Characterization of a novel calcium-binding 90-kDa glycoprotein (BM-90) shared by basement membranes and serum

The protein BM-90 was solubilized from the mouse Engelbreth-Holm-Swarm tumor with neutral buffers in molar yields lower (15-30%) than found for other basement membrane proteins (e.g. laminin, BM-40). The purified protein was shown to be rich in cysteine (5 mol%) and to change in SDS electrophoresis from an 84-kDa position to a 95-kDa one upon reduction. BM-90 was also shown to be a calcium-binding protein. The N-terminal sequence of BM-90, as well as those of several internal peptides, showed no identity with any known protein sequences, indicating that it is a new protein. Specific radioimmunoassays showed no or only minor cross-reactions with other known basement membrane proteins. Immunological assays demonstrated BM-90 to be present in neutral salt extracts from mouse heart and kidney, in serum (20-40 micrograms/ml) and in the medium of various cultured cells (0.1-1 microgram/ml). The protein in these samples was identical in size to BM-90 purified from the tumor, indicating that negligible degradation occurs during purification. An extracellular matrix localization of BM-90 was shown by immunofluorescence for Reichert's membrane, lens capsules and other basement membranes. Thus, BM-90 appears to be a novel basement membrane protein whose functions remain to be studied.     

In vitro production of Reichert's membrane by mouse embryo-derived parietal endoderm cell lines

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth-Holm-Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.     

Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for collagen affinity

We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.     

In vitro production of Reichert's membrane by mouse embryo-derived parietal endoderm cell lines

We report the isolation of eight independent cell lines from preimplantation mouse embryos, which have a parietal endoderm phenotype. When grown as aggregates, these cell lines produce large amounts of a basement membrane matrix, that contains laminin, nidogen, heparan sulfate proteoglycan, collagen IV, and BM-40. The biosynthetic profiles of all eight cell lines are very similar to parietal endoderm cells in vivo which synthesize Reichert's membrane. The structure of the matrix produced by the parietal endoderm cell lines (PEC lines) resembles more closely Reichert's membrane than the Engelbreth—Holm—Swarm (EHS) tumor in susceptibility to proteolytic degradation. Since these cell lines produce large quantities of basement membrane they will be useful for structural and functional comparison of a Reichert's membrane matrix with the basement membrane produced by the EHS tumor.     

Calcium-dependent binding of basement membrane protein BM-40 (osteonectin, SPARC) to basement membrane collagen type IV

Basement membrane protein BM-40, prepared from the mouse Engelbreth-Holm-Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM-40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium-dependent binding of BM-40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal-ion-chelating or chaotropic agents, high salt and reduction of disulfide bonds in BM-40. Fragment studies indicated that domains III (alpha-helix) and/or IV (EF hand) of BM-40 possess the binding site(s) for collagen IV, while the N-terminal domains I and II provide the major antigenic determinants. A major BM-40-binding site on collagen IV was dependent on a triple-helical conformation and could be localized to a pepsin fragment from the central portion of the triple-helical domain, in agreement with electron microscopic visualization of BM-40--collagen-IV complexes.     

Immunochemical and tissue analysis of protease generated neoepitopes of BM-40 (osteonectin, SPARC) which are correlated to a higher affinity binding to collagens.

Proteolytic cleavage at single sites in the extracellular calcium-binding module of BM-40/SPARC/osteonectin either by an unknown endogenous protease (L197-L198) or several matrix metalloproteinases (E196-L197) was previously shown to enhance collagen binding activity 10-fold. Polyclonal rabbit antibodies were now obtained against synthetic peptide antigens containing either an N-terminal L197 or L198 and characterized by radioimmunoassay, ELISA, immunoblots and immunohistology. These neoepitope-specific antibodies reacted with proteolytically processed but not with uncleaved mouse and human BM-40. The cross-reaction between the two different neoepitopes was < 1%, indicating the immunodominant role of the N-terminal residues. Analysis of a basement membrane producing mouse tumor demonstrated extensive cleavage at the L198 site, which correlated with a calcium-dependent binding to the matrix. A variable degree of this cleavage was also detected in BM-40 obtained from adult mouse bone and several other tissues. Negligible or much lower levels of conversion were detected at the MMP-specific L197 site, however. Immunogold staining of mouse heart and a basement membrane-producing mouse tumor showed a distinct extracellular labeling for BM-40 and the L198 neoepitope but only a very weak reaction for the L197 neoepitope. This strongly indicates that these neoepitopes are generated in vivo and emphasizes a specific biological role for the proteolytic activation of BM-40.     

Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies.

Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another.     

The extracellular matrix glycoproteins BM-90 and tenascin are expressed in the mesenchyme at sites of endothelial-mesenchymal conversion in the embryonic mouse heart

Abstract. BM-90 is a novel glycoprotein initially isolated from the extracellular matrix of a mouse tumor. We here studied the expression of BM-90 during embryonic development of the mouse heart and compared its expression pattern with that of tenascin and laminin. Distribution was studied by immunofluorescence using antibodies specifically raised against mouse BM-90, laminin and tenascin. Some expression of BM-90 was seen in myocardial basement membranes at early developmental stages, but expression abruptly decreased from these sites at day 12 of embryogenesis. Laminin B chains were also found in the muscle basement membranes early but did not decrease with advancing development. The most striking observation was the markedly enriched expression of BM-90 in the endocardial cushion tissue (ECT). The ECT is derived from mesenchymal cells converted from endothelium and they will form the cardiac valves and septa. In the ECT, BM-90 showed considerable co-distribution with tenascin, but tenascin expression was more focal and did not mark all areas of the ECT. Northern blot data show that BM-90 and tenascin were produced by the developing heart. With antibodies detecting A, B1 and B2 chains of mouse laminin, no immunoreactivity was seen in the ECT. Our data thus show clear-cut differences in the molecular composition of the ECT and muscle basement membranes in the developing heart. The focal expression of BM-90 in the ECT suggests that BM-90 could be involved in epithelial-mesenchymal transitions.     

Differences in the solubility and susceptibility to proteolytic degradation of basement-membrane components in adult and embryonic mouse tissues

We have studied susceptibility of basement membranes in a variety of tissues to solubility in guanidine hydrochloride and to proteolytic degradation by trypsin and thermolysin. Unfixed sections from embryonic and adult mouse tissues and the EHS tumor were subjected to solvent buffers or digested with enzymes. The retention or disappearance of the basement-membrane components nidogen, laminin, collagen IV, and heparan sulfate proteoglycan was subsequently assayed by immunofluorescence. Our data showed that in all tissues nidogen was the most readily solubilized component and the most susceptible to proteolytic degradation. With few exceptions, nidogen in embryonic tissues was more susceptible to degradation than that in adult tissues, and this correlated well with the susceptibility of the other basement-membrane components to be degraded. We conclude that basement membranes differ quite markedly in their solubility and their susceptibility to proteolytic degradation and that these properties reflect differences in their molecular structure.     

Glomerular basement membrane proteoglycans are derived from a large precursor

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.     

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.     

Identification of the precursor protein to basement membrane heparan sulfate proteoglycans

The precursor protein of a basement membrane specific heparan sulfate proteoglycan has been identified as a 400,000 Mr polypeptide. Antibodies against large and small forms of this proteoglycan, isolated from a basement membrane (Engelbreth-Holm-Swarm, EHS) tumor, immunoprecipitated the same 400,000 protein from pulse-labeled EHS cells. The proteoglycan precursor protein was not recognized by antibodies against other basement membrane components or by antibodies to the cartilage proteoglycan. Furthermore, heparan sulfate proteoglycan purified from the EHS tumor blocked the immunoprecipitation of the precursor protein. Pulse-chase studies with [35S]methionine showed the precursor protein was converted to a proteoglycan. Pulse-chase studies with 35SO4 showed the large, low density proteoglycan appeared first and was degraded to a smaller, high density proteoglycan. We propose that the precursor protein is used after very little or no modification in the assembly of a large, low density heparan sulfate proteoglycan and that a portion of the population of these macromolecules are subsequently degraded to a smaller form.     

Solubilization of protein BM-40 from a basement membrane tumor with chelating agents and evidence for its identity with osteonectin and SPARC

Up to 80% of the calcium-binding protein BM-40 could be extracted from a tumor basement membrane with a physiological buffer containing 10 mM EDTA. About half of its amino acid sequence was determined by Edman degradation demonstrating identity with cDNA deduced sequences of bone osteonectin and SPARC.     

Establishment and characterization of a parietal endoderm-like cell line derived from Engelbreth-Holm-Swarm tumor (EHSPEL), a possible resource for an engineered basement membrane matrix.

Engelbreth-Holm-Swarm (EHS) tumor produces large amounts of basement membrane (BM) components, which are widely used as cell culture substrates mimicking BM functions. EHS tumor arose spontaneously in an ST/Eh strain mouse and has been propagated by transplantation. In the present study, we established a cell line, EHSPEL (EHS Parietal Endoderm-Like), which can be cultured ex vivo and preserves the capacity to form tumors in vivo. EHSPEL cells secreted large amounts of laminin-1 into the medium and deposited BM components onto dishes. To further characterize EHSPEL cells, their gene expression profile was compared to those of parietal endoderm cells from Reichert's membrane at embryonic day 13.5, differentiated F9 embryonal carcinoma cells, and PYS-2 parietal endoderm cells. These analyses outlined not only common features of parietal endoderm-like cells that underlie the efficient production of BM components, but also germline cell-like features of EHSPEL cells, at least some of which may play crucial roles in their capacity to form tumors that accumulate abundant BM components in vivo. Karyotyping of EHSPEL cells using chromosome painting probes showed a large number of interchromosomal rearrangements and partial chromosome hyperploidy. Exogenous introduction of a human laminin-alpha(4)-EGFP fusion protein into EHSPEL cells resulted in the production and deposition of human-mouse-hybrid laminin-8. This strategy should be applicable for creating efficient systems to produce chimeric laminins as well as BM-like gels with modified biological activity.     

Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115- residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.     

Immunohistochemical evidence for the intracellular formation of basement membrane collagen (type IV) in developing tissues

Antibodies to type IV collagen obtained from the basement membrane of the mouse EHS tumor were incubated with sections of rat incisor teeth and other tissues for immunostaining by direct or indirect methods. In all locations, the immunostaining was pronounced in basement membranes in which it was restricted to the "basal lamina" layer, from which "bridges" often extended to nearby basal laminae. Usually no immunostaining was detectable in the cells associated with the basement membranes. However, examination of the capillaries at the posterior extremity of the rat incisor tooth, where tissues are at an early stage of development, showed immunostaining not only of the basement membrane, but also of the endothelial cells. The staining was localized in rough endoplasmic reticulum cisternae, some Golgi saccules and their peripheral distensions, and structures believed to be secretory granules. These findings suggest that the synthesis of type IV collagen proceeds along the classical secretory pathways through rough endoplasmic reticulum and Golgi apparatus. At the same time, immunostaining was usually lacking in the cells of the capillaries that had migrated about 2 mm away from the posterior end of the incisor tooth and also in the cells of most other tissues examined, even though the associated basal laminae were reactive. It is, therefore, presumed that the production of type IV collagen may be high in cells at an early stage of development and that any later production and turnover of basement membrane collagen can only be minimal.     

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.     

Recombinant expression and properties of the human calcium-binding extracellular matrix protein BM-40.

A cDNA construct (approximately 1 kb) of human BM-40 in a plasmid with the cytomegalovirus promoter and enhancer was used to produce several stable clones by transfecting two human cell lines (293, HT 1080). These clones showed a high expression of exogenous 1-kb BM-40 mRNA and no or only little endogenous 2.2-kb mRNA. These clones also secreted BM-40 at high rates (5-50 micrograms ml-1 day-1) into serum-free culture medium as shown by electrophoresis, radioimmunoassay and metabolic labelling. Transfection with the plasmid and overexpression of BM-40 had no effect on cell spreading, proliferation rate and adhesion patterns to extracellular matrix substrates. Recombinant human BM-40 was purified by anion-exchange chromatography and showed the expected N-terminal sequence and amino acid composition. The protein was also identical or similar to authentic BM-40 purified from the mouse Engelbreth-Holm-Swarm tumor in hexosamine content, electrophoretic mobility, circular dichroism and binding activity for calcium and collagen IV. Reduction of both authentic and recombinant BM-40 decreased binding activity which indicates correct formation of disulfide bonds in the recombinant protein. A specific and sensitive radioimmunoassay for human BM-40 was shown to be useful for detecting small quantities of the protein in human cell culture medium and blood. No significant cross-reaction was, however, detected between human and mouse BM-40.     

Characterization of a novel glycoprotein isolated from the basement membrane matrix of the Engelbreth-Holm-Swarm tumor

A previously undescribed protein has been isolated and purified from the extracellular matrix of the Engelbreth-Holm-Swarm (EHS) tumor, a murine tumor that synthesizes an extensive matrix composed of basement membrane molecules. Molecular characterization of the molecule determined that it is a glycoprotein with internal disulfide bonds and an isoelectric point of 6.0. Electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the glycoprotein migrated as a diffuse band with a molecular weight of approximately 72,000-80,000. The amino acid composition was significantly different from known basement membrane components. Polyclonal antibodies that specifically recognize the glycoprotein localized it to the kidney glomerular basement membrane. These antibodies did not cross-react with either known basement membrane components (laminin, type IV collagen, and heparan sulfate proteoglycan), with 70K "culture shock" protein or with components of normal mouse serum (including mouse transferrin, albumin, or alpha-fetoprotein), when analyzed by "Western" immunoblots. Our data indicate that the glycoprotein is synthesized by the EHS tumor cells and is present at relatively high levels in the EHS tumor matrix.