Isolation and characterization of hydroxyproline-containing proteins secreted by a murine carcinoma cell culture

A collagenous protein was isolated from a murine carcinoma cell culture, which has been shown to synthesize basement membrane. The molecular weight of this protein was estimated to be 155 000. It eluted from carboxymethyl-cellulose in the region near the alpha 1 and beta 11 components of calf skin collagen. 63--69% of the peptide-bound prolines were hydroxylated, and the 4-/3-hydroxyproline ratios ranged from 12 : 1 to 14 : 1. About 95% of the hydroxylysines in the peptide were glycosylated, and almost all of them were in the glucosylgalactosyl dissacharide form. Judging from the posttranslational characteristics, this collagenous protein is probably of basement membrane type.     

Type IV collagen synthesis by cultured human microvascular endothelial cells and its deposition into the subendothelial basement membrane

Cultured microvascular endothelial cells isolated from human dermis were examined for the synthesis of basement membrane specific (type IV) collagen and its deposition in subendothelial matrix. Biosynthetically radiolabeled proteins secreted into the culture medium were analyzed by sodium dodecyl sulfate gel electrophoresis after reduction, revealing a single collagenous component with an approximate Mr of 180 000 that could be resolved into two closely migrating polypeptide chains. Prior to reduction, the 180 000 bands migrated as a high molecular weight complex, indicating the presence of intermolecular disulfide bonding. The 180 000 material was identified as type IV procollagen on the basis of its selective degradation by purified bacterial collagenase, moderate sensitivity to pepsin digestion, immunoprecipitation with antibodies to human type IV collagen, and comigration with type IV procollagen purified from human and murine sources. In the basement membrane like matrix elaborated by the microvascular endothelial cells at their basal surface, type IV procollagen was the predominant constituent. This matrix-associated type IV procollagen was present as a highly cross-linked and insoluble complex that was solubilized only after denaturation and reduction of disulfide bonds. In addition, there was evidence of nonreducible dimers and higher molecular weight aggregates of type IV procollagen. These findings support the suggestion that the presence of intermolecular disulfide bonds and other covalent interactions stabilizes the incorporation of the type IV procollagen into the basement membrane matrix. Cultured microvascular endothelial cells therefore appear to deposit a basal lamina-like structure that is biochemically similar to that formed in vivo, providing a unique model system that should be useful for understanding microvascular basement membrane metabolism, especially as it relates to wound healing, tissue remodeling, and disease processes.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Interactions of basement membrane components

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

Biosynthesis of two subunits of type IV procollagen and of other basement membrane proteins by a human tumor cell line

The major collagenous component secreted into the medium of cultured HT-1080 tumor cells was identified as type IV procollagen by specific antibodies and characteristic ratios of incorporated labeled 3-hydroxyproline and 4-hydroxyproline. The disulfide-bonded molecules consisted of two subunits, pro-alpha 1(IV) and pro-alpha 2(IV) chains with apparent molecular weights of 180 000 and 165 000. No conversion of the procollagen to collagen or to procollagen intermediates was detected in the cell cultures. The two subunits apparently represent different gene products, since enzymatic digestion of the separated chains produced quite different peptide maps. Pepsin degraded native type IV procollagen successively into several fragments, some still disulfide-linked, giving rise to a complex set of polypeptide chains (Mr = 30 000-140 000). This agrees with similar diverse patterns produced by pepsin from authentic type IV collagens. The ratio between the pro-alpha 1(IV) and pro-alpha 2(IV) chains varied in several experiments between 1.3 and 1.8, suggesting that the two chains belong to different triple-helical molecules. The cells also produced distinct amounts of fibronectin (subunit Mr = 230 000) and of the basement membrane glycoprotein laminin. The latter showed three subunits with Mr = 220 000, 210 000, and 400 000. A further disulfide-bonded, non-collagenous polypeptide (Mr = 160 000) was detected but not yet identified. Immunofluorescence demonstrated these proteins within the cells but not in a pericellular matrix. The production of basement membrane components by HT-1080 cells and lack of interstitial collagens disagree with the original classification of the cell line as a fibrosarcoma.     

Isolation and identification of basement membrane collagen purified from dissociated rabbit renal tubules

In the present study collagens were isolated and identified from morphologically pure basement membrane material. Preparations of rabbit renal tubules devoid of contaminating glomeruli were obtained by homogenization and sieving of kidney cortices. Cellular material was removed by sequential detergent solubilization and the purity of the resultant tubular basement membrane was verified by transmission electron microscopy. The collagenous component of this ultrastructurally pure starting material was isolated by limited pepsinization and salt precipitation. Polyacrylamide gel electrophoresis of this collagen under nonreducing conditions resulted in four major bands: 300,000 (γ component), 100,000 (100K), 80,000 (80K), and 50,000 (50K). Individual collagen fractions of each of these molecular weights were then isolated from preparative polyacrylamide gels. Identification by their electrophoretic properties and cyanogen bromide peptide patterns leads us to believe that: (i) the 100K is composed of the C chain of type IV collagen; (ii) the 80K and 50K are derived from the genetically distinct D chain of type IV collagen; (iii) the γ component is structurally related to the 100K, 80K, and 50K; and (iv) A and B chains (type V collagen) are not major components of rabbit renal tubular basement membranes.     

Characterization of lens capsule collagen: evidence for the presence of two unique chains in molecules derived from major basement membrane structures

A collagen fraction representing two-thirds of the collagenous sequences in bovine lens capsules has been isolated following limited pepsin digestion and purified by DEAE- and carboxymethyl-cellulose chromatography in native form. The denaturation products of this collagen contain two types of components. The more acidic components (C and 50K₁) are, respectively an α-chain-sized collagenous polypeptide and a mixture of smaller molecular weight proteolytic cleavage products of the C chain. The more basic components (80K and 50K₂) represent, respectively, a collagenous polypeptide with an apparent M_r = 80,000 and a mixture of smaller molecular weight components derived through proteolysis of the 80K component. The C chain and 80K components are unique with respect to chromatographic properties, amino acid composition, and cyanogen bromide cleavage products. These data are interpreted to indicate that lens capsule basement membrane collagen molecules collectively contain at least two genetically distinct collagen chains: the C chain representing the collagenous domain of one type of chain and the 80K component representing the major portion of the collagenous domain of a second type of chain, designated the D chain.     

Type VIII collagen from bovine Descemet's membrane: structural characterization of a triple-helical domain

Bovine corneal Descemet's membrane (DM) was subjected to limited pepsin digestion. Soluble native collagens were fractionated by differential salt precipitation, and a mixture of type V collagen and collagenous fragments with a chain Mr of 50,000 (50K) was obtained at a concentration of 1.5 M NaCl. Further purification of the 50K collagen by molecular sieve and high-performance liquid chromatography resulted in the isolation of two-non-disulfide-bonded polypeptides, 50K-A and 50K-B, which were susceptible to several neutral proteases, including bacterial collagenase. By the criteria of peptide mapping, amino acid composition, and N-terminal sequence analysis, 50K-A and 50K-B were structurally dissimilar, although both chains contained Gly-X-Y repeats. 50K-A and 50K-B were immunologically and structurally distinct from collagen type I, III, IV, V, and VI. Immunohistochemical studies of bovine ocular tissue showed preferential distribution of the collagen containing the 50K fragment in the DM, with a more disperse arrangement of apparently interconnecting fibrils in the corneal stroma. Type VIII collagen isolated from the culture medium of metabolically radiolabeled bovine corneal endothelial (BCE) cells and its pepsin-resistant Mr 50 000 domain(s) both cross-reacted with antisera to 50K polypeptides from the corneal DM. Additionally, the CNBr peptide maps of pepsin-resistant Mr 50 000 polypeptides of type VIII collagen isolated from BCE cells and bovine corneal DM were highly similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nonhelical,fibronectin-binding basement-membrane collagen from endodermal cell culture

A novel method of affinity chromatography on insolubilized collagen-binding fragments of fibronectin was utilized to isolate a random-coil collagenous protein from culture media of mouse teratocarcinoma-derived endodermal cells. These cells also produced another collagenous protein, which did not bind to fibronectin but could be isolated by differential salt precipitation. The affinity-purified collagen differs from its conventionally isolated counterpart in that it is not triple-helical in structure, its polypeptides are not disulfide-crosslinked and it has affinity for fibronectin in its native state. Both collagens resemble previously characterized type IV basement-membrane collagens with respect to their amino acid composition, cyanogen bromide peptides, chain size, immunological reactivity and tissue localization. The random-coil collagen is directly active in promoting the attachment of some lines of cells, but for attachment of the endodermal cells addition of fibronectin is required. This suggests that the presence of nonhelical, fibronectinbinding collagen may have biological significance in the interaction of cells with the extracellular matrix.     

Assay for radiolabeled type IV collagen in the presence of other proteins using a specific collagenase

An enzymatic assay is described which quantitates radiolabeled type IV basement membrane collagen in the presence of large amounts of other proteins. A partially purified neutral protease is used which cleaves type IV collagen into fragments at 37°C which are not precipitated at 1.3% (final concentration) trichloroacetic acid-tannic acid. The kinetics of type IV collagen digestion by this enzyme are not significantly altered by the presence of a 10-fold excess of type III collagen. [¹⁴C]Tryptophan-labeled control proteins prepared from fibroblast cultures are not degraded significantly by this protease in the presence of 2.5 mmN-ethylmaleimide. The proportion of type IV collagen in a mixture of labeled placenta collagenous proteins was calculated after separate digestions with the type IV collagenolytic activity and bacterial collagenase: this value compared favorably with the proportion of type IV collagen estimated by gel electrophoresis.     

Degradation of intact basement membranes by human and murine tumor enzymes

Homogenates from malignant tumors, obtained from surgery specimens or from transplants of Walker 256 carcinosarcoma in rats, contained an enzyme activity capable of degrading intact 3H-acetylated basement membranes from bovine lens. The enzyme activity from murine tumor was purified about 7500-fold by (NH4)2SO4 fractionation, ion exchange and gel chromatography. The apparent molecular weight of the purified enzyme was approximately 50,000. The rate of degradation of 3H-labelled basement membrane by the murine tumor enzyme was reduced by addition of excess type IV collagen, but not of excess type I, type III or type V collagen. These results suggested specificity of this enzyme for type IV collagen. Inhibitors of serine proteinases, thiol proteinases and soybean trypsin inhibitor were without effect on the enzyme activity. Chelators such as 1,10-phenanthroline or EDTA reduced the activity to control levels, indicating that the enzyme activity was due to a metalloproteinase. Chromatographic and electrophoretic separation of the enzymatic products from 3H-labelled basement membrane and type IV collagen indicated that the enzyme activity was due to a type IV collagenase. The use of basement membrane in the native physiological state as a substrate for the study of basement membrane-degrading activity by homogenates of solid malignant tumors offers an in vitro model for the investigation of the metastatic potential of these tumors.     

Procollagen production by rat hepatocytes in primary culture

Hepatocytes were obtained from rat liver and maintained in primary culture for periods up to 14 days. Collagen synthesis was maximal after 3–5 days and declined thereafter. The rate of collagen production was appox. one-tenth that observed by the rat skin fibroblasts of the same animals after 3–5 passages. Type I procollagen, the major macromolecular collagenous species, was identified as a 450 000 dalton molecule which was converted to 120 000 dalton, denatured, reduced procollagen chains. Prior pepsin digestion of the native procollagen released 95 000 dalton collagen chains identified as α1(I) and α2(I) by co-migration with carrier rat skin type I collagen chains. The production of type III procollagen was also tentatively identified by DEAE-cellulose chromatography. This material was isolated and identified with type-specific antibodies developed against the amino-terminal extension peptide of bovine skin type III procollagen. The relative distribution of type I:type III procollagen was estimated at 7:3 similar to the ratio previously found in whole rat liver. No evidence of type IV or type V procollagen biosynthesis was observed. These results suggest that rat hepatocytes in primary culture are capable of interstitial type I and type III collagen biosynthesis in a ratio similar to that found in their parent hepatic tissue in situ. They also suggest that the less abundant type IV (basement membrane-associated) or type V are nor major collagenous products of these cells.     

Type IV collagen modulates angiogenesis and neovessel survival in the rat aorta model

Summary Type IV collagen is a major basement membrane component that has been implicated in the regulation of angiogenesis. The purpose of this study was to evaluate the effect of type IV collagen on the angiogenic response of native endothelial cells in three-dimensional vascular organ culture. Rings of rat aorta were cultured under serum-free conditions in gels of type I collagen with or without type IV collagen. In the absence of type IV collagen, aortic rings generated neovessels, which proliferated until day 9 and gradually regressed during the second and third weeks of culture. Type IV collagen promoted neovessel elongation and survival in a dose-dependent manner. Microvascular length increased by 43, 57, and 119% over control values in cultures treated with 3, 30, and 300 μg/ml type IV collagen, respectively. When used at high concentrations (300 μg/ml) type IV collagen stabilized the neovascular outgrowths and prevented vascular regression. Type IV collagen also promoted the formation of neovessels, but significant stimulatory effects were observed only at an intermediate concentration (30 μg/ml) and were no longer significant at the high concentration (300 μg/ml). The observation that type IV collagen has dose-dependent effects on vascular elongation, proliferation, and stabilization, supports the concept that the developing basement membrane of neovessles acts as a solid-phase regulator of angiogenesis, whose function varies depending on the concentration of its molecular components.     

Isolation and partial characterization of a novel basement membrane collagen

A guanidine-HCl extraction of lens capsule basement membrane dissolves collagenous material. This material was fractionated on an Agarose A-5M column. Fractions 1, 2 and 3 were further purified and partially characterized immunochemically and by amino acid analysis. Fraction 3 has a molecular weight of 55,000 when compared with collagen type I standard. The CNBr peptide pattern and composition of fraction 3 are different from those of alpha 1 (IV) 95K and alpha 2 (IV) 95K chains. The results described suggest the presence of a new chain in lens capsule basement membrane.     

Partial purification and characterization of a neutral protease which cleaves type IV collagen

A neutral protease has been extracted from the media of cultured metastatic tumor cells and purified approximately 1000 times after sequential ammonium sulfate fractionization, concanavalin A column chromatography, and molecular sieve chromatography. The protease has an apparent molecular weight of 70 000--80 000, is inactive at acid pH, requires trypsin activation, and is inhibited by ethylene-diaminetetraacetic acid but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide, or soybean trypsin inhibitor. The enzyme produces specific cleavage products for both chains of pro type IV collagen isolated without pepsinization and apparently cleaves at one point in a major pepsin-extracted chain of placenta type IV collagen. The partially purified enzyme fails to significantly degrade other collagens or fibronectin under digestion conditions in which specific reaction products are produced for type IV collagen. The existence of this enzyme is significant since previously described animal collagenases fail to degrade type IV collagen. Such a type IV specific collagenase could play a role in tumor invasion and may be secreted by other cells such as endothelial cells, epithelial cells, and immune cells.     

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

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

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

Assembly of chick and bovine lens-capsule collagen.

Chick-embryo and adult bovine lens-capsular epithelia in organ culture synthesized 4-hydroxy[3H]proline-containing polypeptides when incubated in the presence of [3H]proline. These collagenous polypeptides of apparent Mr 180 000, 175 000 and 160 000 became incorporated with time into aggregates of higher molecular size. The formation of such aggregates was inhibited when the tissues were labelled in the presence of beta-aminopropionitrile, thereby implicating lysine-derived cross-links in aggregate formation. When the tissues were incubated in the presence of tunicamycin, the collagenous polypeptides synthesized exhibited increased electrophoretic mobilities on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The addition to lens-capsule incubation medium of alpha alpha'-bipyridine led to the synthesis of underhydroxylated type IV collagen, also of increased electrophoretic mobility. Extended pulse-chase experiments indicated that such underhydroxylated collagen did not participate in aggregate formation, but was at least as stable as fully hydroxylated non-cross-linked collagen synthesized in the presence of beta-aminopropionitrile. Native type IV collagen, recovered from the culture medium when capsules were incubated with [3H]proline for 24h, was purified by ion-exchange chromatography. Separations conducted on CM-cellulose under denaturing and nondenaturing conditions suggested that the alpha 1(IV) and alpha 2(IV) chains occur in the same heterologous triple helix. Densitometric analyses of appropriate fluorograms indicated that these two polypeptides occur in a 2:1 ratio, suggesting that lens-capsule collagen is synthesized as a triple-helical molecule of composition [alpha 1(IV)]2 alpha 2(IV).     

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.     

Isolation of a type IV collagen binding protein from human platelets.

In order to study the molecular basis of platelet interaction with collagen IV of the basement membrane separating the arterial endothelium from the underlying subendothelial connective tissue, the possibility of presence of platelet membrane protein with affinity to type IV collagen was examined by subjecting the platelet membrane extract to affinity chromatography on collagen IV-sepharose. Urea (4 M) eluate was found to contain a protein with an apparent mol. wt of 68 kDa. The radioiodinated protein was isolated and used to test its specificity. By dot blot assay on nitrocellulose disks and solid-phase assays, the 68 kDa protein was found to bind with high affinity to collagen IV. Lack of significant binding to fibronectin and laminin when compared to albumin control indicated its high specificity for collagen. The radioiodinated protein was inserted into egg yolk lecithin liposomes. While these liposomes attached to microtitre plates coated with collagen IV, there was no significant binding to fibronectin or laminin coated wells, suggesting the membrane associated character of the protein as well as its specificity for collagen. These results indicate that presence of a 68 kDa protein in platelet membrane which interacts with very high specificity to collagen IV.