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.     

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.     

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.     

Fine structure of the glomerular basement membrane and immunolocalization of five basement membrane components to the lamina densa (basal lamina) and its extensions in both glomeruli and tubules of the rat kidney

Electron microscopic immunostaining was used to examine the localization of type IV collagen, laminin, entactin , heparan sulfate proteoglycan, and fibronectin within the basement membranes of the rat kidney. In preliminary experiments, various methods of processing formaldehyde-fixed kidney were compared using antilaminin antiserum and the indirect immunoperoxidase method. Little or no laminin immunostaining of the glomerular basement membrane was present in sections unless they had been frozen-thawed; and even in this case, the immunostaining was light in comparison to that of basement membranes in adjacent tubules. However, when frozen-thawed sections were treated with 0.5% sodium borohydride, immunostaining was then as strong in glomerular as in tubular basement membranes. Accordingly, this treatment was applied to frozen-thawed sections before immunostaining for any of the substances under study. Immunostaining of the glomerular basement membrane for each of the five substances was fairly uniform throughout the lamina densa (also called basal lamina), but uneven in the lamina lucida interna and externa (also called lamina rara interna and externa) in which stained bands extended from the lamina densa. Similarly in the basement membranes of tubules, immunostaining for the five substances was localized to the lamina densa and bands extending into the lamina lucida. When the ultrastructure of the glomerular basement membrane was examined, three structures were found: (1) a network of 4-nm-thick "cords," which seems to be the main component; the cords are closely packed in the lamina densa and more loosely arranged in the lamina lucida interna and externa; (2) straight, hollow 7-10-nm-thick structures referred to as " basotubules "; and (3) 3.5-nm elements composed of minute paired rods, referred to as "double pegs." The distribution of the cords, but not that of the other two structures, was related to the immunostaining pattern. It is concluded that (1) to fully reveal the antigenicity of the glomerular basement membrane, frozen-thawed sections must be treated with sodium borohydride prior to immunostaining, possibly because this basement membrane is more compact than the others; and (2) in both glomerular and tubular basement membranes, type IV collagen, laminin, entactin , heparan sulfate proteoglycan and fibronectin are colocalized in the lamina densa and its extensions to the laminae lucidae . Since the distribution of the cords corresponds to that of immunostaining, it is likely that the five substances are present within the cords.     

Light microscopic immunolocalization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes of a variety of rat organs

Immunohistochemical methods were used to determine whether type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan were present in diverse basement membranes. Antisera or antibodies against each substance were prepared, tested by enzyme-linked immunosorbent assay, and exposed to frozen sections of duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth from rats aged 20 days to 34 months. Bound antibodies were then localized by indirect or direct peroxidase methods for examination in the light microscope. Immunostaining for type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan was observed in all of the basement membranes encountered. Fibronectin was also found in connective tissue. In general, the intensity of immunostaining was strong for type IV collagen and laminin, moderate for heparan sulfate proteoglycan, and weak for fibronectin. The pattern was similar in the age groups under study. Very recently the sulfated glycoprotein, entactin, was also detected in the basement membranes of the listed tissues in 20-day-old rats. It is accordingly proposed that, at least in the organs examined, type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan, and entactin are present together in basement membranes.     

Changes in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

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.     

Immunoelectron microscopy of endothelial cells in rat incisor suggests that most basement membrane components are produced by young cells, whereas heparan sulfate proteoglycan is produced by both young and old cells

When periodontal capillaries of rat incisor tooth were immunostained for four basement membrane components (laminin, collagen IV, fibronectin, heparan sulfate proteoglycan), all four were detected in the secretory organelles of endothelial cells located within 3 mm of the tooth's proximal end, but only the proteoglycan was observed in cells located 4 mm away and beyond (Experiment I). [3H]-Thymidine autoradiography revealed that the endothelial cells located at the tooth's proximal end were young and actively dividing, whereas those located 4 mm or more away were older and generally quiescent (Experiment II). Since immunostaining of a cell's secretory organelles for a given substance indicates production of this substance, the first experiment shows that endothelial cells at the proximal end produce the four basement membrane components. The second experiment discloses that these cells are young. As for the endothelial cells located 4 mm or more beyond the proximal end, the first experiment reveals that they produce only heparan sulfate proteoglycan, while the second shows that they are relatively old. Production of laminin, collagen IV, and fibronectin only by young cells implies that these substances are long-lived and stable components of basement membrane, whereas production of the proteoglycan by both young and old cells implies that it is labile and continually replaced.     

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.     

Location of the integrin complex and extracellular matrix molecules at the chicken myotendinous junction

Summary The distribution of several extracellular matrix macromolecules was investigated at the myotendinous junction of adult chicken gastrocnemius muscle. Localization using monoclonal antibodies specific for 3 basal lamina components (type IV collagen, laminin, and a basement membrane form of heparan sulfate proteoglycan) showed strong fluorescent staining of the myotendinous junction for heparan sulfate proteoglycan and laminin, but not for type IV collagen. In addition, a strong fluorescent stain was observed at the myotendinous junction using a monoclonal antibody against the subunit of the chicken integrin complex (antibody JG 22). Neither fibronectin nor tenascin were concentrated at the myotendinous junction, but instead were present in a fibrillar staining pattern throughout the connective tissue which was closely associated with the myotendinous junction. Tenascin also gave bright fluorescent staining of tendon, but no detectable staining of the perimysium or endomysium. Type I collagen was observed throughout the tendon and in the perimysium, but only faintly in the endomysium. In contrast, type III collagen was present brightly in the endomysium and in the perimysium, but could not be detected in the tendon except when associated with blood vessels and in the epitendineum, which stained intensely. Type VI collagen was found throughout the tendon and in all connective tissue partitions of skeletal muscle. The results indicate that one or more molecules of the integrin family may play an important role in the attachment of muscle to the tendon. This interaction does not appear to involve extensive binding to fibronectin or tenascin, but may involve laminin and heparan sulfate proteoglycan.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by a human malignant cell line

The basal lamina components laminin, heparan sulfate proteoglycan (HSPG), and type IV collagen were synthesized and codeposited in the extracellular matrix (ECM) by a cultured human cell line from gestational choriocarcinoma (JAR). Laminin and HSPG formed a noncovalent complex detected by the coimmunoprecipitation of HSPG with laminin from cell lysates and culture media. The complex was stable in the cell lysis buffer that contained detergents (1% Triton X-100, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate) and sodium chloride (from 0.15 to 1.0 M), but was dissociated by adding 8 M urea to the detergent lysates. Even though JAR cells produced roughly equal amounts of HSPG and chondroitin sulfate proteoglycan, only HSPG complexed with laminin, suggesting a specific interaction between these basal lamina components. The laminin-HSPG complex was deposited and retained in the ECM. This was shown biochemically by isolating an enriched fraction of ECM from JAR cells cultured on native type I collagen gels. At steady state, more than half (52%) of the laminin-HSPG in the culture was recovered in the ECM fraction, in contrast to 16% of the total laminin and 29% of the total type IV collagen, which were secreted to a greater extent than laminin-HSPG into the culture medium. The retention of the laminin-HSPG complex in the ECM suggests that it may participate in the assembly of the basal lamina-like extracellular matrix deposited by JAR cultures. Omission of ascorbate from the culture medium abolished the ECM deposition of type IV collagen but had little effect on the deposition of laminin or laminin-HSPG. This demonstrates that the stable deposition of laminin-HSPG and laminin in the collagen-based choriocarcinoma cultures is not dependent on an assembled network of type IV collagen.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

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.     

Connective tissue of the livers of newborn and adult marmosets (Callithrix jacchus)

Immunofluorescence microscopic and electron microscopic investigations revealed components of the matrix and of the basal lamina (collagen type I, III, IV and V, BL-heparan sulfate and fibronectin) in the sinus wall (Disse's space) of the livers of newborn and adult marmosets (Callithrix jacchus). Collagen type I was missing in both the two age groups. Small amounts of laminin were present in the livers of newborn and absent in those of adult animals, whereas collagen type III occurred in the form of delicate fibres. Light microscopic inspection showed a continuous distribution of all other components in the sinus wall. The amount of collagen type III and V increased depending on the age. Electron microscopic investigations revealed single or bundled fibrils (20-30 nm) and filaments (10-12 nm). After addition of tannic acid, plaques of a fine-filamentous network and incorporated granules were observed. After addition of resting Ruthenium Red, electron-dense granules (20-60 nm) were irregularly distributed in the structureless space, resting on collagenous fibrils and cell membranes. The fibrils were allocated to collagen type III, the filaments to collagen type V. The plaques were supposed to contain heparan sulfate, collagen type IV and fibronectin. The absence of a Lamina densa of the basal lamina was attributed to the absence of laminin which probably plays an important role in the formation of this layer. Differences in the distribution pattern of the matrix components and thus a functional mosaic of the permeability of Disse's space were assumed. The complete absence of collagen type I and laminin in the lobules makes the adult marmoset liver especially suited for studies on the importance of this collagen type under pathological conditions, since both components are expressed in this way.     

Distribution of connective tissue proteins in chick muscle spindles as revealed by monoclonal antibodies: a unique distribution of brachionectin/tenascin

The distribution of chick muscle spindles of eight connective tissue proteins (collagen types I, IV, V, and VI, laminin, heparan sulfate, fibronectin, and brachionectin/tenascin) was examined by immunofluorescent histochemistry. Intrafusal fibers were surrounded by layers of collagen type VI and fibronectin, and by an external lamina containing collagen type IV, laminin, and heparan sulfate. Most of these layers displayed a different pattern of staining at the sensory region of the equator than at the polar region. The crescent-like sheath that caps each intrafusal fiber and sensory terminal at the equator was strongly positive for collagen type I and weakly positive for collagen type V. The outer spindle capsule contained laminin, heparan sulfate, collagen types IV and VI, brachionectin/tenascin, fibronectin, and to a lesser degree also collagen types I and V. Brachionectin/tenascin had the narrowest distribution of any of the connective tissue macromolecules studied. It was found only in the outer capsule and in the coverings of blood vessels and nerves associated with the outer capsule.     

Immunogold quantitation of laminin, type IV collagen, and heparan sulfate proteoglycan in a variety of basement membranes

A series of basement membranes was immunolabeled for laminin, type IV collagen, and heparan sulfate proteoglycan in the hope of comparing the content of these substances. The basement membranes, including thin ones (less than 0.3 micron) from kidney, colon, enamel organ, and vas deferens, and thick ones (greater than 2 micron), i.e., Reichert's membrane, Descemet's membrane, and EHS tumor matrix, were fixed in formaldehyde, embedded in Lowicryl, and treated with specific antisera or antibodies followed by anti-rabbit immunoglobulin bound to gold. The density of gold particles, expressed per micron2, was negligible in controls (less than or equal to 1.1), but averaged 307, 146, and 23, respectively, for laminin, collagen IV, and proteoglycan over the thick basement membranes (except for Descemet's membranes, over which the density was 16, 5, and 34, respectively) and 117, 72, and 64, respectively, over the lamina densa of the thin basement membranes. Lower but significant reactions were observed over the lamina lucida. Interpretation of the gold particle densities was based on (a) the similarity between the ultrastructure of most thick basement membranes and of the lamina densa of most thin basement membranes, and (b) the biochemical content of the three substances under study in the EHS tumor matrix (Eur J Biochem 143:145, 1984). It was proposed that thick basement membranes (except Descemet's) contained more laminin and collagen IV but less heparan sulfate proteoglycan than the lamina densa of thin basement membranes. In the latter, there was a fair variation from tissue to tissue, but a tendency towards a similar molar content of the three substances.     

The basement-membrane-like matrix of the mouse EHS tumor: II. Immunohistochemical quantitation of six of its components

The presence of six substances--laminin, type IV collagen, heparan sulfate proteoglycan, entactin, fibronectin, and the amyloid P component--was investigated immunohistochemically in the matrix of the Engelbreth-Holm-Swarm (EHS) mouse tumor after it had been fixed in formaldehyde (with or without a brief preliminary glutaraldehyde fixation), embedded in Lowicryl K4M, and sectioned for processing through the protein A-gold sequence. Enumeration of the number of gold particles per square micrometer of matrix sections demonstrated that the six substances were present in distinct amounts. The results for each substance were fairly consistent throughout the matrix in three experiments. Furthermore, the available evidence indicated that, with the exception of the amyloid P component, the substances were associated with the cord network of the tumor matrix. Finally, the use of a reconstituted basement membrane containing known amounts of laminin, type IV collagen, and heparan sulfate proteoglycan as a standard, led to the conclusion that, in the tumor matrix, the relative content of laminin to type IV collagen to the proteoglycan was in a ratio of 1:0.6:0.03, suggesting molar ratios of approximately 1:1:0.2, respectively.     

Formation of a supramolecular complex is involved in the reconstitution of basement membrane components

Basement membrane macromolecules, including type IV collagen, laminin, and heparan sulfate proteoglycan, do not aggregate when incubated alone. Rather, precipitation occurs in the presence of equimolar amounts of laminin and type IV collagen but variable amounts of heparan sulfate proteoglycan. This interaction requires native laminin and type IV collagen. Heparan sulfate proteoglycan increases the precipitation of laminin particularly in the presence of type IV collagen. Fibronectin does not cause type IV collagen to precipitate. These studies show that the components of basement membrane interact in a highly specific manner and suggest that such interactions may be involved in the deposition of basement membrane in situ.