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

Abstract

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 Cl–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 Cl–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 Cl–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. Copyright © 1989, Wiley Blackwell. All rights reserved