Generation of truncated forms of the NG2 proteoglycan by cell surface proteolysis.

Abstract

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 300 kDa. In the present study we have characterized three molecular forms of the NG2 core protein expressed by different cell lines. Many cell lines that express the full length 300-kDa NG2 core protein also release a 290-kDa form into the medium. This species lacks the cytoplasmic domain but contains almost the entire ectodomain. Two core protein species, the intact 300-kDa form and a truncated 275-kDa form, are expressed at the surface of an NG2-transfected cell line U251NG52. The 275-kDa species lacks the cytoplasmic domain and at least 64 amino acids of the ectodomain. Mild trypsinization of B49 cells also generates the 275-kDa species, suggesting that this component is produced by proteolysis of the 300-kDa form. Conversion of the 300-kDa species to the 275-kDa form in U251NG52 cells is stimulated by reagents such as phorbol esters, which activate protein kinase C. Phorbol esters are also known to induce expression of metalloproteinases such as collagenase and stromelysin, which could be responsible for cleavage of the 300-kDa core protein. Although B49 cells do not spontaneously produce the truncated 275-kDa species, use of monoclonal antibodies against NG2 to block the interaction between NG2 and type VI collagen results in the appearance of the 275-kDa component in these cells. Thus the interaction between NG2 and type VI collagen, which contains a Kunitz-type proteinase inhibitor sequence in the alpha 3 chain, may protect the proteoglycan against proteolysis. This is consistent with the observed deficiency of U251NG52 cells in anchoring type VI collagen at the surface.

Cytoskeletal Reorganization Induced by Engagement of the NG2 Proteoglycan Leads to Cell Spreading and Migration

“…Rabbit antibodies and mouse mAbs against rat NG2 have been described previously (Nishiyama et al, 1991(Nishiyama et al, , 1995Tillet et al, 1997), as has a rabbit antibody against the L1 cytoplasmic domain . mAbs against the human ␤1 integrin subunit (GIBCO-BRL, Gaithersburg, MD), rat CD44 (PharMingen, La Jolla, CA), fascin (DAKO, Carpenteria, CA), ␤-actin (Sigma, St. Louis, MO), and vinculin (Sigma) were obtained commercially.…”

“…Samples of the extract were then fractionated by SDS-PAGE on 3-20% gradient gels and transferred by electroblotting onto Immobilon P membranes (Millipore, Bedford, MA). Blocking and probing of these membranes were performed as previously described (Nishiyama et al, 1995;Grako et al, 1999). Immunoreactive bands were visualized using an ECL chemiluminescence kit (Amersham Life Science, Buckinghamshire, England).…”

Matrix Metalloproteinase-14 Both Sheds Cell Surface Neuronal Glial Antigen 2 (NG2) Proteoglycan on Macrophages and Governs the Response to Peripheral Nerve Injury

“…In the presence of MMP-14, the 300-kDa NG2 was transformed into the major 290-and minor 260 -275-kDa species concomitantly with a significant increase in the 160-kDa NG2. According to the earlier data, the 290-and 260 -275-kDa species represent the shed soluble and shed membrane NG2, respectively (18,37). In the presence of TIMP-2 or GM6001, MMP-14 proteolysis of NG2 was fully repressed.…”

“…Expression of the EBNA-1 gene is controlled by the CMV promoter and is high-level and constitutive. The specificity of the five NG2 antibodies we used (which were directed against the individual portions of the NG2 ectodomain) was described earlier (37,38). Thus, the rabbit polyclonal EC, D2, and D3 antibodies were raised against the purified individual EC, D2, and D3 domain constructs, respectively.…”

“…Because MMPs normally cleave the C-terminal portion of NG2 according to previous data by others (18,37,43), we tested whether MMP-14 cleaved the similar region of NG2. For this purpose, we compared the proteolysis of the 1-2223 EC construct versus the 1-1465 EC⌬3 truncation lacking the C-terminal D3 domain.…”