Neuronal Pentraxin 2 Binds PNNs and Enhances PNN Formation

Figure 3

Nptx2 binds to the PNN. (a) Sequential buffer treatment of homogenized brain tissue was applied to purify the GAGs in the four separate samples. The GAGs were randomly biotinylated to allow for detection with streptavidin alkaline phosphatase on a custom-made NPTX2 ELISA plate. NPTX2 binds preferentially to PNN GAGs (). (b) To investigate which PNN GAGs can bind NPTX2, an ELISA was performed with biotinylated samples of purified GAGs. The applied GAGs were chondroitin sulphates (CS) A, B, C, D, and E, hyaluronan (HA), and heparan sulphate (HS). NPTX2 binds significantly to HA and CS-E. ELISA results were analysed with ANOVA (). (c) To investigate which of the PNN GAGs are responsible for the binding of NPTX2 to PNN GAGs, an NPTX2 ELISA bound by the PNN GAG fraction was treated with the enzymes chondroitinase ABC, Streptomyces hyaluronidase, and testicular hyaluronidase for one hour. All these enzymes were able to significantly reduce the signal of the bound PNN GAGs, which indicates that all enzymes break GAG bonds responsible for the binding of PNN GAGs to NPTX2. This result indicates that both, CS-E and HA in the PNN GAG sample, bind to NPTX2 on the plate (). (d) To confirm whether PNN glycan binding to NPTX2 could be blocked in a competition ELISA, one set of wells was treated with unbiotinylated HA before exposure to biotinylated PNN glycans. The hyaluronan indeed blocked the PNN glycans from binding (). (e) Sequential buffer treatment of homogenized rat brain tissue was applied to purify extracellular molecules (1), membrane molecules (2), molecules contained by ionic interactions (3), and PNN molecules (4). The samples were run on a Western blot, alongside a commercial NPTX2 control sample, at 120 V on a 4-12% gel. Nptx2 was found in samples 2, 3, and 4. This result suggests that Nptx2 is located on the membrane, contained by ionic interactions and in the PNN. (f–h) Cortical neurons from E18 rat brains were cultured to reach 35 DIV. Neurons were treated with chondroitinase ABC or Streptomyces hyaluronidase for 30 minutes. Neurons were fixed with 4% PFA for 20 minutes. After blocking for 1 hour, neurons were incubated with WFA and his-tag antibody to stain for the his-tagged NPTX2. Appropriate secondary antibodies were applied, and three wash steps were performed after each step. Representative images of neurons are shown (h). The relative Nptx2 his-tag intensity (f) and WFA PNN intensity (g) were significantly reduced after treatment with chondroitinase ABC or Streptomyces hyaluronidase (, ANOVA, , resp.). These findings indicate that both enzymes successfully removed the PNN from the neuronal surface and this removal of the PNN resulted in a removal of the exogenous Nptx2 protein from the neuronal surface ().

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