Characterization of proteolytic fragments of the laminin-nidogen complex and their activity in ligand-binding assays

Some 12 new nidogen and laminin fragments were purified from elastase, thrombin and trypsin digests and characterized by their sizes (22 kDa to greater than 300 kDa), subunit patterns on electrophoresis, partial amino acid sequences, content of specific epitopes and their binding to laminin or nidogen structures in radioligand assays. This permitted the various fragments to be ordered along the dumbbell-shaped structure of nidogen and to compare them with previously described nidogen fragments arising by endogenous proteolysis. Two nidogen fragments (E-50, E-90; 50 kDa and 90 kDa) remain associated with a large laminin fragment in elastase digests of the complex and could be dissociated with 2 M guanidine.HCl. Recombination studies demonstrated Kd = 10-20 nM for this interaction. Nidogen fragments devoid of binding activity included the tryptic peptide T-40 (40 kDa) corresponding to the rod-like domain and several larger fragments extending more to the N-terminus of nidogen. An N-terminal thrombin fragment of about 50 kDa was also inactive. Together the data show a lack of laminin binding to the N-terminal globule and rod of nidogen and provide indirect evidence that this activity is located within or close to its C-terminal globular domain. Nidogen-binding structures of laminin were obtained as two large fragments (greater than 300 kDa), P1X and E1X. They correspond to the short arm structure of laminin with one (E1X) or two (P1X) arms decreased in size to the inner rod-like segment. Shortening in E1X is mainly due to the B1 chain segment including the central globular domain which was identified as a new laminin fragment E10. Binding of E1X and P1X to nidogen was comparable to that of laminin while much lower activity was found for other laminin fragments. A 10-fold lower binding potential was also observed for the laminin-nidogen complex whose structure can now be defined in more precise molecular terms.     

Enzyme induction in soybean infected by Phytophthora megasperma f.sp. glycinea

Laminin, the glycoprotein of basement membranes, consists of two subunits of 200,000 (α) and 400,000 (β) M_r on gel electrophoresis after reduction. We evaluated the relative proteolytic susceptibility of the two subunits using a variety of serine proteases. Human α-thrombin degraded the β subunit without altering the density or size of the α subunit. Chymotrypsin, plasmin, and cathepsin G all degraded both the β and α subunits producing limited digestion products. Chymotrypsin and cathepsin G both produced two major fragments of 160,000 and 130,000 M_r whereas plasmin produced two fragments of 180,000 and 140,000 M_r. Time course digestion studies demonstrated that the 400-kd β subunit was digested much more rapidly than the α subunit, and suggested that the major fragments (greater than 100,000 M_r) produced by chymotrypsin, plasmin, and cathepsin G were derived from the α subunit. The latter supposition was confirmed by first digesting laminin with thrombin to completely remove the β subunit, followed by digestion with chymotrypsin, cathepsin G, or plasmin. We conclude that the β subunit of laminin is highly protease labile. In contrast, the α subunit contains a large region resistant to serine proteases. Electron microscopic studies of the purified fragment of laminin derived from digestion with cathepsin G demonstrated that the protease resistant region of the α subunit contained three arms of similar appearance (32 nm) and included the intersection of the three short arms of the laminin molecule.     

The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival

The survival of cultured chick sympathetic neurons and the outgrowth of neurites were stimulated by the basement membrane protein laminin coated onto polyornithine culture substrates. The survival-potentiating activity was dependent on the presence of nerve growth factor. Both effects of laminin could be completely inhibited by affinity-purified antibodies against laminin fragment 3, the product of a limited proteolysis that corresponds to the heparin-binding globular domain at the end of the long arm of the laminin molecule. Antibodies against other laminin fragments were inactive, including those against previously determined cell-binding domains. A large laminin fragment, E8, was produced by brief elastase digestion and shown to consist of fragment 3 and an adjacent rod-like structure. Although lacking the cell binding domains, fragment E8 potentiated both neuronal survival and neurite outgrowth, and these effects could be blocked by antibodies against fragment 3. Weak survival and neurite potentiating activity was also detected in another fragment corresponding to the short arms of laminin, but as these effects were not inhibited by any of the antibodies tested they probably arose de novo during proteolysis. The heparin-binding domain of laminin is therefore responsible for its effects on neurons.     

Enzymatic and Molecular Biochemical Characterizations of Human Neutrophil Elastases and a Cathepsin G-like Enzyme

Human neutrophil elastase (HNE, EC 3. 4. 21. 37) is a causative factor of inflammatory diseases, including emphysema and rheumatoid arthritis. Enzymatic characterization is important for the development of new drugs involved in the regulation of this enzyme. In this study, we investigated the enzymatic and biochemical properties of five different elastolytic enzymes, with a molecular mass between 24 kDa and 72 kDa. Three elastases, molecular masses of 27, 29, 31 kDa, might be elastase isozymes that have the same NH₂-terminal amino acid sequences of Ile-Val-Gly-Gly-Arg-Arg-Ala. The 24-kDa enzyme, which showed the identical NH₂-terminal amino acid sequences to elastase, was a degraded fragment of native elastase. The elastolytic activity was conserved at the 6/7 domain of the NH₂-terminal region. The inhibitory characteristics of PMSF, DipF were the same as those of native elastases. The 72-kDa molecule, which showed elastolytic activity, might be a trimer formed between native elastases (31 kDa and 29 kDa) and a cathepsin G-like enzyme, which did not show elastolytic activity but enhanced the elastolytic activity of neutrophil elastase. Although this cathepsin G-like enzyme showed weak cathepsin G activity, it has distinguishable NH₂-terminal sequences of Ile-Val-Gly-Gly-Ser-Arg-Ala- from those of elastase or cathepsin G. The potentiation of elastolytic activity could be a result of the trimerization of native elastase with a cathepsin G-like enzyme, and was then weakly inhibited by serine protease inhibitors, such as PMSF, DipF. Therefore, we suggest the cathepsin G-like enzyme to be a novel enzyme, which has an important role in the development of inflammation.     

Identification of protease-sensitive sites in Human Endothelial-Monocyte Activating Polypeptide II protein

The cleaved approximately 22-kDa form of Endothelial-Monocyte Activating Polypeptide [mature (m)EMAP II] functions as a potent inhibitor of tumor growth. Although the anti-tumor effect of mEMAP II has been described, little is known regarding the cleavage of mEMAP II from its precursor form (pEMAP II). We determined that pEMAP II is expressed at the cell membrane surface and proteinases MMP-9, elastase, and cathepsin L release protein fragments consistent with mEMAP II molecular mass. MMP-9 and elastase generate a approximately 25-26 kDa spanning fragments, while cathepsin L generates a approximately 22 kDa fragment. Although several fragments are processed from pEMAP II within a 44 AA residue stretch, cathepsin L cleaves pEMAP II within 4 amino acids of the determined N-terminal sequence, suggesting that this region is sensitive to proteinases.     

Binding of Ca2+ influences susceptibility of laminin to proteolytic digestion and interactions between domain-specific laminin fragments

Ca2+ was found to influence the patterns of limit digests of laminin obtained with various neutral proteases. In the presence of Ca2+, larger fragments were obtained from the central part of laminin than in its absence. This was interpreted as being due to a stabilization of the central short-arm domains of laminin by bound Ca2+. When proteolytic fragments were tested for their ability to aggregate, only large fragments containing intact short arms were active, indicating an important role for these domains in laminin self-aggregation.     

Localization of viral-envelope-glycoprotein-binding sites in fibronectin.

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.     

The high-affinity binding of laminin to cells. Assignation of a major cell-binding site to the long arm of laminin and of a latent cell-binding site to its short arms

The laminin proteolytic fragments 1 (derived from the intersection of the short arms of the cruciform laminin molecule) and 8 (derived from the laminin long arm) bind to distinct receptors on HT-1080 human fibrosarcoma cells; both fragments are shown here to inhibit the high-affinity binding of laminin to these cells. Inhibition of binding between fragment 8 and laminin was competitive, whereas that between fragment 1 and laminin was noncompetitive. This indicates that laminin and fragment 8 most probably share the same cellular receptors, whereas laminin and fragment 1 bind to distinct receptors, inhibition being due to steric hindrance. Surprisingly, fragment 1-4 (corresponding to the complete short arms of laminin) neither bound to HT-1080 cells nor inhibited the binding of laminin or fragment 1. After treatment of fragment 1-4 with pepsin, however, the smaller subfragment 1 was liberated, which could then bind to the cells, and so was shown to block the binding of laminin and fragment 1. We conclude that native laminin bound to HT-1080 cells via the fragment-8-binding site near the end of its long arm. Although these cells also have distinct receptors for the short arm fragment 1, this receptor-binding site was not used as it appeared to be latent within the native laminin molecule.     

Human urinary proteinase inhibitor: inhibitory properties and interaction with bovine trypsin

The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.     

Two distinct cell-binding domains in laminin can independently promote nonneuronal cell adhesion and spreading [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

Two subfragments of laminin, E8, a major part of the long arm, and E1-4, the three short arms, promote cell adhesion and spreading. Three distinct types of adhesive behavior are seen in short term (1 h) assays, typified by secondary murine fibroblasts, adherent only on fibronectin; secondary murine myoblasts, adherent on fibronectin, laminin, and the E8 fragment; and Rugli human glioblastoma cells, adherent on fibronectin, laminin, E8, and E1-4. E8-specific polyclonal antibodies block myoblast adhesion to E8 and to laminin with identical concentration dependence; Rugli binding to E8 but not to laminin is also totally blocked by these antibodies. Heating of E8 and laminin to approximately 60 degrees C abolishes cell attachment-promoting activity for myoblasts. Adhesion of Rugli cells to E8 is also lost, but on laminin the attachment-promoting activity remains constant. This is due to an increase in the activity of E1-4 fragment as it is heated. Thus, major sites for initial cell adhesion to and spreading on laminin lie within the E8 and E1-4 fragments, but not all cells binding to laminin will bind to both fragments. These data may tentatively be explained by the existence of more than one type of receptor for laminin at the cell surface; one is needed for each fragment.     

Cryptic fragment alpha4 LG4-5 derived from laminin alpha4 chain inhibits de novo adipogenesis by modulating the effect of fibroblast growth factor-2

Cleavage of the extracellular matrix (ECM) by proteolysis unmasks cryptic sites and generates novel fragments with biological activities functionally distinct from those of the intact ECM molecule. The laminin G-like (LG)4-5 fragment has been shown to be excised from the laminin α 4 chain in various tissues. However, the functional role of this fragment has remained unknown to date. To investigate this, we prepared α 4 LG1-3 and α 4 LG4-5 fragments by elastase digestion of recombinant α 4 LG1-5, and examined their effects on de novo adipogenesis in mice at the site of injection of basement membrane extract (Matrigel) and fibroblast growth factor (FGF)-2. Although the addition of whole α 4 LG1-5 suppressed adipogenesis to some extent, the α 4 LG4-5 fragment could strongly suppress adipogenesis at a concentration of less than 20 n m . Addition of the α 4 LG4 module, which contains a heparin-binding region, had a suppressive effect, but this was lost in mutants with reduced heparin-binding activity. In addition, antibodies against the extracellular domain of syndecan-2 and -4, which are known receptors for the α 4 LG4 module, suppressed adipogenesis. Thus, these results suggest that the cryptic α 4 LG4-5 fragment derived from the laminin α 4 chain inhibits de novo adipogenesis by modulating the effect of FGF-2 through syndecans.     

Laminins and other strange proteins.

Laminins are large multidomain proteins of the extracellular matrix (ECM) with important functions in the development and maintenance of cellular organization and supramolecular structure, in particular in basement membranes. Each molecule is composed of three polypeptide chains, A (300-400 kDa) and B1 and B2 (180-200 kDa), which together form the characteristic cross-shaped laminin structure with three short arms and one long arm. Many different domains have been identified in laminin by sequence analysis, structural investigations, and functional studies. Each short arm is formed by homologous N-terminal portions of one of the three chains. Structurally, each short arm contains two or three globular domains which are connected by rows of manyfold-repeated Cys-rich "EGF-like" domains. In all three chains this region is followed by a long heptad repeat region similar to those found in many alpha-helical coiled-coil proteins. These parts of the three laminin chains constitute a triple-stranded coiled-coil domain, which forms the extended rodlike structure of the long arm. This is the only domain in the protein which is made up of more than one chain and consequently serves the function of chain assembly. The two B chains are terminated by the coiled-coil domain, but the A chain contains an additional C-terminal segment which accounts for five globular domains located at the tip of the long arm. Several important functions of laminin have been assigned to individual domains in either the short arms or terminal regions of the long arm.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of Ca2+ binding in the self-aggregation of laminin-nidogen complexes

Laminin-nidogen complexes were found to aggregate in the presence of divalent cations in a manner dependent on ion concentration. This effect shows a selectivity for Ca2+, as half-maximal aggregation is achieved already at about 10 microM Ca2+, while Mg2+ induces aggregation at 10-fold higher ion concentrations and always to a lesser extent. When binding of Ca2+ to laminin-nidogen complexes was measured by equilibrium dialysis, a total of about 16 binding sites with dissociation constants in the range of 5-300 microM could be identified. At 50 microM Ca2+, where the aggregation is maximal, only two to three Ca2+ ions are bound to laminin-nidogen complexes, indicating that the aggregation reaction is induced by the binding of Ca2+ to a small number of sites and possibly to a single distinct site. Analysis of Ca2+ binding to various proteolytic fragments of laminin allowed the tentative localization of a high affinity binding site to a large fragment comprising two of the short arms connected by the central part of the laminin molecule.     

The N terminus of laminin A chain is homologous to the B chains

A major proteolytic fragment (E1/E1-4) of the basement membrane protein laminin, comprising the three short arms with some terminal globules missing, was isolated by elastase digestion, and partial protein sequence data were determined for several tryptic peptides. Sequences which corresponded to A-chain structures were used to synthesize oligonucleotides for the construction and screening of a primer-extended cDNA library from mouse PYS-2 cells. A clone of 1.1 kb was obtained and shown by sequencing to correspond to the 5' end of the 10-kb mRNA of the A chain of laminin. The clone contains 77 nucleotides of 5' untranslated sequence and a region coding for 334 amino acids, including a presumptive signal peptide of 24 amino acids. The sequence is 30% homologous to the corresponding N-terminal part of the B1 chain of laminin, suggesting the same structure for both domains. The data present further evidence for a recent structural model which postulates that each of the three laminin polypeptide chains forms a distinct short arm.     

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.     

Separate domains of the insulin receptor contain sites of autophosphorylation and tyrosine kinase activity

We have studied the structure and function of the solubilized insulin receptor before and after partial proteolytic digestion to define domains in the beta-subunit that undergo autophosphorylation and contain the tyrosine kinase activity. Wheat germ agglutinin purified insulin receptor from Fao cells was digested briefly at 22 degrees C with low concentrations (5-10 micrograms/mL, pH 7.4) of trypsin, staphylococcal V8 protease, or elastase. Autophosphorylation of the beta-subunit was carried out before and after digestion, and the [32P]phosphoproteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, detected by autoradiography, and analyzed by tryptic peptide mapping by use of reverse-phase high-performance liquid chromatography. Mild trypsin digestion reduced the apparent molecular mass of the beta-subunit from 95 to 85 kDa, and then to 70 kDa. The 85-kDa fragment was not immunoprecipitated by an antibody directed against the C-terminal domain of the beta-subunit (alpha Pep-1), indicating that this region of the receptor was lost. The 85-kDa fragment contained about half of the [32P]phosphate originally found in the beta-subunit, and tryptic peptide mapping showed that two major tryptic phosphopeptides (previously called pY2 and pY3) were removed. Three other tryptic phosphopeptides (pY1, pY1a, and pY4) were found in the 85- and 70-kDa fragments. Treatment of the intact receptor with staphylococcal V8 protease also converted the beta-subunit to an 85-kDa fragment that did not bind to alpha Pep-1, contained about 50% of the initial radioactivity, and lacked pY2 and pY3. Elastase rapidly degraded the receptor to inactive fragments between 37 and 50 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neutrophil elastase cleaves laminin-332 (laminin-5) generating peptides that are chemotactic for neutrophils

Proteolytic processing of laminin-332 by matrix metalloproteinase (MMP)-2 and MMP-14 has been shown to yield fragments that are promigratory for epithelial cells. During acute and chronic inflammation, proteases are elaborated by neutrophils and macrophages that can degrade basement membranes. We investigated the susceptibility of laminin-332 to degradation by the following neutrophil and macrophage proteases: neutrophil elastase (NE), cathepsin G, proteinase-3, and MMPs-2, -8, -9, and -12. Protease-specific differences were seen in the capacity to cleave the individual chains of laminin-332. NE and MMP-12 showed the greatest activity toward the gamma2 chain, generating a fragment similar in size to the gamma2x fragment generated by MMP-2. The digestion pattern of laminin-332 by degranulated neutrophils was nearly identical to that generated with NE alone. Digestion by supernatants of degranulated neutrophils was blocked by an inhibitor of NE, and NE-deficient neutrophils were essentially unable to digest laminin-332, suggesting that NE is the major neutrophil-derived protease that degrades laminin-332. In vivo, laminin gamma2 fragments were found in the bronchoalveolar lavage fluid of wild-type mice treated with lipopolysaccharide, whereas that obtained from NE-deficient mice showed a different cleavage pattern. In addition, NE cleaved a synthetic peptide derived from the region of human laminin gamma2 containing the MMP-2 cleavage site, suggesting that NE may generate laminin-332 fragments that are also promigratory. Both laminin-332 fragments generated by NE digestion and NE-digested laminin gamma2 peptide were found to be chemotactic for neutrophils. Collectively, these data suggest that degradation of laminin-332 by NE generates fragments with important biological activities.     

The alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin

This study was undertaken to determine the roles of individual alpha/beta 1 integrin heterodimers in promoting cellular interactions with the different attachment-promoting domains of laminin (LN). To do this, antibodies to the integrin beta 1 subunit or to specific integrin alpha subunits were tested for effects on cell attachment to LN, to elastase fragments E1-4 and E1, derived from the short arms and core of LN's cruciform structure, and to fragment E8 derived from the long arm of this structure. The human JAR choriocarcinoma cells used in this study attached to LN and to fragments E1 and E8. Attachment to E1-4 required a much higher substrate coating concentration, suggesting that it is a poor substrate for JAR cell attachment. The ability of cells to attach to different LN domains suggested the presence of more than one LN receptor. These multiple LN receptors were shown to be beta 1 integrin heterodimers because antibodies to the integrin beta 1 subunit inhibited attachment of JAR cells to LN and its three fragments. To identify the individual integrin alpha/beta 1 heterodimers that mediate interactions with these LN domains, mAbs specific for individual beta 1 heterodimers in human cells were used to study JAR cell interactions with LN and its fragments. An anti-alpha 6/beta 1-specific mAb, GoH3, virtually eliminated cell attachment to E8 and partially inhibited attachment to E1 and intact LN. Thus the major alpha 6/beta 1 attachment domain is present in fragment E8. An alpha 1/beta 1-specific mAb (S2G3) strongly inhibited cell attachment to collagen IV and partially inhibited JAR attachment to LN fragment E1. Thus, the alpha 1/beta 1 heterodimer is a dual receptor for collagen IV and LN, interacting with LN at a site in fragment E1. In combination, the anti-alpha 1- and anti-alpha 6-specific antibodies completely inhibited JAR cell attachment to LN and fragment E1. Thus, the alpha 1/beta 1 and alpha 6/beta 1 integrin heterodimers each function as LN receptors and act together to mediate the interactions of human JAR choriocarcinoma cells with LN.     

Identification and characterization of a 43-kilodalton laminin fragment from the "A" chain (long arm) with high-affinity heparin binding and mammary epithelial cell adhesion-spreading activities

A recently described procedure of reduction and carboxymethylation followed by heparin-Sepharose chromatography [Arumugham et al. (1988) Connect. Tissue Res. 18, 135-147] was used to characterize high-affinity heparin binding fragments of the laminin "A" chain. Two laminin fragments of Mr 53K and 43K selectively bound to the heparin-Sepharose column from the chymotrypsin digest of laminin, indicating that these fragments originate from the "A" chain. Without reduction and carboxymethylation but in the presence of 2.0 M urea, the heparin-Sepharose-bound material from the chymotrypsin laminin digest contains all the attachment-promoting activity for normal mouse mammary epithelial cells. The reduced 200-kDa intact three short arm fragment, fragments of Mr 70K-160K obtained either from laminin or from the reduced 200-kDa three short arm fragment, and the 53-kDa heparin binding fragment were all inactive in promoting the adhesion of mouse mammary epithelial cells. The mammary epithelial cell adhesion and spreading properties of laminin are associated with the high-affinity heparin binding 43-kDa fragment. The mammary epithelial cells attach to the 43-kDa fragment substrate and synthesize laminin, collagen type IV, and desmoplankins I and II as are the cells attached to laminin substrate and to the cells grown on tissue culture dishes. The biologically active 43-kDa fragment is generated from laminin, but not from the three short arm fragment. These results suggest that normal mouse mammary epithelial cells interact with laminin through a single site which is present in the 43-kDa heparin binding fragment located on the long arm of the "A" chain.