Structure, composition, and assembly of basement membrane

Basement membranes are thin layers of matrix separating parenchymal cells from connective tissue. Their ultrastructure consists of a three-dimensional network of irregular, fuzzy strands referred to as "cords"; the cord thickness averages 3-4 nm. Immunostaining reveals that the cords are composed of at least five substances: collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin. Collagen IV has been identified as a filament of variable thickness persisting after the other components have been removed by plasmin digestion or salt extraction. Heparan sulfate proteoglycan appears as sets of two parallel lines, referred to as "double tracks," which run at the surface of the cords. Laminin is detected in the cords as diffuse material within which thin wavy lines may be distinguished. The entactin and fibronectin present within the cords have not been identified as visible structures. The ability of laminin, heparan sulfate proteoglycan, fibronectin, and entactin to bind to collagen IV has been demonstrated by visualization with rotary shadowing and/or biochemical studies. Incubation of three of these substances-collagen IV, laminin (with small entactin contamination), and proteoglycan-at 35 degrees C for 1 hr resulted in a precipitate that was sectioned for electron microscopic examination and processed for gold immunolabeling for each of the three incubated substances. Three structures are present in the precipitate: 1) a lacework, exclusively composed of heparan sulfate proteoglycan in the form of two parallel lines, similar to double tracks; 2) semi-solid, irregular accumulations, composed of the three initial substances distributed on a cord network; and 3) convoluted sheets, which are also composed of the three initial substances distributed on a cord network but which, in addition, have the uniform appearance and thickness of the lamina densa of basement membrane. Hence these sheets are closely similar to the main component of authentic basement membranes.     

Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. Affinity fractionation on columns of platelet factor 4, plasma fibronectin, and octyl-sepharose

Both newly formed and long-term culture-generated substratum adhesion sites, generated by EGTA-mediated detachment of Balb/c SVT2 cells, were extracted with an eta-octyl-beta-D-glucopyranoside buffer containing salt and several protease inhibitors under conditions which result in maximal solubilization of the sulfate-radiolabeled proteoglycans. Because of the functional importance of heparan sulfate proteoglycans in the fibronectin-dependent cell-substratum adhesion processes of these cells, these proteoglycans were fractionated on affinity columns of octyl-Sepharose or of the heparan sulfate-binding proteins platelet factor 4 or plasma fibronectin. These affinity matrices resolved a number of both binding and nonbinding classes of heparan sulfate proteoglycan from both types of adhesion sites. In particular, the platelet factor 4 column could resolve several proteoglycans with differing binding affinities. Approximately twice as much heparan sulfate proteoglycan from newly formed sites bound to all three matrices as proteoglycan from longterm sites. The proteoglycan which bound to one matrix was then tested for binding to a second matrix; this approach resolved a number of biochemically distinct species. For example, one-half of the fibronectin-Sepharose-binding fraction from the long-term sites could also bind to platelet factor 4-Sepharose; however, over 90% of the fibronectin-binding fraction from newly formed sites could bind to platelet factor 4. A major portion of the octyl-Sepharose-binding fractions of the original extracts could bind to fibronectin-Sepharose. These studies indicate that some of these proteoglycans have overlapping affinities for fibronectin, platelet factor 4, and octyl-Sepharose and that a portion of the heparan sulfate proteoglycan from these adhesion sites cannot bind to any of these affinity matrices. These results are discussed with regard to the functional significance of these various heparan sulfate proteoglycans in mediating adhesion to extracellular matrices containing fibronectin or platelet factor 4.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Biological activity of a proteoglycan form of macrophage colony-stimulating factor and its binding to type V collagen.

Two different types of macrophage colony-stimulating factors (M-CSF) were found, one with an apparent molecular mass of 85 kDa and the other greater than 200 kDa. The high molecular mass M-CSF was identified as a proteoglycan carrying chondroitin sulfate glycosaminoglycan and was designated as the proteoglycan form of M-CSF (PG-M-CSF). In this study, we compared the biological activity of the 85-kDa M-CSF and PG-M-CSF and examined the binding properties of these two M-CSF to certain extracellular matrix proteins, i.e. types I-V collagen and fibronectin, using a modified enzyme-linked immunosorbent assay. PG-M-CSF was capable of supporting the formation of murine macrophage colonies, and pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate, did not alter its colony-stimulating activity. The specific activity of PG-M-CSF was similar to that of the 85-kDa M-CSF. The 85-kDa M-CSF had no apparent affinity for the extracellular matrix proteins examined, whereas PG-M-CSF had an appreciable binding capacity to type V collagen, but did not bind to types I, II, III, and IV collagen or to fibronectin. Pretreatment of PG-M-CSF with chondroitinase AC completely abolished the binding of the species to type V collagen. Addition of exogenous chondroitin sulfate inhibited the binding of PG-M-CSF to type V collagen in a dose-dependent manner. These data indicated that the interaction between PG-M-CSF and type V collagen was mediated by the chondroitin sulfate chain of PG-M-CSF. PG-M-CSF bound to type V collagen could stimulate the proliferation of bone marrow macrophages, indicating that the matrix protein-bound PG-M-CSF retained its biological activity. This interaction between PG-M-CSF and type V collagen implies that the role of PG-M-CSF may be distinct from that of 85-kDa M-CSF.     

Lack of heparan sulfate proteoglycan in a discontinuous and irregular placental basement membrane

A discontinuous basement membrane of variable width that surrounds spongiotrophoblast cells of rat placenta was examined for the presence of type IV collagen, laminin, a heparan sulfate proteoglycan, entactin, and fibronectin using monospecific antibodies or antisera and the indirect peroxidase technique. At the level of the light microscope, the basement membrane was immunostained for type IV collagen, laminin, entactin, and fibronectin. Heparan sulfate proteoglycan immunostaining, however, was virtually absent even after pretreatment of sections with 0.1 N acetic acid, pepsin (0.1 microgram/ml) or 0.13 M sodium borohydride. Examination in the electron microscope confirmed the lack of immunostaining for heparan sulfate proteoglycan, whereas the other substances were mainly localized to the lamina densa part of the basement membrane. The absence of heparan sulfate proteoglycan in this discontinuous and irregular basement membrane even though type IV collagen, laminin, entactin, and fibronectin are present, suggests that heparan sulfate proteoglycan may have a structural role in the formation of basement membrane.     

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of binding sites for laminin, heparan sulfate proteoglycan and fibronectin on basement membrane (type IV) collagen

Rotary shadowing electron microscopy was used to examine complexes formed by incubating combinations of the basement membrane components: type IV collagen, laminin, large heparan sulfate proteoglycan and fibronectin. Complexes were analyzed by length measurement from the globular (COOH) domain of type IV collagen, and by examination of the four arms of laminin and the two arms of fibronectin. Type IV collagen was found to contain binding sites for laminin, heparan sulfate proteoglycan and fibronectin. With laminin the most frequent site was centered approximately 81 nm from the carboxy end of type IV collagen. Less frequent sites appeared to be present at approximately 216 nm and approximately 291 nm, although this was not apparent when the sites were expressed as a fraction of the length of type IV collagen to which they were bound. For heparan sulfate proteoglycan the most frequent site occurred at approximately 206 nm with a less frequent site at approximately 82 nm. For fibronectin, a single site was present at approximately 205 nm. Laminin bound to type IV collagen through its short arms, particularly through the end of the lateral short arms and to heparan sulfate proteoglycan mainly through the end of its long arm. Fibronectin bound to type IV collagen through the free end region of its arms. Using a computer graphics program, the primary laminin binding sites of two adjacent type IV collagen molecules were found to align in the "polygonal" model of type IV collagen, whereas with the "open network" model, a wide meshed matrix is predicted. It is proposed that basement membrane may consist of a lattice of type IV collagen coated with laminin, heparan sulfate proteoglycan and fibronectin.     

Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: electron microscopic localization

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules.     

The incubation of laminin, collagen IV, and heparan sulfate proteoglycan at 35 degrees C yields basement membrane-like structures

Three basement membrane components, laminin, collagen IV, and heparan sulfate proteoglycan, were mixed and incubated at 35 degrees C for 1 h, during which a precipitate formed. Centrifugation yielded a pellet which was fixed in either potassium permanganate for ultrastructural studies, or in formaldehyde for Lowicryl embedding and immunolabeling with protein A-gold or anti-rabbit immunoglobulin-gold. Three types of structures were observed and called types A, B, and C. Type B consisted of 30-50-nm-wide strips that were dispersed or associated into a honeycomb-like pattern, but showed no similarity with basement membranes. Immunolabeling revealed that type B strips only contained heparan sulfate proteoglycan. The structure was attributed to self-assembly of this proteoglycan. Type A consisted of irregular strands of material that usually accumulated into semisolid groups. Like basement membrane, the strands contained laminin, collagen IV, and heparan sulfate proteoglycan, and, at high magnification, they appeared as a three-dimensional network of cord-like elements whose thickness averaged approximately 3 nm. But, unlike the neatly layered basement membranes, the type A strands were arranged in a random, disorderly manner. Type C structures were convoluted sheets composed of a uniform, dense, central layer which exhibited a few extensions on both surfaces and was similar in appearance and thickness to the lamina densa of basement membranes. Immunolabeling showed that laminin, collagen IV, and proteoglycan were colocalized in the type C sheets. At high magnification, the sheets appeared as a three-dimensional network of cords averaging approximately 3 nm. Hence, the organization, composition, and ultrastructure of type C sheets made them similar to the lamina densa of authentic basement membranes.     

Chondroitin sulfate proteoglycan (PG-M-like proteoglycan) is involved in the binding of hyaluronic acid to cellular fibronectin

Preparations of cellular fibronectin from chick embryonic fibroblasts have previously been shown to have hyaluronate-binding activity. However, gel filtration and CsCl isopycnic centrifugation of fibronectin preparations showed that the binding activity was associated with molecules with a density and a molecular weight higher than those of fibronectin. An immunoprecipitation assay using antibodies to the chondroitin sulfate proteoglycan (PG-M) from the mesenchyme of chick embryo limb bud showed that the hyaluronate-binding activity of fibronectin preparations was precipitable with this antibody. The immunoprecipitation analyses also showed that fibronectin preparations as well as conditioned culture medium and extracts of chick embryonic fibroblasts contained a chondroitin sulfate proteoglycan, the protein-enriched core molecules from which were identical to those from PG-M with respect to electrophoretic mobility and immunological reactivity. This proteoglycan was purified from conditioned culture medium and extracts of fibroblasts by dissociative CsCl isopycnic centrifugation. The proteoglycans from medium or extracts gave core derivatives with electrophoretic mobility identical to those from PG-M, and they had equal hyaluronate-binding activities. These results, taken together, suggest that most, if not all, of the hyaluronate-binding activity in preparations of chick cellular fibronectin is due to a proteoglycan identical to PG-M. This proteoglycan was also found to bind directly to fibronectin and to type I collagen, but not to laminin or type IV collagen. It is possible that the fibroblast proteoglycan mediates interactions between hyaluronate, fibronectin, and type I collagen, thereby participating in formation of the pericellular matrix of fibroblasts.     

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.     

Laminin promotes formation of cord-like structures by Sertoli cells in vitro

Basement membranes are thin extracellular matrices which contact epithelial cells and promote their adhesion, migration, differentiation, and morphogenesis. These matrices are composed of collagen IV, heparan sulfate proteoglycan, laminin, and entactin as well as other minor components. Sertoli cells, like most epithelial cells, are in contact at their basal surface with a basement membrane. When cultured within three-dimensional basement membrane gels (Matrigel), Sertoli cells reorganize into cords that resemble testicular seminiferous cords found in the in vivo differentiating testis. Anti-laminin and anti-entactin antisera inhibit this cord morphogenesis by Sertoli cells whereas antisera against type IV and type I collagen, heparan sulfate proteoglycan, fibronectin, and preimmune sera had no effect. The RGD (RGDS-NH2) sequence, found in the cell binding domain of the integrin family of cell adhesion molecules as well as in the A chain of laminin and in entactin, effectively inhibited Sertoli cell cord formation at a concentration of 1.0 mg/ml but was unable to prevent Sertoli cell attachment at concentrations as high as 2.0 mg/ml. A synthetic pentapeptide from a cell-binding domain of the B1 chain of laminin. YIGSR-NH2, inhibited cord formation at a concentration of 0.25 mg/ml, but Sertoli cells were still adherent to the basement membrane matrix. At concentrations greater than 0.50 mg/ml, Sertoli cells detached. Antiserum against the YIGSR-NH2-containing sequence was also effective in inhibiting cord formation by Sertoli cells. Ligand (YIGSR-NH2 peptide) blot analysis of Sertoli cell lysates revealed an interaction with a major band at 60 kDa and with minor bands at 39 and 127 kDa. Furthermore, in Western blot analysis the anti-67-kDa laminin-binding protein antibody recognized a 59- to 60-kDa protein in Sertoli cells. The data indicate that laminin is involved in both Sertoli cell attachment and migration during formation of histotypic cord structures by these cells in culture. Two separate laminin cell-binding domains appear to be involved in Sertoli cell cord morphogenesis in vitro and are likely to participate in the formation of seminiferous cords in vivo.     

Basement membrane matrix in vitro: focal binding of exogenous fibronectin to the matrix of teratocarcinoma-derived endodermal cells

Interaction of exogenous fibronectin with the basement membrane-like PYS-2 cell matrix, lacking fibronectin and hyaluronic acid but containing heparan sulfate proteoglycan, was studied in vitro. Both human plasma fibronectin and fibronectin in fetal calf serum bound to PYS-2 matrix; also, fragments of fibronectin containing heparin-binding domains but lacking the collagen-binding domain bound to the matrix. In immunoelectron microscopy the bound fibronectin was found as 20-40 nm globules or patches. Distribution of fibronectin differed from that of laminin and correlated best with that of heparan sulfate proteoglycan. The results suggest that the binding of fibronectin to basement membrane matrices is not due to random adherence but involves specific interactions with other components.     

A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.     

Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of fibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (alpha 1 subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM.     

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.     

Binding of von Willebrand factor to the small proteoglycan decorin

The small proteoglycan decorin plays an important role in the organisation of the extracellular matrix by binding to several components, including collagen and fibronectin. In this work, we report the dose-dependent and saturable interaction of decorin with the adhesive glycoprotein, von Willebrand factor (VWF). This interaction was mediated by the glycosaminoglycan side chain of decorin and was critically regulated by the degree of sulfation, but not by the amount of iduronic acid. Both chondroitin sulfate and dermatan sulfate, in addition to heparin, were found to bind VWF equally well. Although soluble decorin prevented VWF binding to heparin, purified VWF-A1 domain failed to interact with the proteoglycan. These results identify VWF as a new partner for the small proteoglycan, decorin, in the structural organisation of the extracellular matrix.     

Localization of laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan in chick retinal pigment epithelium basement membrane during embryonic development

Type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin were localized in the basement membrane (BM) of chick retinal pigment epithelium (RPE) during various stages of eye development. At different times over a 4-17 day period after fertilization, chick embryo eyes were dissected, fixed in periodate-lysine-paraformaldehyde, and 6 micron frozen sections through the central regions of the eye were prepared. Sections were postfixed in -20 degrees C methanol and stained immediately by indirect immunofluorescence using sheep anti-mouse laminin, sheep antimouse type IV collagen, rabbit anti-mouse heparan sulfate proteoglycan, and mouse monoclonal anti-porcine plasma fibronectin. Fluorescein-labeled F(ab')2 fragments of the appropriate immunoglobulins (IgGs) were used as secondary antibodies. Laminin could be readily demonstrated in the BM of the RPE during all stages of development. The staining for type IV collagen, fibronectin, and heparan sulfate proteoglycan HSPG) was less intense than that for laminin, but was also localized in the BM along the basal side of the RPE. In addition to staining the BM, antiserum to HSPG, gave a diffuse labeling from day 9 onward, above the RPE extending into the region of the photoreceptors. Whereas the intensity of staining generally increased between day 4 and day 17 of development, the distribution of the different BM components did not change. Hence the presence of type IV collagen, laminin, fibronectin, and HSPG in the BM of RPE in vivo during all the stages of development investigated supports the concept that these macromolecules are important basic components of this, and other, BMs. Furthermore, these results indicate that the composition of the BM of RPE cells in vivo is similar to the BM material deposited by RPE cells in vitro (Turksen K, Aubin JE, Sodek JE, Kalnins VI: Collagen Rel Res, 4:413-426, 1984) and that the in vitro cultures can therefore serve as a useful model for studying BM formation.