Macromolecular organization of basement membranes. Characterization and comparison of glomerular basement membrane and lens capsule components by immunochemical and lectin affinity procedures

The macromolecular components of bovine glomerular basement membrane (GBM) and lens capsules (anterior and posterior) solubilized by sequential extractions with denaturing agents were quantitated and characterized by polyacrylamide gel electrophoresis, CL-6B filtration, and DEAE-cellulose chromatography with the help of immunochemical techniques. Laminin, entactin, fibronectin, and heparan sulfate proteoglycan were primarily recovered (over 80%) from both basement membranes in a guanidine HCl extract which contained only a limited amount of the total protein (10-14%); most of the remainder of these noncollagenous components could be solubilized by the guanidine in the presence of reducing agent. Although a portion of the Type IV collagen could be obtained by these treatments, effective extraction of this protein depended on exposure to sodium dodecyl sulfate under reducing conditions. Immunoblot analysis revealed a remarkably similar pattern for GBM and lens capsule Type IV collagens with prominent bands of Mr = 390,000, 210,000, and 190,000 being evident. Fibronectin was present in much greater amounts in GBM than lens capsule while the reverse was true for entactin. In both GBM and lens capsules, the entactin (Mr = 150,000) exceeded laminin; the latter protein on immunoblotting was found to contain primarily the alpha-subunit (Mr = 200,000). The size of the heparan sulfate proteoglycan from anterior (Mr = 400,000) and posterior lens capsule (Mr greater than 500,000) was substantially larger than that from GBM (Mr = 200,000). During DEAE-cellulose chromatography under nonreducing conditions in a denaturing solvent, a portion of the Type IV collagen coeluted with the proteoglycan from these membranes. Considerable Bandeiraea simplicifolia I binding activity (alpha-D-galactose specific) was observed in GBM and lens capsule extracts and column fractions which could not be accounted for by laminin alone. Several components which reacted with this lectin were seen on transblots and among these Type IV collagen was identified. In contrast to the basement membranes from bovine tissues, the constituents from human GBM did not react with the B. simplicifolia I lectin.     

Kinetic analysis of the reaction catalyzed by rat-liver 3-hydroxy-3-methylglutaryl-coenzyme-A reductase using two specific inhibitors.

Laminin was recently characterized as being a major non-collageneous protein of the basement membrane matrix produced by a mouse tumor. It was extracted from the tumor matrix with neutral buffer and purified under non-denaturing conditions. Rabbit and guinea pig antisera raised against laminin or a large pepsin fragment P1 of laminin showed strong binding to both laminin and peptide P1 but only a weak reaction with reduced and alkylated laminin. The major antigenic determinants of laminin were located in a disulfide knot, comprising one third of the molecule, which resisted degradation by pepsin or cyanogen bromide. Minor antigenic determinants shared by the native and reduced protein could also be identified. The data were interpreted as showing that laminin consists of conformationally rigid as well as more flexible domains. Absorption studies with mouse kidney homogenate indicated that authenic basement membranes contain a protein immunologically identical to laminin. Tissues from other species contain a related protein which exhibits partial cross-reaction with mouse laminin. Together with immunofluorescence data the findings demonstrate that laminin, like type IV collagen, occurs in most basement membranes of the body.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

The connective tissue of the rat lung: electron immunohistochemical studies

The ultrastructural distribution of specific connective-tissue components in the normal rat lung was studied by electron immunohistochemistry. Three of these components were localized: type I collagen, fibronectin and laminin. Type I collagen was present not only in major airways and vascular structures, but also in alveolar septa. Laminin was found in all basement membranes, and only in basement membranes, demonstrating once more that this glycoprotein is an intrinsic component of the basement membrane. Fibronectin was found free in the interstitium and on the surfaces of collagen fibers. The basement membranes of bronchial, glandular and endothelial cells of large vessels lacked fibronectin; however, capillary endothelial and occasionally epithelial alveolar basement membranes contained some fibronectin in an irregular, spotty distribution. This localization suggests that in the lung, as in other tissues, fibronectin is not an intrinsic component of the basement membrane, but rather a stromal and plasma protein. Only basement membranes in the alveolar parenchyma contained "trapped" plasma fibronectin.     

Identification of a cell surface-binding protein for the core protein of the basement membrane proteoglycan

We have identified a protein(s) on the surface of hepatocytes that binds to the core protein of the heparan sulfate proteoglycan of basement membranes. These cells attached and spread on substrates prepared from the basement membrane heparan sulfate proteoglycan (HSPG) and its core protein (HSPG-core). Three proteins (Mr = 38,000, 36,000, and 26,000) were found to bind to a HSPG-core affinity column using extracts of iodinated hepatocytes, whereas proteins extracted from isolated membranes contained primarily the larger protein (Mr = 38,000). Similar results were obtained using a solid phase binding technique using labeled HSPG-core. Binding of HSPG-core to the protein (Mr = 38,000) was not altered by the presence of an excess of heparin, heparan sulfate, fibronectin, laminin, or collagen IV but was reduced by unlabeled HSPG-core. Similar studies showed that the binding protein (Mr = 3,0000) was present in extracts from the membranes of Engelbreth-Holm-Swarm tumor cells, Madin-Darby canine kidney cells, COS cells, melanoma cells, and rat kidney epithelial cells but not in fibroblasts. The protein was found in increased amounts in 3T3 cells treated with retinoic acid. These observations suggest that a variety of cells that contact basement membrane contain the proteoglycan-binding protein.     

Immunohistochemical distribution of laminin-5 gamma2 chain and its developmental change in human embryonic and foetal tissues

Immunohistochemical distribution of laminin gamma2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin gamma2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5-6 weeks of gestation. Between 6-7 and 12-13 weeks, laminin gamma2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin gamma2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin gamma2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin gamma2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its gamma2 chain in the morphogenesis of human tissues.     

Immunohistochemical Distribution of Laminin-5 γ 2 Chain and its Developmental Change in Human Embryonic and Foetal Tissues

Immunohistochemical distribution of laminin γ 2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin γ 2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5–6 weeks of gestation. Between 6–7 and 12–13 weeks, laminin γ 2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin γ 2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin γ 2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin γ 2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its γ 2 chain in the morphogenesis of human tissues.     

Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes.

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.     

Laminin forms an independent network in basement membranes [published erratum appears in J Cell Biol 1992 Jun;118(2):493]

Laminin self-assembles in vitro into a polymer by a reversible, entropy-driven and calcium-facilitated process dependent upon the participation of the short arm globular domains. We now find that this polymer is required for the structural integrity of the collagen-free basement membrane of cultured embryonal carcinoma cells (ECC) and for the supramolecular organization and anchorage of laminin in the collagen-rich basement membrane of the Engelbreth-Holm-Swarm tumor (EHS). First, low temperature and EDTA induced the dissolution of ECC basement membranes and released approximately 80% of total laminin from the EHS basement membrane. Second, laminin elastase fragments (E4 and E1') possessing the short arm globules of the B1, B2, and A chains selectively acted as competitive ligands that dissolved ECC basement membranes and displaced laminin from the EHS basement membrane into solution. The fraction of laminin released increased as a function of ligand concentration, approaching the level of the EDTA-reversible pool. The smaller (approximately 20%) residual pool of EHS laminin, in contrast, could only be effectively displaced by E1' and E4 if the collagenous network was first degraded with bacterial collagenase. The supramolecular architecture of freeze-etched and platinum/carbon replicated reconstituted laminin gel polymer, ECC, and collagenase-treated EHS basement membranes were compared and found to be similar, further supporting the biochemical data. We conclude that laminin forms a network independent of that of type IV collagen in basement membranes. Furthermore, in the EHS basement membrane four-fifths of laminin is anchored strictly through noncovalent bonds between laminin monomers while one-fifth is anchored through a combination of these bonds and laminin-collagen bridges.     

Laminin is induced in astrocytes of adult brain by injury.

Laminin is a high mol. wt. non-collagenous matrix glycoprotein, confined in adult tissues to basement membranes. In normal rat brain we found laminin mainly in vessel walls but, after injury, induced by stereotaxic injection of a neurotoxin, laminin immunoreactivity appeared also in reactive astrocytes, which are characteristically positive for the glial fibrillary acidic protein (GFAP). Laminin was first detected in GFAP-immunoreactive glial cells 24 h after injury. Four days later the majority of reactive astrocytes in the gray matter were positive for laminin and the laminin immunoreactivity, but not that of GFAP, gradually subsided within a month. Fibronectin, the other major matrix glycoprotein, was found only in capillary structures both in normal and lesioned brain tissue. The results indicate that mature astrocytes have the potential to produce laminin and suggest a role for this glycoprotein in brain regeneration.     

Mouse heart laminin. Purification of the native protein and structural comparison with Engelbreth-Holm-Swarm tumor laminin

Laminin was selectively extracted from different mouse tissues using EDTA-containing buffer. By immunoblotting with an antiserum raised against mouse Engelbreth-Holm-Swarm (EHS) tumor laminin, such extracts could be shown to contain laminin-like molecules with a low apparent proportion of A chain to B chains. Native laminin was purified from mouse heart tissue and was shown to have an aberrant polypeptide composition as compared to mouse EHS tumor laminin. Most prominently, mouse heart laminin contains an Mr 300,000 polypeptide which is not antigenically related to the A or the B chains. Furthermore, nonreducible polypeptide components were seen with apparent Mr values of 600,000 and 900,000. The Mr 600,000 component contains epitopes shared with both EHS tumor laminin and the Mr 300,000 polypeptide and possibly represents a covalently cross-linked complex of an A or B chain with the Mr 300,000 chain.     

Isolation of a tumor cell laminin receptor

BL6 murine melanoma cells contain approximately 110,000 cell surface binding sites for the basement membrane glycoprotein laminin. Treatment of isolated melanoma cell plasma membranes with detergent yields a single class of laminin receptor. The receptor was purified 900 fold by laminin affinity chromatography. The isolated receptor has a Mr of 67,000 and binds laminin with high affinity: kd = 2 nm. The binding affinity of the isolated receptor was similar to that of the plasma membranes or the whole cells. Such a laminin receptor, isolated here for the first time, could facilitate the interaction of metastasizing tumor cells with the basement membrane.     

Migration by haptotaxis of a Schwann cell tumor line to the basement membrane glycoprotein laminin

Laminin is a large (greater than 850-kdalton) glycoprotein that is localized within basement membranes. Recent work has indicated that this protein is present within the endoneurium of mouse sciatic nerve. Furthermore, it has been shown that a rat Schwannoma cell line, RN22F, produced laminin and that laminin promoted the attachment of these cells to bacterial plastic. This report presents evidence that RN22F cells migrate in vitro to laminin in a concentration-dependent fashion. Laminin was extracted from the mouse EHS tumor and purified by molecular sieve and heparin-agarose affinity chromatography. The migration of Schwannoma cells to laminin, as assessed in a microwell modified Boyden chamber, was inhibited in a dose-dependent manner by affinity-purified antilaminin antibody. Zigmond-Hirsch checkerboard analysis experiments indicated that laminin stimulated both random and directed movement of RN22F cells. Additionally, reversal of the laminin gradient in the chambers also stimulated RN22F migration in a concentration-dependent manner, suggesting that directed migration of RN22F cells was due to a substratum-bound laminin (haptotaxis) as opposed to cell movement in response to fluid-phase laminin (chemotaxis). Binding studies using [3H]laminin demonstrated that laminin bound to the filter surface under the assay conditions used, and support the contention that cells are migrating to substrate-bound material. Furthermore, RN22F cells were shown to migrate on filters coated with laminin in the absence of additional fluid-phase laminin. The magnitude of this response could be altered by changing the relative density of bound laminin. In contrast, fibronectin promoted only marginal migration of RN22F cells. Collectively, these observations indicate that haptotaxis may be a mechanism by which laminin may guide cells during development and raise the possibility that it may be involved in peripheral nervous system myelination.     

Laminin and fibronectin in normal and malignant neuroectodermal cells

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Protease treatment combined with immunohistochemistry reveals heterogeneity of normal and neoplastic basement membranes

Heterogeneity of normal tissue and neoplastic basement membranes was investigated immunohistochemically with monoclonal antibodies and polyclonal antisera to laminin and collagen type IV. Cryostat sections of normal and neoplastic human tissues were digested with bacterial protease or trypsin. The duration of digestion and the concentration of enzyme were varied to determine whether laminin and collagen type IV could be removed differentially from basement membranes from distinct anatomic sites. After digestion, the residual antigenicity of glycoprotein was assessed immunohistochemically. Laminin could be removed more easily from all tissues than could collagen IV, and also much more easily from malignant tumors than from benign tumors or normal tissues. On the basis of susceptibility to proteolytic digestion, basement membranes from normal human tissues were classified as susceptible (e.g., heart and smooth muscle of gastrointestinal tract and uterus), moderately resistant (e.g., nerve, skeletal muscle, epithelial basement membrane of skin, smooth muscle of arteries), and very resistant (e.g., glomerulus). Differential susceptibility to proteolytic digestion most likely reflects quantitative and possibly also qualitative differences in the composition of basement membranes.     

Isolation of a human laminin B2 (LAMB2) cDNA clone and assignment of the gene to chromosome region 1q25----q31

Laminin B2 is one of the three polypeptide chains of laminin, a large, complex glycoprotein synthesized by a variety of cells and specifically deposited in basement membranes. A cloned cDNA that encodes the human laminin B2 chain was isolated and characterized. The human laminin B2 gene (LAMB2) was assigned to region 1q25----q31 by (1) hybridization of the probe to DNA from a panel of human x mouse somatic cell hybrids containing different human chromosomes and (2) in situ hybridization to isolated metaphase chromosomes.     

Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma

We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.     

Ultrastructural localization of fibronectin and laminin in the basement membranes of the murine kidney

Affinity-purified rabbit antibodies specific for two large noncollagenous gycoproteins--laminin and fibronectin--were used to study the distribution of these proteins in normal murine kidneys. Immunofluorescence staining of conventional frozen sections demonstrates fibronectin within mesangial areas of the glomerulus. Laminin is also found in mesangial areas. However, it also appears to be distributed in typical basement membranelike patterns on glomerular and tubular basement membranes and Bowman's capsule. At the ultrastructural level, by labeling 600-800-A thick frozen sections with a three-stage procedure consisting of specific antibodies, biotinyl sheep anti-rabbit IgG, and avidin-ferritin conjugates, fibronectin is present ony in the mesangial matrix and is specifically localized to areas immediately surrounding mesangial cell processes. Laminin, on the other hand, is found uniformly distributed throughout tubular basement membranes, the mesangial matrix, and Bowman's capsule. In glomerular basement membranes, laminin labeling is restricted to the lamina rara interna and adjacent regions of the lamina densa.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.