Role Of Heparanase In NGF Induced PC12 Neuritogenesis And Structure, Chain Conformation And Biological Avtivity Study On Heparan Sulfate Mimetic Prepared From Polysaccharide

Heparanase, an endo-β-D-glucuronidase, can cleave heparin / HS chains of heparan sulfate protein glycan in specific intra chain sites, which participates in degradation and remodeling of the extracellular matrix (ECM) and basement membrane (BM). Its enzymatic activity is associated with tumor metastasis and inflammation. Nevertheless, it is also expressed in normal cells, including leukocytes, astrocytes, neurons and white matter glia of rat spinal cord. During development, heparanase has been detected in the early mouse embryo, the chick vascular and nervous systems. However, there is very few reports on study of the role played in the neuritogenesis.PC12 differentiate into sympathetic neuron like cells in response to nerve growth factor (NGF). Phenotypic changes associated with this process include the biosynthesis of neurotransmitters, the acquisition of electrical excitability, and the growth of axon-like extensions called neuritis through a process known as neuritogenesis, which is a complex phenomenon that involves multiple interactions between the growing neurite and the extracellular matrix. Because heparanase is also expressed in neuron and participates in remodeling of the extracellular matrix, we try to investigate the role of heparanase in NGF induced PC12 neuritogenesis.We find that: (1) RT-PCR experiment showed that NGF (50ng/ml) promoted heparanase mRNA notably; (2)Heparanase inhibitor suramin bloked NGF induced PC12 neuritogenesis; (3) In NGF (25 ng/ml) induced PC12 neuritogenesis (72 h) essay, human heparanase Overexpression by its gene stable transfected in PC12 cells stable PC12 cells enhanced the neurite outgrowth, while cell with heparanase silence impaired the process. (4) Western blot experiments demonstrated that p38 MAPK phosphorylation was induced in the human heparanase stable transfected cell line, while this signaling was attenuated in siRNA stable transfected PC12 cell line.Heparanase can cleave heparin / HS chains of heparan sulfate protein glycan in specific intra chain sites, releasing HS fragments. HS is a linear copolymer comprising a repeating sulfate substituted glucosamine - hexuronic acid. To date, more than 100 functionally versatile proteins have been reported to interact with the highly charged groups of HS, which plays basic role in proliferation, differentiation and morphogenesis. This prompts us to develope the mimetics of HS and to study its activity.A water soluble glucan, PLB-2C, was isolated from the water extract of the root of Pueraria lobata (Willd) Ohwi using anion-exchange and gel permeation chromatography. Its structure was investigated by gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), infrared (IR) spectra, and nuclear magnetic resonance (NMR) spectroscopy of heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) techniques. The results indicated that PLB-2C was a linear glucan composed ofα-(1→6)-D-Glcp. Chain conformation study showed that the polysaccharide took random coil compact conformation. PLB-2CS, sulfated derivative of PLB-2C was prepares by chlorosulfonic acid-pyridine method. ¹³C NMR demonstrated that PLB-2CS was substituted at 2-0, 3-0, 4-0 positions. In neurite outgrowth essay, neither PLB-2C nor PLB-2CS could promote PC12 neuritogenesis. Howerver, in vitro cell viability assay by MTT method, PLB-2CS at 0.1, 1, and 5 mg/ml, could attenuate PC12 cell damage significantly caused by hydrogen peroxide.Based on aforementioned results, we deduced that heparanase may enhance PC12 neuritogenesis induced by NGF via the MAPK p38 pathway. PLB-2CS, failed to promote the neuritogenesis. This raises the possibility that the growth factor binding to HS may contribute to neuritogenesis. PLB-2CS has antioxidant activity indicates that they may be antioxidants.