Dissection of laminin by cathepsin G into its long-arm and short-arm structures and localization of regions involved in calcium dependent stabilization and self-association.
Native laminin-nidogen complex isolated from mouse Engelbreth-Holm-Swarm tumor was treated with purified cathepsin G or leucocyte elastase, two neutral serine proteases which play a role in inflammatory processes accompanied by degradation of basement membranes. Both enzymes were found to be more active than porcine pancreatic elastase. In the absence of Ca2+, laminin fragments produced by leucocyte elastase resembled those formed by the pancreatic enzyme but at physiological concentrations of Ca2+ cleavage by cathepsin G was much more selective. Initially laminin (900 kDa) was cleaved at two major sites only with similar rates leading to three fragments. Fragment C1-4 (about 550 kDa) comprises the intact three short arms of the molecule and fragment C8-9 (about 350 kDa) contains the entire triple-coiled region by which its three chains are assembled and the major part of the terminal globular domain of the long arm. The remaining C-terminal region of this domain was recovered as fragment C3 of about 50 kDa. Stabilization against proteolytic attack was restricted to the region of fragment C1-4 and only this fragment exhibited strong Ca2+ dependent self-association similar to that of intact laminin or of its complex with nidogen. The associative properties of fragment C1-4 were dramatically diminished upon removal of the tip of one of the short arms comprising fragment 4. In addition, this provides a clear assignment of the important laminin function to a distinct domain in one of its short arms. The new fragment C8-9 may be employed for exploring the properties and possible functions of the upper long-arm region which so far has not been available as a fragment.
Bruch
M
M
Abteilung Biophysikalische Chemie, Biozentrum der Universität, Basel, Switzerland.
Landwehr
R
R
Engel
J
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology*; D006801:Humans; D007797:Laminin / Q000378:metabolism* / Q000648:ultrastructure; D019272:Leukocyte Elastase; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases
D007797:Laminin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C029967:nidogen; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534096:Ctsg protein, mouse; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D002118:Calcium