Research The Biological Activities For Glycosaminoglycan Of NG2 Proteoglycan Polygoni Multiflori Caulis Extracts Inhibit MMPs And Influence Cell Morphology

The understanding for the significances of the biological function of the proteoglycan have been increased rapidly in the past few years. Many different core protein take uniform or different Glycosaminoglycan, these Glycosamin- oglycan have altitudinal variety in strcture,and this leading to specialities with linking to protein.In the studying of the proteoglycan, heparan sulfate is at large. It has manifold biological activity.Experiment confirmation, heparan sulfate could regulate singnals transfer by combine to growth factor and soluble celluar factor.This intensify the knowledge of singles transform.Up to the minute research indicate, heparan sulfate act on tumour cell growth and metastasis.Different sequence motifs of heparan sulfate has biological activity of acceleration or inhibitor growth.The NG2 chondroitin sulfate proteoglycan is expressed by immature progenitor cells in several different developmental lineages, including oligodendrocyte progenitors, chondroblasts, and pericytes/smooth muscle cells.NG2 is also expressed by a number of different types of tumors, including melanomas, glioblastomas, chondrosarcomas,and myeloid leukemias. Engagement of the NG2 proteoglycan can induce cell cytoskeleton reorganization that leads to cell spreading and migration.NG2 is a large chondroitin sulfate proteoglycan of 400-800KD with a core protein size of 300KD,it is a membrane-spanning protein that interacts with macromolecules on both sides of the plasma membrane. Outside parts of NG2 cell plasma membrane is large,include 2195 amino acid,And 76 amino acid is in the cytoplasm.NG2 is modiffied with a single chondroitin sulfate at Ser999 of core protein.The localization patterns of NG2 on both well-spread and motile cells point to an interaction of the proteoglycan with the actin cytoskeleton. Furthermore, engagement of NG2 by the substratum triggers cytoskeletal rearrangements that lead to cell spreading and migration.The nature of these cytoskeletal changes suggests the involvement of the rho family GTPases rac and cdc42 in NG2-activated processes.By studying the signaling pathways through which NG2activates these small GTPases.One possible mechanism could involve an interaction of NG2 with PDZ domain-containing scaffolding proteins in the cytoplasm.On the cell surface, NG2 interacts with a variety of diverse ligands, including extracellular matrix(ECM) component, growth factors and kringle domain proteins. Kringle-dependent binding of NG2 to plasminogen and angiostatin may provide key mechaanisms for regulating angiogenesis. The NG2-bound form of plasminogen exhibits an increased rate of conversion to plasmin, whereas binding of NG2 to angiostatin appears to neutralize the inhibitory effect of angiostatin on endothelial cell proliferation. Tested several growth factors, and among these NG2 was found to selectively bind to platelet-derived growth factor AA(PDGF-AA) and basic fibroblast growth factor(bFGF). NG2 may therefore act as an auxiliary cell surface recepor for these two factors, potentiating their ability to interact with and activate their respective receptor tyrosine kinases. With respect to NG2-interactive ECM components.the best-studied is type VI collagen, which may interact with NG2 to mediate processes such as cell migration, cell proliferation and protection from apoptosis.It is play an essential role of these character for cell proliferation and migration. In the case of heparan sulfate proteoglycans, the heparan sulfate chains play a key role in interactions with ligands. For example, bFGF binding to this class of proteoglycans, along with subsequent activation of bFGF receptors, it is highly dependent on the presence of the heparan sulfate chains. By contrast, Little is know about a role for the NG2 chondroitin sulfate chain in any of the aforementioned paradigms.The functions for the proteoglycan in the molecular pathological processes were also gradually unveiled in critical diseases such as cancer and arthritis. The part of this paper study focuses on the chondroitin sulfate proteoglycan NG2. It show very important function for the aspects on enhancing the ability of cell migration and modulating the reaction to cell and cellular factor. It is play an essential role of these character for cell proliferation and migration.Engagement of the NG2 proteoglycan can induce cell cytoskeleton reorganization that leads to cell spreading and migration.Little is know about the function of the chondroitin sulfate glycosaminoglycan moietythat links to Ser999 of NG2 core protein. As a result of glycosaminoglycan has mightiness varieties leads to the specificity of action on various proteins. So it is very necessary to gain insight of the biological activity on this chains component of NG2 chondroitin sulfate proteoglycan. A site-directed mutation of the wild type NG2 chondroitin sulfate proteoglycan was carried out by performing sequential PCR reactions using overlapping primers encoding the mutated sequence, resulting the change of serine to alanine at amino acid position999 in the NG2 core protein. Expression constructs pcDNA3.1+/NG2, pcDNA3.1+/NG2/S999A were made. Transfection of wild-type pcDNA3.1+/NG2 and mutant forms pcDNA3.1+ /NG2/ S999A of NG2 and expression vector pcDNA3.1+ in U251 human asyrocytoma cell were performed. The NG2 transfectants were isolated. Western Blot analysis showed the NG2/S999A transfected U251 human asyrocytoma cell lack chondroitin sulfate glycosaminoglycan chain. Cell migration assays were performed using these three U251 transfectants. The result suggests NG2 play a role in the cell migration. Furthermore, it indicates the chondroitin sulfate glycosaminoglycan chain of NG2 could modulate this process. This is the first time that the function of glycosaminoglycan chain of the NG2 is linked to cell migration.Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptinases known to degrade the components of the extracellular matrix (ECM), they have emerged as critical molecules that mediate the remodeling of ECM in both physiological and pathological conditions. Based on their substrate specificity and primary structure, the MMP family can be subdivided into six groups: collagenases, gelatinases, stromelysins, Matrilysins, membrane type (MT) and heterogeneous subgroup. MMPs can degrade ECM components, other proteases, clotting factors, chemotactic molecules, latent growth factors, growth-factor binding proteins, adhesion molecules, cell surface receptors etc. Thereby, MMPs mediate the synthesis and secretion of cytokine, growth factor, hormone, cell-cell adhesion molecules receptors and control cell migration, proliferation, morphogenesis and apoptosis and regulate tumor expansion, angiogenesis and dissemination. In vivo, it is important that the activity of these enzymes is kept under tight control. The activity of MMPs is controlled at the following three levels: transcription, activation of the latent proenzymes, and inhibition by their endogenous inhibitors, the TIMPs. The development of many diseases, such as tumor, rheumatoid arthritis, osteoarthritis and cardiovascular diseases, all relate to the abnormal of MMPs activities. Therefore, the regulation of MMPs activities is significant to the treatment of these diseases. From last 1990's, the development of MMPs inhibitors are more and more rapidly. According the different sources of MMPs inhibitors, they can divided into four groups: endogenous tissue inhibitors of metallo- proteinases (TIMPs); matrix metalloproteinase inhibitors (MMPIs) that may be derived from natural resources such as herbs, plants, fruits, and other agriculture products; synthetic MMPs inhibitors; some small peptide screening from Phage display libraries. Currently, the research focus on the matrix metalloproteinase inhibitors (MMPIs ) that may be derived from natural resources such as herbs, plants, fruits, and other agriculture products and the synthesis MMPs inhibitors. However, because of the incomplete understanding of the function of MMPs, non-specific and side effects of MMPs inhibitors, although earlier studies can lead to highly effectively inhibit the activity of MMPs. and effectively prevent tumor growth and metastasis, but the results are not satisfactory clinical treatment of cancer, eventually led to the failure of the latter part of the clinical examination. Along with the continuous deepening of the understanding of MMPs function, the researching and screening MMPs inhibitors with high specificity and low toxicity are becoming increasingly important. According to the reports, currently, some research institutions have isolated new and potentially beneficial compounds which have been shown to exhibit some degree of MMPI activities from herbs, plants, fruits, and other agriculture products, but they are far less potent and specific than the TIMP family. These natural compounds include long chain fatty acids, epigallocatechin gallate (EGCG) and other polyphenols, flavonoids, and a variety of other natural compounds.So, it become great important event to screen MMP inhibitors(MMPIs) from herbal to search effective inhibiters medicine targeting to MMPs. The second part report Polygoni Multiflori Caulis, the classification of traditional Chinese medicine (TCM) could effectively inhibit MMPs. Extracts of it have a strong inhibitory effect (IC50?1μg/ml) to MMP-16 .Polygoni Multiflori Caulis, was further extracted by organic reagents and obtained five extracts (petroleum ether extract, chloroform extract, ethyl acetate extract, n-butyl alcohol extract and final water extract) with herb. The ethyl acetate extract, n-butyl alcohol extract and water extract of Polygoni Multiflori Caulis inhibit MMP-9, MMP-14 and MMP-16, and among these extract the n-butyl alcohol extract and water extract inhibit the form of HT1080 cell pseudopodia. Influence HT1080 cell morphology and migration. The isolation of MMPs inhibitors from TCM is significant to research the treatment mechanism of TCM. And expect to find the effective forerunner compound of MMPIs.Meanwhile, it is more important to help developing the new drug targeting to MMPs.