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...

No abstract

SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.

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.

Electron microscopy after rotary shadowing and negative staining of the large chondroitin sulphate proteoglycan from rat chondrosarcoma, bovine nasal cartilage and pig laryngeal cartilage demonstrated a unique multidomain structure for the protein core. A main characteristic is a pair of globular domains (diameter 6-8 nm), one of which forms the N-terminal hyaluronate-binding region. They are connected by a 25 nm-long rod-like domain of limited flexibility. This segment is continued by a 280 nm-long polypeptide strand containing most chondroitin sulphate chains (average length 40 nm) in a brush-like array and is terminated by a small C-terminal globular domain. The core protein showed a variable extent of degradation, including the loss of the C-terminal globular domain and sections of variable length of the chondroitin sulphate-bearing strand. The high abundance (30-50%) of the C-terminal domain in some extracted proteoglycan preparations indicated that this structure is present in the cartilage matrix rather than being a precursor-specific segment. It may contain the hepatolectin-like segment deduced from cDNA sequences corresponding to the 3'-end of protein core mRNA [Doege, Fernandez, Hassell, Sasaki & Yamada (1986) J. Biol. Chem. 261, 8108-8111; Sai, Tanaka, Kosher & Tanzer (1986) Proc. Natl. Acad. Sci. 83, 5081-5085; Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259].