IMMUNOLOGICAL IDENTIFICATION OF 2 PROTEOGLYCAN FRAGMENTS DERIVED FROM NEUROCAN, A BRAIN-SPECIFIC CHONDROITIN SULFATE PROTEOGLYCAN

Neurocan is a brain-unique chondroitin sulfate proteoglycan (CSPG) whose expression and proteolytic cleavage are developmentally regulated. One of the proteolytic products (C-terminal half) is known to be a CSPG with a 150 kDa core glycoprotein (CSPG-150). To identify the N-terminal half of neurocan, we raised an anti-neurocan polyclonal antibody (PAb 291) using a synthetic peptide whose amino acid sequence matched a part of the N-terminal half of neurocan. Western blots showed that PAb 291 recognized two CSPGs, one with a 220 kDa core glycoprotein (CSPG-220, namely neurocan) and one with a 130 kDa core glycoprotein (CSPG-130) isolated from young rat brains. CSPG-130 was co-purified along with CSPG-220 by PAb 291-immunoaffinity column chromatography. The amino acid sequence of the N-terminus of the immunopurified CSPG-130 was exactly the same as the N-terminal sequence of CSPG-220. These results suggest that not only the C-terminal half (CSPG-150) but also the N-terminal half (CSPG-130) of CSPG-220 exists in a CSPG form in rat brain. Using PAb 291 and monoclonal antibody 1G2 (MAb 1G2) which recognizes CSPG-150 in addition to CSPG-220, we found that the contents of CSPG-130 and CSPG-150 in the rat brain reached maximum levels around the time of birth. Both CSPG-130 and 150 were observed, while CSPG-220 was hardly detectable in extracts from the adult rat brain. Immunohistochemical investigation showed that the PAb 291 antigen had a similar distribution pattern to the MAb 1G2 antigen. PAb 291 and MAb 1G2 are proposed to be useful tools for studying the mechanism and biological significance of the cleavage of neurocan.