No abstract

Laminin, obtained from a tumor basement membrane, was treated with neutral proteases (trypsin, chyinotrypsin, elastase, subtilisin, Stuphylucoccus UUYPU.Y protease), which all produced similar fragment patterns upon prolonged digestion. The patterns were different from those obtained for fibronectin, which showed a much higher susceptibility to proteolysis by the same enzymes. Four large fragments could be purified which accounted together for more than half of the mass of laminin. They were found to differ in size, amino acid composition, spectral properties and antigenicity. The largest fragment 1 (Mr 260000-300000) was rich in half-cystine (120 residues/1000) and showed a circular dichroism spectrum indicating the absence of c! helix and p structure. Fragment 3 ( M , = 50000) possessed fl structure and was able to bind onto heparin-Sepharose. Fragments 2 ( M , = 50000) and 4 ( M , = 75000) were related structures and their relative yields depended on the protease used. They showed spectra similar to those of fragment 1 . Electron microscopy revealed that fragment 1 consists of three rodlike elements (length 26 nm) connected to each other at one end. The other fragments appeared as globules (fragment 3), short rods (fragment 2) or globules connected to a short rod (fragment 4). Data obtained from limited proteolysis indicated that fragment 1 and 4 (or 2) are in close proximity to each other in the three short arms of laminin, which in its intact form has the shape of an asymmetric cross. The long arm appeared to be readily degraded by proteases.Circular dichroism studies of native laminin indicated about 55 y;, aperiodic structures, 15 "G 0 structure and 30% x helix. The x helix was readily destroyed by proteolysis and showed a sharp, reversible transition at 5 X C.Stability of these structures was decreased by reduction of disulfide bonds or by increasing concentrations of guanidine. Heat denaturation rendered laminin susceptible to plasmin, which did not degrade the native protein.Cleavage occurred mainly within the 440000-M, polypeptide chain of laminin and was accompanied by a partial loss of the long arm.Laininin has been identified as an abundant non-collagenous glycoprotein of basement membranes [1 -31 and is produced by a variety of cells including epithelial, embryonic and tumor cells [2,4]. Other studies indicated that laminin serves in the basement membrane as an adhesive protein capable of interacting with cell sufaces [5 -71, collagen type IV [5] and heparin or heparan sulfate [8]. A multitude of biological functions is frequently associated with large and elongated proteins. Laininin has in fact a multidomain structure with the shape of an asymmetric cross consisting of three short arms and one long arm [9]. The major elements of this structure are rod-like segments and seven globular domains located at the ends and along the arms. Pepsin degrades about 70';; of laininin into small peptides but releases also two large fragments P1 and P2 [lo], which appear as rod-like structures on electron micro...

Intima collagen was studied by electron microscopy (rotary shadowing and negative staining) and by analytical ultracentrifugation. It was found that the monomeric unit (Mr 170 000) consists of a 105 nm-long triple helix terminated by a small globular domain (Mr about 30 000) at one end and a large globular domain (Mr about 40 000) at the other end. The monomer was produced by selective reduction of interchain disulphide bridges. Before reduction, dimers, tetramers and larger filamentous structures were found. Dimers are lateral staggered aggregates of two monomers aligned in an anti-parallel fashion. This gives rise to an inner 75 nm-long region of two slightly intertwisted triple helices flanked by the large globular domains. The outer triple-helical segments (length 30 nm) with the small globular domains at their ends emerge at both sides of this structure. Interchain disulphide bridges are probably located in the vicinity of the large domains. Only the outer segments could be degraded by bacterial collagenase. In tetramers the outer segments of two dimers are covalently linked, forming a scissors-like structure. In the fibrous forms several tetramers are assembled end-to-end with an overlap between the outer segments. The molecular masses and sedimentation coefficients were calculated for these various forms from the electron-microscopically observed dimensions and agreed with results obtained by ultracentrifugation. The unique structure of intima collagen suggests that it originates from a microfibrillar component and that it can be considered a unique collagenous protein, for which we propose the designation type VI collagen.

Three new laminin fragments, E8, E9 and 25K with mol. wt. 50 000‐280 000, were prepared from a limited elastase digest of laminin and from tissue extracts. They were similar with respect to their rod‐like structure, a high alpha‐helix content, the assembly from two chain segments and immunological cross‐reactivity. Two of the fragments (E8 and E9) possess in addition globular domains which lack alpha‐helices. Chemical, immunological and physical data together with sequence analysis strongly indicate that the alpha‐helical segments are assembled in coiled‐coil structures which are located in the rod of the long arm of laminin. These data give new insights into the overall structure of the protein.

Intima collagen was obtained from pepsin digests of human placenta in two forms, which differ to some extent in the size of their constituent polypeptide chains (Mr 50 000-70 000). These chains are connected by disulphide bonds to large aggregates. The aggregates are arranged in a triple-helical conformation with a remarkably high thermal stability (Tm 41-62 degrees C) and are resistant to further proteolytic digestion. Reduction of as little as 5% of the disulphide bonds produces mainly monomeric triple helices (Mr about 160 000) with Tm 32 degrees C. Partially reduced material can be separated into triple-helical and non-collagenous domains by proteolysis. Pepsin releases a collagenous component with chains of Mr 38 000. Bacterial collagenase liberates two non-collagenous segments (Mr 15 000-30 000) rich in cystine. Treatment with collagenase before reduction separates intima collagen into a large fragment composed of collagenous (Tm 41 degrees C) and non-collagenous structures and a single non-collagenous segment. The data support the electron-microscopical model of intima collagen [Furthmayr, Wiedemann, Timpl, Odermatt & Engel (1983) Biochem. J. 211, 303-311], indicating that the basic unit of the fragment consists of a continuous triple helix joining two globular domains.