd bar). Pretreatment of the PNN GAGs with ChABC (3rd bar) or Hep I and III (4th bar) only partially removes the inhibition. However, co-treatment of both ChABC and heparitinases restores the Sema3A-AP binding to the immobilized heparin, Binding affinity determined by AP activity measured as absorbance at 405 nm and presented as a percentage of binding relative to reading in Sema3A-AP only sample (equals 100%), n = 4, mean ± S.D., two-tailed paired t test: *, p < 0.05, ***, p < 0.001.

FIGURE 4.

FIGURE 4.

Sema3A binding to PNN GAGs is inhibited by blocking the CS-E structure. A, slot blot assay using anti CS-E phage display antibody GD3G7 (26) toward CS-E, CS-B, and bovine aggrecan. The result demonstrates that although the GD3G7 antibody specifically recognizes CS-E and detects a CS-E-like structure in aggrecan, it does not bind to CS-B. B and C, ELISA of Sema3A-AP interaction toward CS-E and PNN GAGs, with and without the addition of anti-CS-E antibody GD3G7. GD3G7 effectively blocks the binding of Sema3A-AP to both CS-E and PNN GAGs by ∼60%. Binding affinity was determined by AP activity measured and presented as absorbance at 405 nm, n = 3, mean ± S.D. D, a direct ELISA of Sema3A-AP interaction toward PNN GAGs as in C using various concentrations of GD3G7 and a nonspecific control phage display antibody MPB49V. It shows a concentration-dependent blocking of GD3G7 toward Sema3A-AP binding to the PNN-GAGs. n = 3, mean ± S.D.

FIGURE 5.

FIGURE 5.

Solubilization of PNNs, CRTL1, and Sema3A with different buffers from adult rodent brains. A, adult rat cortical sections were sequentially washed with buffers of increasing denaturing power and stained with biotinylated WFA PNNs. Treatment with Triton X-100 (Tx-100) and 1

m

NaCl leads to a decrease in the WFA staining in the neurites (arrowheads) but not on the soma (arrows). Scale bar equals 50 μm. B, Western blot of the buffers collected after the sequential extraction from mouse brain homogenates. Although the majority of CRTL 1 is released with 6

m

urea buffer, most of the Sema3A is released with Triton X-100 buffer, to a much lesser extent in the 1

m

NaCl or 6

m

urea buffers.

FIGURE 6.

FIGURE 6.

Release of Sema3A from brain tissue after enzymatic degradation of GAG chains. The released Sema3A was detected by Western blot of the extracted soluble fractions after enzyme treatment. A, Sema3A is released with high salt (1

m

NaCl) wash. B, whereas digestion with ChABC and chondro-6-sulfatase causes an increase in the release of Sema3A from the brain tissue, digestion of hyaluronan using hyaluronidase, chondro-4-sulfatases on CS-A, and Hep I and III does not increase the release Sema3A (C) or quantification of the signal in the Western blots in B. Relative release is presented as a ratio of Sema3A signal from the enzyme-treated brain homogenate against the nonenzyme-treated samples, n = 2–4, mean ± S.D., two-tailed paired t test: *, p = 0.054; **, p < 0.01.

FIGURE 7.

FIGURE 7.

Co-immunoprecipitation (co-IP) of brain CSPG(s) with Sema3A-GFP. A and B, full-length Sema3A-GFP recovered from cell lysate binds to heparin. Recombinant Sema3A-GFP in media and cell lysate were analyzed by Western blotting (WB) (A) and used in direct ELISA (B). A, Western blotting of collected cell media and lysate from Sema3A-GFP-transfected cells. N-terminal (65 kDa) and full-length recombinant Sema3A-GFP (95 kDa) were detected by anti-GFP or anti-Sema3A antibody. This suggests that the recombinant Sema3A-GFP is cleaved by normal cellular processing giving a fragment corresponding to the wild type 65 kDa N-terminal fragment (25). B, direct interaction with Sema3A-GFP in media and lysate toward heparin. Because of extensive cleavage of the secreted Sema3A-GFP, we applied conditioned media as well as lysate in the ELISA. Binding to heparin-coated wells was detected when using the lysate, containing mainly full-length Sema3A-GFP. Binding affinity was measured and presented as absorbance at 405 nm, n = 3, mean ± S.D. C, PNN PGs isolated from the adult rat brain was incubated with and without Sema3A-GFP lysate (1st and 2nd lanes), followed by IP against GFP using anti-GFP antibody on protein A magnetic beads. The beads were then treated with ChABC, and the supernatant was analyzed using SDS-PAGE and Western blot assay. 2-B-6 antibody was used to detect the stub on the released CSPG(s) after the enzyme treatment. Two bands slightly smaller than 460 kDa observed in the Sema3A-treated samples suggest that Sema3A-GFP is binding to one (or more) high molecular weight CSPG(s) in the brain homogenates. The size of the band suggests that the CSPGs could be aggrecan. 5% of the input was added as a control after chondroitinase treatment (3rd lane).

FIGURE 8.

FIGURE 8.

Combination of Sema3A and PNN-GAGs confers a stronger inhibition on DRG culture that is alleviated with anti-CS-E antibody. Substrate inhibition of DRG-neurite outgrowth by PNN GAGs and Sema3A. Adult rat DRG explants were cultured on substrate containing laminin, Sema3A, and/or PNN GAGs and subsequently stained with βIII tubulin for the neurites. Outgrowth measurement is presented as an average of 10 neurites per explant, 2–10 explants per condition from two separate experiments. A, measurement of neurite outgrowth of DRG explants on 25 μg/ml PNN GAGs in combination with increasing amounts of Sema3A. DRG explants cultured on PNN-GAGs showed a reduction in neurite length when compared with laminin control. Further reduction was observed when the explants were cultured with increasing concentration of Sema3A (1:50 and 1:10) suggesting Sema3A confers a stronger inhibition upon PNN GAGs on neurite inhibition. Increasing amounts of Sema3A demonstrated a dose-dependent inhibition. Data are presented as mean ± S.E. B, classification of neurite outgrowth ability of the DRG explants according to the number of neurites per explant. Explants cultured on PNN GAGs showed a decrease in the percentage of explants bearing >20 neurites. This inhibition was even stronger when the explants were cultured in the presence of both PNN GAGs and Sema3A. The majority of explants project less than three neurites. The data are presented as mean ± S.E. for each class. C, images of the DRG explants cultured on “laminin,” “laminin plus PNN GAGs,” “laminin, PNN GAGs, Sema3A, and control antibody (MPB49V),” and “laminin, PNN GAGs, Sema3A, and anti-CS-E antibody” (GD3G7). Scale bar equals 200 μm. D, quantification of neurite outgrowth of the DRG explants culture with the presence of anti-CS-E antibody. Explants cultured on PNN GAGs or Sema3A project significantly shorter neurites than the laminin control (1st to 3rd bars). A combination of PNN GAGs and Sema3A leads to a stronger inhibition on the outgrowth (4th bar), and this inhibition is ameliorated when the PNN GAGs were treated with ChABC (5th bar), Hep I and III (to a lesser extent, bar 6) and anti-CS-E GD3G7 antibody. Treatment with control MPB49V antibody does not remove the inhibition from the substrates. n = 60–100, mean ± S.D., two-tailed paired t test, ***, p < 0.0001.

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