DETERMINATION OF A NATIVE PROTEOLYTIC SITE IN MYELIN-ASSOCIATED GLYCOPROTEIN

Myelin-associated glycoprotein (MAG) is a transmembrane structural pro tein that is thought to be involved in the formation and/or maintenanc e of the myelin sheath. MAG is proteolyzed at a discrete location near its transmembrane domain by a calcium activated myelin-associated cys teine protease in the central nervous system. The soluble proteolysis product, dMAG, can be found in the cerebrospinal fluid. The proteolysi s of MAG may be involved in the molecular mechanism of demyelination, as the proteolytic degradation of myelin proteins has been observed in disease states. The site for the proteolysis of MAG to dMAG was ident ified. This was accomplished by developing a protocol for the purifica tion of soluble dMAG and by protein sequencing of short peptides conta ining the carboxy-terminus of dMAG. The results from these experiments indicated that the native proteolytic site in MAG was located extrace llularly and occurred between residues 512 (Ala) and 513 (Lys), with a large hydrophobic residue at the P-2 position (Trp-511). This finding in turn indicated that the protease for which MAG was a substrate had cathepsin L-like activity. Cathepsin L-like activity in myelin was co nfirmed by peptidolysis experiments using known cathepsin L substrates . Additional experiments are in progress to determine the identity of this protease.