Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes.
ABSTRACT: The presence of laminin in authentic basement membranes was examined at the level of a large pepsin-resistant fragment P1. This strongly antigenic fragment has been recently isolated from a mouse tumour basement membrane. By using antibodies to mouse laminin P1 for identification it was possible to isolate a homologous fragment P1 (Mr about 250 000) and a related component Pa (Mr about 70 000--90 000) from pepsin digests of human placenta and kidney. The fragments were in half-cystine (90--130 residues/1000) and carbohydrate and showed strong binding to concanavalin A. Reduction of disulphide bonds produced several smaller peptide chains, indicating a complex pepsin cleavage. Immunological assays demonstrated partial antigenic identity between laminin fragments obtained from mouse and human tissue, and suggested that fragment Pa may originate from a protein not completely identical with laminin. The results showed that laminin is an abundant component of tissue rich in basement membranes, which has been previously suggested by immunohistological studies.
Project description:Arterial basement-membrane-like material was isolated from rabbit aortic myomedial cell cultures by sonication and differential centrifugation. Isolated basement-membrane-like material was shown to be free of both cellular and matrix contaminants, on the basis of determinations of DNA, RNA, cholesterol, phosphorus and (Na+ + K+)-activated ATPase, combined with electron microscopy. Amino acid analyses showed that arterial basement-membrane-like material was composed of predominantly non-collagenous amino acids. Evaluated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, reduced basement-membrane-like material comprised six major and about 30 minor components in the Mr range 10 000-600 000. One of the major peptides (Mr 225 000) was disulphide-linked. Periodic acid-Schiff staining of gels indicated that most high-molecular-weight components were glycoproteins. Two-dimensional gel electrophoresis resolved reduced basement-membrane-like material into more than 100 components, with pI from 5 to 7. The disulphide-linked Mr-225 000 peptide appeared heterogeneous, with pI of 5.6-6.0, and was considered to represent fibronectin. All major peptides were of non-collagenous nature, on the basis of their susceptibility to pepsin and resistance to collagenase. Purified myomedial basement-membrane-like material contained collagenous peptides, as indicated by the presence of hydroxyproline and hydroxylysine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pepsin-treated and reduced basement-membrane-like material revealed five high-molecular-weight collagenous components appearing in the Mr range 105 000-375 000 relative to type I collagen standards.
Project description: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.
Project description:Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.
Project description:The globular domain IVa (about 250 residues) of the laminin alpha1 chain was obtained in recombinant form from mammalian cell clones. It was prepared either with (alpha1IVa-R) or without (alpha1IVa) an adjacent cell-adhesive RGD site which seems to be masked in laminin-1. The recombinant products could be visualized as globular structures by rotary shadowing, were resistant to trypsin and shared immunological epitopes with laminin-1, indicating folding into a native structure. Sequence analysis of pepsin fragments demonstrated the insertion of the globular domain into an epidermal growth factor-like scaffold which is characteristic of the extracellular laminin domain IV (L4) module. Only little immunological cross-reaction was found, however, with other L4 modules from perlecan and different laminin isoforms. Fragment alpha1IVa-R, but not fragment alpha1IVa, bound to alphaVbeta3 integrin, although to a distinctly lower level than a laminin fragment where the RGD site is fully exposed. The fragments also had no or only little cell attachment activity. This confirmed previous predictions that the globular domain alpha 1IVa masks the RDG site in laminin-1. Domain alpha 1IVa showed, in addition, a weak binding activity for the basement-membrane protein fibulin-1.
Project description:Connective tissue cells synthesize and secrete a group of matrix metalloproteinases (MMPs), all of which are capable of degrading the extracellular-matrix components. One of them, MMP-3 (stromelysin) has been shown to degrade purified basement-membrane components, collagen IV and laminin [Okada, Y., Nagase, H. & Harris, E. D., Jr. (1986) J. Biol. Chem. 261, 14245-14255]. Here we report that MMP-3 degrades collagen IV and laminin in intact basement membranes from bovine glomeruli (GBM) and bovine anterior-lens capsules (LBM). Degradation products were analysed by SDS/polyacrylamide-gel electrophoresis to determine the number and sizes of polypeptide fragments. Immunoblotting techniques were used to identify the origins of the fragments, i.e. collagen IV or laminin. The fragments of collagen IV were further mapped using specific antibodies that recognize the N-terminal (7 S) domain, the C-terminal (NC-1) domain, or the major triple-helical region between the terminal domains. Degradation of collagen IV was extensive; many fragments were found, from both GBM and LBM, in the Mr range 25,000-380,000. A large fragment of laminin (Mr greater than 380,000) was found in the GBM digests without reduction, but it dissociated into 220,000-Mr chains upon reduction. The results suggest that MMP-3 plays an important role in the catabolism of basement membranes.
Project description:Purified viral-envelope glycoproteins from influenza A virus were found to bind to two fragments of the fibronectin molecule. Human plasma fibronectin was digested by leucocyte cathepsin G, and three different fragments, of Mr 30000, 40000 and 12000-140000, with specific binding functions were isolated. Micelles of radiolabelled influenza A glycoprotein were allowed to bind to these fragments immobilized on polystyrene micro-titre wells. The C-terminal 120000-14000-Mr fragments that carry the cell-binding activity bound viral proteins most efficiently, whereas the 40000-Mr gelatin-binding fragment bound considerably less. The N-terminal 30000-Mr Staphylococcus aureus-binding fragment was negative in the assays. Laminin, a basement-membrane protein, also bound viral proteins, though less effectively than fibronectin. The binding was abolished if laminin or fibronectin fragments were pretreated with neuraminidase. This suggests that the sialic acids in the sugar moieties of these glycoproteins are involved in the binding. The affinity of viral-envelope glycoproteins for certain domains of fibronectin and for laminin may play a role in virus-cell interactions.
Project description:Antisera against mouse and human basement-membrane type IV collagen showed in radioimmunoassays distinct binding with large pepsin fragments obtained from the C-terminal portions of alpha 1 (IV)- and alpha 2 (IV)-chains. These reactions were specific for each constituent polypeptide chain. The data were confirmed by immunoadsorption, allowing the separation of antibodies with restricted chain specificity. Inhibition assays with CNBr peptides demonstrated the different localizations of antigenic determinants, which were either species-specific or shared by the human and mouse antigens.
Project description:The dynamic interplay between the extracellular matrix and embryonic stem cells (ESCs) constitutes one of the key steps in understanding stem cell differentiation in vitro. Here we report a biologically-active laminin-111 fragment generated by matrix metalloproteinase 2 (MMP2) processing, which is highly up-regulated during differentiation. We show that the ?1-chain-derived fragment interacts via ?3?1-integrins, thereby triggering the down-regulation of MMP2 in mouse and human ESCs. Additionally, the expression of MMP9 and E-cadherin is up-regulated in mouse ESCs--key players in the epithelial-to-mesenchymal transition. We also demonstrate that the fragment acts through the ?3?1-integrin/extracellular matrix metalloproteinase inducer complex. This study reveals a previously unidentified role of laminin-111 in early stem cell differentiation that goes far beyond basement membrane assembly and a mechanism by which an MMP2-cleaved laminin fragment regulates the expression of E-cadherin, MMP2, and MMP9.
Project description:The N-terminal sequences of mouse laminin alpha3B and alpha5 chains have been completed and demonstrate the presence of a signal peptide followed by a complete laminin N-terminal (LN) module (domain VI). These signal peptides were released after recombinant production of larger fragments comprising domains VI/V (45-65 kDa) from this region yielding properly folded proteins, which were secreted from HEK-293-EBNA cells. Pepsin digestion of these fragments yielded products of 25-35 kDa, which consisted only of domain V. The alphaVI/V fragments were able to inhibit self-assembly of laminin-1, with those from the alpha3B and alpha5 chains being more active than those from alpha1 and alpha2 chains. Domain V fragments, however, showed a reduced activity, indicating the major contribution of the LN module in inhibition. These interactions were confirmed by surface-plasmon-resonance assays demonstrating moderate affinities (K(d)=0.02 to >6 microM) for the binding to laminin-1. This indicated that laminins containing alpha3B or alpha5 chains should also be able to form non-covalent networks by polymerization. The LN modules also showed heparin binding in affinity chromatography, which was strongest for alpha1/alpha2, moderate for alpha3B, whereas no binding was observed for alpha5. They all bound to heparan sulphate chains of perlecan and to sulphatides, with a lower variability in binding activity. Specific antibodies were raised against alpha3BVI/V and alpha5VI/V and were shown to stain basement membrane zones in various mouse tissues. These antibodies also allowed the identification of a new laminin assembly form 5B consisting of alpha3B, beta3 and gamma2 chains.
Project description:Laminin ?5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin ?5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ?6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin ?5. Expression of human LAMA5 did not affect the timing of the mouse laminin ?1-?5 isoform switch, or that for mouse laminin ?1-?2. Immunoelectron microscopy showed that human laminin ?5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin ?5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin ?5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein.