Study The Biological Activity Of The Glycosaminoglycan (GAG) Chain In NG2 Proteoglycan

NG2 is a chondroitin sulfate proteoglycan that is expressed on dividing progenitor cells of several lineages including glia, muscle, and cartilage. It is an integral membrane proteoglycan with a core glycoprotein of 300kDa. Many cell lines that express the full length 300-kDa NG2 core protein also release a 290-kDa form into the medium. The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O₂A progenitor cells. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids).The engagement of NG2 can trigger the cytoskeletal rearrangements necessary for changes in cell morphology and motility. The ectodomain of NG2 may interact with several types of extracelluar ligands, including FN, collgen II V VI, lamine, tenasin. NG2 can interact with the actin cytoskeleton.NG2 is uniformly and abundantly expressed in most melamona lesions and has been implicated in tumor invasion. NG2 acts as a coreceptor forα₄β₁ integrin to modulate cell adhesion and spreading mechanisms dependent on the small Rho family GTPase Cdc42 and adaptor protein p130^cas.Krigele-dependent binding NG2 to plasminogen and angiostatin may provide key mechanisms 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 neutrelize the inhibitory effect of angiostatin on endonthelial cell proliferation. NG2 is modified with a single chondroitin sulfate chain at Ser₉₉₉An AG to GC switch at nucleotide positions 3064-3065 encoding the wild type NG2 proteoglycan was made by sequential PCR reactions using overlapping primers, resulting in change of serine to alanine at amino acide position in the NG2 core protein. Expression constructs pcDNA3.1(+)NG2 Ser₉₉₉ and pcDNA3.1(+)NG2Ala999 were made. Transfection of wild type and mutant forms of NG2 and expression vector pcDNA3.1(+) in U251 cell were performed with LipofectAmine.The NG2 transfectants were isolated, identified by Western Blot and immnofluorescense. The pcDNA3.1(+)-NG2Ala999 transfected U251 cells lack the CS-GAG chain. Cell migration and extension assays were performed with these three U251 transfections. The results suggest that NG2 plays a role in promoting the cell migration and extension. Furthermore, it indicates that the CS-GAG of NG2 could modulate this process.