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HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix

View Article: PubMed Central - PubMed

ABSTRACT

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

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HB-GAM prevents PTPσ binding to chondroitin sulfates of aggrecan.Extracellular domain of wild type and mutant PTPσ (80 nM) was mixed with different concentrations of HB-GAM. The mutant PTPσ lacks the CS binding site. The mixture was added to wells coated with aggrecan (10 μg/ml) and bound PTPσ was measured with HPR conjugated goat anti-human IgG (FC part) using the O-phenylendiamine dihydrochloride colorimetric assay.
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f4: HB-GAM prevents PTPσ binding to chondroitin sulfates of aggrecan.Extracellular domain of wild type and mutant PTPσ (80 nM) was mixed with different concentrations of HB-GAM. The mutant PTPσ lacks the CS binding site. The mixture was added to wells coated with aggrecan (10 μg/ml) and bound PTPσ was measured with HPR conjugated goat anti-human IgG (FC part) using the O-phenylendiamine dihydrochloride colorimetric assay.

Mentions: The transmembrane tyrosine phosphatase PTPsigma (PTPσ) regulates neurite outgrowth and acts as a nexus for multiple protein and proteoglycan interactions. It was recently shown to mediate CSPG inhibition of neurite outgrowth through binding to the CS side chains of CSPGs192021. Since HB-GAM has strong binding affinity to the CS and HS chains of proteoglycans, we hypothesized that HB-GAM might compete with PTPσ for CS binding thus reducing the CSPG inhibition. We devised solid phase binding assays mimicking conditions of the neurite outgrowth experiments using PTPσ ectodomain and a PTPσ mutant lacking the CS binding site19. The mutant PTPσ displayed little if any binding to substrate-bound aggrecan compared to the wild-type PTPσ. Binding of the wild-type PTPσ to substrate-bound aggrecan was dose-dependently inhibited by HB-GAM (Fig. 4). Furthermore, we found that HB-GAM displayed binding to CS but not to PTPσ in a solid phase binding assay showing that HB-GAM binds to aggrecan and not to PTPσ(Supplementary Fig. S4f), and PTPσ was not found as an HB-GAM-binding component in bead assays using CNS neurons (Table 1). Altogether, the data revealed a competition between HB-GAM and PTPσ for the CS chains on aggrecan.


HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix
HB-GAM prevents PTPσ binding to chondroitin sulfates of aggrecan.Extracellular domain of wild type and mutant PTPσ (80 nM) was mixed with different concentrations of HB-GAM. The mutant PTPσ lacks the CS binding site. The mixture was added to wells coated with aggrecan (10 μg/ml) and bound PTPσ was measured with HPR conjugated goat anti-human IgG (FC part) using the O-phenylendiamine dihydrochloride colorimetric assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037378&req=5

f4: HB-GAM prevents PTPσ binding to chondroitin sulfates of aggrecan.Extracellular domain of wild type and mutant PTPσ (80 nM) was mixed with different concentrations of HB-GAM. The mutant PTPσ lacks the CS binding site. The mixture was added to wells coated with aggrecan (10 μg/ml) and bound PTPσ was measured with HPR conjugated goat anti-human IgG (FC part) using the O-phenylendiamine dihydrochloride colorimetric assay.
Mentions: The transmembrane tyrosine phosphatase PTPsigma (PTPσ) regulates neurite outgrowth and acts as a nexus for multiple protein and proteoglycan interactions. It was recently shown to mediate CSPG inhibition of neurite outgrowth through binding to the CS side chains of CSPGs192021. Since HB-GAM has strong binding affinity to the CS and HS chains of proteoglycans, we hypothesized that HB-GAM might compete with PTPσ for CS binding thus reducing the CSPG inhibition. We devised solid phase binding assays mimicking conditions of the neurite outgrowth experiments using PTPσ ectodomain and a PTPσ mutant lacking the CS binding site19. The mutant PTPσ displayed little if any binding to substrate-bound aggrecan compared to the wild-type PTPσ. Binding of the wild-type PTPσ to substrate-bound aggrecan was dose-dependently inhibited by HB-GAM (Fig. 4). Furthermore, we found that HB-GAM displayed binding to CS but not to PTPσ in a solid phase binding assay showing that HB-GAM binds to aggrecan and not to PTPσ(Supplementary Fig. S4f), and PTPσ was not found as an HB-GAM-binding component in bead assays using CNS neurons (Table 1). Altogether, the data revealed a competition between HB-GAM and PTPσ for the CS chains on aggrecan.

View Article: PubMed Central - PubMed

ABSTRACT

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

No MeSH data available.


Related in: MedlinePlus