Recombinant Human Collagen XV Regulates Cell Adhesion and Migration

The C-terminal end of collagen XV, restin, has been the focus of several studies, but the functions of full-length collagen XV have remained unknown. We describe here studies on the production, purification, and function of collagen XV and the production of a monoclonal N-terminal antibody to it. Full-length human collagen XV was produced in insect cells using baculoviruses and purified from the cell culture medium. The yield was 15 mg/liter of cell culture medium. The collagen XV was shown to be trimeric, with disulfide bonds in the collagenous region. Rotary shadowing electron microscopy revealed rod-like molecules with a mean length of 241.8 nm and with a globular domain at one end. The globular domain was verified to be the N-terminal end by N-terminal antibody binding. The molecules show flexibility in their conformation, presumably due to the many interruptions in their collagenous domains. The ability of collagen XV to serve as a substrate for cells was tested in cell adhesion assays, and it was shown that cells did not bind to collagen XV-coated surfaces. When added to the culture medium of fibroblasts and fibrosarcoma cells, however, collagen XV rapidly bound to their fibronectin network. Solid phase assays showed that collagen XV binds to fibronectin, laminin, and vitronectin and that it binds to the collagen/gelatin-binding domain of fibronectin. No binding was detected to fibrillar collagens, fibril-associated collagens, or decorin. Interestingly, collagen XV was found to inhibit the adhesion and migration of fibrosarcoma cells when present in fibronectin-containing matrices.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.     

Human term placenta contains transforming growth factors

Growth factors of apparent molecular weights of 6,000, 10,000, 20,000 and one in excess of 30,000 daltons can be isolated from acid-ethanol extracts of human term placentas. Each size class of growth factor resembles transforming growth factor (TGF) in that it stimulates anchorage independent growth of normal rat kidney cells and competes with EGF for binding to EGF membrane receptors. The 6,000, 10,000 and 20,000 molecular weight polypeptides also resemble TGF in their acid and heat stability, and their requirement for intact disulfide bonds for growth promoting activity. By homologous radioimmunoassay, neither the 6,000 nor 10,000 dalton polypeptide is related to human epidermal growth factor (hEGF). The presence of these TGFs in ample concentrations (approximately 100 ng of EGF equivalents per term placenta for the 10,000 dalton polypeptide) indicates the usefulness of this tissue source for study of human TGFs.     

Fibronectin-Binding Proteins of a Human Oral Spirochete Treponema denticola

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000-daltons (53-kDa) and 72-kDa surface antigenic proteins and a 38-kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38-kDa axial flageller protein.     

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).     

Characterization of U small nuclear RNA-associated proteins

Differential immunoaffinity chromatography using a combination of autoimmune antibodies allows for the rapid bulk separation of specific small nuclear ribonucleoproteins (snRNPs). Passage of a HeLa cell extract over a column constructed of human anti-Sm autoantibodies results directly in the elution of complexes containing the small nuclear RNA species, U1, U2, U4, U5, and U6, and nine major polypeptides of molecular weight 69,000, 32,000, 27,000, 26,000, 18,500, 13,000, 11,000 doublet, and less than 10,000. Passage of crude extracts through a column bearing murine monoclonal antibodies directed against the 69,000 molecular weight (U1)RNP peptide gives an enriched population of U1 snRNP particles in the retained material. When the flowthrough material from the (U1)RNP column is passed through an anti-Sm column, the retained material is enriched in U2, U4, U5 plus U6 snRNP complex. The 69,000, 32,000, and 18,500 molecular weight polypeptides are confined to the U1 fraction while the remaining proteins are recovered in both fractions. The procedure is simple and rapid, producing complexes with a high degree of resolution and in sufficient yield to provide a ready source of snRNP complexes for functional studies.     

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.     

Fibronectin and fibrinogen degradation products stimulate PMN-leukocyte and mast cell degranulation.

The ability of various peptides cleaved by plasmin from human fibrinogen and fibronectin or fibrinogen- and fibronectin- related synthetic peptides to induce histamine release from mast cells and collagenase and elastase from PMN-leukocytes was examined. Low molecular weight fibrinogen degradation products showed dose dependent secretion of collagenase. These peptides (mol. wt. 1.4 kD) at the concentration of 10(-5) M released about 47% of collagenase and 13% of elastase. Synthetic fibrinopeptides A and B had a similar strong collagenase releasing potency and also released histamine from mast cells. Peptides from plasmin digestion of fibronectin containing cell attachment site with sequence Arg-Gly-Asp-Ser and also synthetic peptide reproducing this amino-acid sequence at the concentration of 1000 micrograms/ml released about 50% of collagenase and 55% of elastase from PMN-leukocytes. Moreover peptides containing cell attachment and gelatin binding site induced histamine release from mast cells. The association of fibrinogen and fibronectin degradation with activation of mast cells may motivate the treatment with antihistaminic drugs of all pathological conditions where the intensive protein degradation takes place.     

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.     

Synthesis and retention of fibronectin (LETS protein) by mouse teratocarcinoma cells.

Mouse teratocarcinoma cells in culture were examined for both the synthesis (by metabolic labelling) and surface accumulation (by indirect immunofluorescence) of fibronectin, a glycoprotein with subunits of molecular weight 220000 D known to form part of the extracellular matrix of many cells in vivo. Although lines of both pluripotent and nullipotent embryonal carcinoma cells synthesize the protein and release it into the medium, they do not retain it on their surfaces. Monolayers of the endoderm line PSA5-E both synthesize fibronectin and lay it down in an extracellular network. A line of PYS parietal endoderm cells does not retain surface fibronectin, although it does accumulate other extracellular matrix material. When pluripotent embryonal carcinoma cells differentiate into cystic embryoid bodies, fibronectin accumulates in a basement membrane below the outer endoderm cells (both visceral and parietal-like) and may play a transient role in organizing the inside cells into an epithelial layer.     

Replication of simian herpesvirus SA8 and identification of viral polypeptides in infected cells

The replication of the simian herpesvirus SA8 in Vero cells was examined. The time course of replication of the simian herpesvirus SA8 was found to be similar to that of the herpes simplex viruses. Infectious progeny virions were first detectable by 6 h postinfection and were readily released into the extracellular fluids beginning at 9 h postinfection. All cell lines tested, with the exception of Madin-Darby canine kidney cells, were permissive for SA8. Analysis of SA8-infected cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed over 40 infected cell polypeptides ranging in molecular weight from 158,000 to less than 10,000. Of these proteins, 23 were present in virions. Three classes of infected cell polypeptides could be identified based on the kinetics of their synthesis. Post-translational processing of several SA8-induced proteins was also observed in pulse-chase experiments. Six distinct SA8-specific glycoproteins ranging from 118,000 to 19,500 daltons were also identified in infected cells. Of these glycoproteins, five were present in virions.     

The stimulation of normal human melanocyte proliferation in vitro by melanocyte growth factor from bovine brain

Cell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10(-9) M), hydrocortisone, (5 X 10(-5) M), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10(-10) M), and bovine brain extract (150 micrograms/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor-supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]-Dopa incorporation. The growth factor-supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline-labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co-cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilities of human melanoma.     

Application of sodium dodecyl sulfate-gel electrophoresis to low molecular weight polypeptides

Experiments with nine polypeptides with molecular weights between 2000 and 10,760 confirm the value of sodium dodecy sulfate (SDS)-gel electrophoresis for separating polypeptides in this molecular weight range. In one case, electrophoretic blotting and microsequencing were successfully carried out. However, molecular weight determination in the low molecular weight range (less than 10,000) is much less reliable than that in the conventional molecular weight range (greater than 10,000) for SDS gels. Information provided by suppliers of horse heart myoglobin fragment kits is potentially misleading.     

Dimeric character of fibronectin, a major cell surface-associated glycoprotein

Exposed proteins of cultured chick and human fibroblasts were labeled by lactoperoxidase-catalyzed iodination and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Extracts from both cell types contained the characteristic, heavily labeled band of fibronectin (molecular weight = 2.2 × 10⁵) when analyzed after reduction with 2-mercaptoethanol. Without prior reduction, however, the 2.2×10⁵ molecular weight band was missing and replaced by labeled bands of 4.4×10⁵ and of very high molecular weight. This finding indicates that fibroblast cell-surface fibronectin, like the fibronectin purified from plasma, is composed of two high molecular weight polypeptides hed together by disulfide bonds, and suggests that the dimer may in addition form disulfide-bonded multimers.     

High-level production of human acidic fibroblast growth factor in E. coli cells: inhibition of DNA synthesis in rat mammary fibroblasts at high concentrations of growth factor.

Recombinant human acidic fibroblast growth factor has been produced in E. coli cells at a level of at least 50 mg/l culture. The recombinant and natural acidic fibroblast growth factors are almost identical to one another when tested on rat mammary fibroblasts for their ability to stimulate DNA synthesis, to bind to the high-affinity surface receptors of the cells and to inhibit DNA synthesis when present in the culture medium at high concentrations. The recombinant acidic fibroblast growth factor binds to two cell-surface polypeptides of molecular masses 160 kDa and 140 kDa, which are the same size as the receptors for basic fibroblast growth factor, and it binds preferentially to the smaller polypeptide.     

Factors influencing the release of proteins by cultured schwann cells

Cultured rat schwann cells grown in association with sensory neurons when labeled with [(3)H]leucinem, [(3)H]glucosamine, or [(35)S]methionine release labeled polypeptides into the culture medium. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of more than 20 polypeptides with molecular weights ranging from 15,000 to more than 250,000. Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for approximately 40 percent of the leucine or methionine radioactivity in medium polypeptide. Schwann cells grown in a serum-free defined medium, in which schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides tha cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides. Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE. This change in the pattern of polypeptides release by schwann cells is accompanied by changes in the association between schwann cells and axons. An early step in the establishment of normal axon-schwann cell relations appears to be an inward migration of schwann cells into axonal bundles and spreading of schwann cells along neurites. These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by schwann cells may be important for the development of normal axonal ensheathment.     

Species-specific monoclonal antibodies in the assignment of the gene for human fibronectin to chromosome 2.

Using three different species-specific monoclonal antibodies we have studied, in human-mouse and human-hamster somatic cell hybrids, the correlation between the presence of different human chromosomes and the ability to release human fibronectin into the tissue culture medium. Presence of human fibronectin was determined by an affinity-radioimmunoassay. In addition, tissue culture media of the different hybrids were separated on SDS-polyacrylamide gels, the proteins were blotted onto a nitrocellulose sheet and human fibronectin visualized by an immunoenzymatic technique. Karyology and determination of isoenzyme markers of specific human chromosomes show that the ability to produce human fibronectin segregated with the presence of human chromosome 2.     

Effect of substances of a polypeptide nature isolated from the thymus, cerebral cortex and substantia alba on the cellular and humoral indices of immunity in thymectomized mice]

Effects of low molecular weight polypeptides (M. W. lower 10,000) isolated from the calf thymus, cortex and white matter of the brain by extraction with acetic acid on the cellular and humoral immune responses were studied in experimental thymectomized mature CBA mice. Thymectomy reduced markedly the number of T-cells in the spleen. Accordingly, the ability to generate both Ig M and IgG antibody forming cells as well as humoral antibodies to thymus-dependent antigen, SRBC, was significantly suppressed in the animals. Subcutaneous administration of 1 micron/g (body weight) of the thymus and brain cortex polypeptides during 8 days not only completely restored T-cells population in the spleen and immune responsibility but also elevated these values 1.5-2 fold in comparison with sham controls which had been given saline solution. The preparation from the white matter of the brain lacked biological activity.