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H Sage

Publications and source records attributed to H Sage.

At least 19 recordsLinked to original sources
PubMed1999

The compact conformation of fibronectin is determined by intramolecular ionic interactions.

Fibronectin exists in a compact or extended conformation, depending upon environmental pH and salt concentration. Using recombinant fragments expressed in bacteria and baculovirus, we determined the domains responsible for producing fibronectin's compact conformation. Our velocity and equilibrium sedimentation data show that FN2-14 (a protein containing FN-III domains 2 through 14) forms dimers in low salt. Experiments with smaller fragments indicates that the compact conformation is produced by binding of FN12-14 of one subunit to FN2-3 of the other subunit in the dimer. The binding is weakened at higher salt concentrations, implying an electrostatic interaction. Furthermore, segment FN7-14+A, which contains the alternatively spliced A domain between FN11 and 12, forms dimers, whereas FN7-14 without A does not. Segment FN12-14+A also forms dimers, but the isolated A domain does not. These data imply an association of domain A with FN12-14, and the presence of A may favor an open conformation by competing with FN2-3 for binding to FN12-14.

Animals↗
PubMed1997

The copper- and zinc-containing superoxide dismutase from Escherichia coli: molecular weight and stability.

The periplasmic Cu,Zn superoxide dismutase (Cu,ZnSOD) from Escherichia coli has been shown by sedimentation equilibrium to be a monomer with a molecular weight of approximately 17,000. The enzyme suffered a reversible inactivation when heated to 70 degrees C. This was minimized by added Cu(II) or Zn(II). Heat lability was greater in phosphate than in Tris buffer. The enzyme exhibited a time-dependent inactivation by Hg(II) and this too was greater in phosphate than in Tris. This behavior can be explained by a modest affinity of the enzyme for Cu(II) and Zn(II) which results in a dissociation/association equilibrium. Elevation of the temperature shifts this equilibrium toward dissociation and phosphate sequesters the released metals making them less available for reinsertion at the active site. Hg(II) competes for occupancy of the active site and there were more unoccupied sites in phosphate than in Tris. A parallel was drawn between the E. coli Cu,ZnSOD and FALS varients of human Cu,ZnSOD, which are also relatively unstable and exhibit low affinity for Cu(II).

Azides↗
PubMed1995

Angiogenesis.

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Capillaries↗
PubMed1991

Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro.

The response of endothelial cells to angiogenic stimuli has been shown to be influenced by the extracellular microenvironment. We tested whether thrombospondin, an extracellular matrix protein, modulated the spontaneous formation of cords by endothelial cells in vitro. Despite continued proliferation, a decrease in secreted thrombospondin was detected in cord-containing, as compared with subconfluent, cultures of both aortic and microvascular endothelial cells. Consistent with this trend, mRNA levels of thrombospondin decreased by factors of 16 in aortic and 60 in microvascular cultures that contained endothelial cords. Since thrombospondin was immunolocalized to fibrillar arrays that appeared to be associated with endothelial cords, we added anti-thrombospondin IgG to cord-forming cultures to limit the availability of the protein during this process. In the presence of anti-thrombospondin antibodies, there was a 33-50% increase in cord formation. These results suggest that thrombospondin is an inhibitor of angiogenesis in vitro and are consistent with its proposed roles as a destabilizer of endothelial cell focal contacts and as an inhibitor of endothelial cell proliferation.

Animals↗
PubMed1991

Adhesion, shape, proliferation, and gene expression of mouse Leydig cells are influenced by extracellular matrix in vitro.

Interactions between Leydig cells and the extracellular matrix (ECM) within the interstitial compartment of the mammalian testis have not been characterized. We have examined the influence of ECM on adult mouse Leydig cells by culturing the cells on different ECM substrates. Leydig cells adhere weakly to hydrated gels of type I collagen (including those supplemented with collagen types IV, V, or VIII), or to air-dried films of collagen types I, V, or VIII. In contrast, the cells attach firmly to substrates of purified type IV collagen, fibronectin, or laminin. Leydig cells also attach rapidly and adhere strongly to gelled basement membrane matrix derived from the murine Englebreth-Holm-Swarm sarcoma (Matrigel). Leydig cells assume spherical shapes and form aggregates on thick (1.5-mm) layers of Matrigel; however, on thin (0.1-mm) layers, networks of cell clusters linked by cords of elongated cells are formed within 48 h. Similar networks are formed on thick layers of Matrigel that are supplemented with type I collagen. On substrates with high ratios of collagen I to Matrigel or on untreated tissue culture plastic, Leydig cells flatten and do not aggregate. On substrates that induce rounded shapes, proliferation is inhibited and the cells maintain the steroidogenic enzyme 3 beta-hydroxysteroid dehydrogenase for as long as 2 wk. Under conditions where Leydig cells are flattened, they divide and cease expressing the enzyme. Proliferating Leydig cells also exhibit elevated levels of mRNA for SPARC (Secreted Protein, Acidic and Rich in Cysteine), a Ca2(+)-binding glycoprotein associated with changes in cell shape that accompany morphogenesis and tissue remodeling. Our results indicate that the shape, association, proliferation, and expression of gene products by Leydig cells can be significantly affected in vitro by altering the composition of the extracellular substratum.

Animals↗
PubMed1991

Synthesis of type VIII collagen by epithelial cells of human gingiva.

The composition of the extracellular matrix produced by epithelial cells in contact with metabolically inert substrata was studied with antibodies specific for type VIII collagen. Type VIII collagen was present in the cell layer of cultured gingival epithelial cells, and at the epithelium-substratum interface of an explant culture model for junctional epithelium. Samples of the epithelial attachment apparatus (EAA) in vivo were collected by removing the junctional cells directly attached to the tooth (DAT cells) and the associated EAA matrix. The amount of material collected was sufficient for biochemical analysis of both intra- and extracellular components of the junctional cell-EAA complex. Immunological examination of the samples revealed that type VIII collagen was associated with epithelial cells forming the EAA in vivo. We suggest that this collagen type functions in the extracellular space as an attachment-promoting factor for gingival epithelial cells at the tooth surface.

Adult↗
PubMed1991

Differential expression of extracellular proteins is correlated with angiogenesis in vitro.

Strains of bovine aortic endothelial cells, grown on plastic under conventional culture conditions and in the absence of growth factor supplementation, exhibited a sprouting phenotype and a predisposition toward the formation of cords and tubular structures. We examined endothelial cells at different stages of tube formation. Analysis of metabolically labeled proteins showed that the synthesis of type I collagen was initiated in sprouting cells and during the formation of tubular structures. SPARC (secreted protein, acidic and rich in cysteine) a Ca2(+)-binding protein associated with cellular shape change and morphogenetic processes (Sage H, Vernon RB, Funk SE, Everitt EA, Angello J: J Cell Biol 109:341, 1989), was upregulated during spontaneous tube formation. Levels of messenger RNA for type I collagen and SPARC corroborated the stage-specific increases observed for these proteins. Differential levels of transcription were apparent in multilayered cells directly involved in tube formation, in comparison with cells comprising either the tubes or the confluent monolayers at a distance from the tubes. Analysis of DNA synthesis indicated that multilayered sprouting cells in the proximity of the endothelial tubes were actively proliferating, whereas cells that had been incorporated into tubes showed low levels of DNA synthesis. Immunolabeling studies revealed a dense accumulation of SPARC and type I collagen in the cytoplasm of cells that were situated near the growing tubes. Two other secreted proteins, type III collagen and thrombospondin, were expressed constitutively by subconfluent cultures and were increased in those cells contributing to tube formation. We propose that type I collagen and SPARC are specifically related to the angiogenesis-like phenomenon displayed by bovine aortic endothelial cells in vitro. Type I collagen might facilitate the active migration of endothelial cells, or the stabilization of the resulting tubes, with SPARC directing the re-organization and dynamic assembly of the tubular network.

Animals↗
PubMed1990

Type VIII collagen in murine development. Association with capillary formation in vitro.

Bovine endothelial and human astrocytoma cells, and a limited number of other normal and malignant cells, synthesize three chains that have been identified as type VIII collagen (180 kDa, 125 kDa, and 100 kDa). Digestion with pepsin converts these forms to major fragments of 65 kD (based on globular protein standards). In this study we have examined the structure and distribution of type VIII collagen in developing mice by immunohistological and immunoblotting techniques. Temporal and tissue-specific expression was observed in embryonic heart, cranial mesenchyme, and placental capillaries. Western blotting of embryonic and neonatal tissues showed major species of 125 and 65 kDa in the brain, placenta, heart, lung, and thymus. The predominant band in pepsin-treated tissues was 60-70 kDa, with additional forms of 250 and 150 kDa in neonatal heart and lung. Type VIII collagen was also synthesized by endothelial cells, forming capillary tubes in vitro. We suggest that type VIII collagen functions in cellular organization and differentiation, and that its various forms reflect not only tissue-specific processing but the presence of several related chains.

Aging↗
PubMed1989

SPARC: a Ca2+-binding extracellular protein associated with endothelial cell injury and proliferation.

SPARC (Secreted Protein that is Acidic and Rich in Cysteine) is a Ca2+-binding, stress-related protein released in vitro by both malignant and normal cells derived from all primordial germ layers. It is specifically elevated in endothelial cells as a result of "culture shock" (characterized by high levels of proliferation and migration) and exhibits density-dependent secretion. Exposure of bovine aortic endothelial cells to endotoxin results in a 70-100% increase in secreted protein, with a three-fold increase in SPARC. Immunofluorescence histochemistry on mouse tissues revealed (a) a preferential association of SPARC with highly proliferative cells (e.g., gut epithelia, mammary gland, and epidermis), (b) a cell surface or stromal location in thymus, lung, and salivary gland, (c) staining of epididymidal epithelium and testicular cells, and (d) an association with somites of 14 d mouse embryos. We envision SPARC as an extracellular modulator of Ca2+ and other cation-sensitive proteins/proteinases, that facilitates cellular proliferation in response to injury and to developmental signals.

Animals↗
PubMed1989

SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca+2-dependent binding to the extracellular matrix.

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a Ca+2-binding glycoprotein that is differentially associated with morphogenesis, remodeling, cellular migration, and proliferation. We show here that exogenous SPARC, added to cells in culture, was associated with profound changes in cell shape, caused rapid, partial detachment of a confluent monolayer, and inhibited spreading of newly plated cells. Bovine aortic endothelial cells, exposed to 2-40 micrograms SPARC/ml per 2 x 10(6) cells, exhibited a rounded morphology in a dose-dependent manner but remained attached to plastic or collagen-coated surfaces. These round cells synthesized protein, uniformly excluded trypan blue, and grew in aggregates after replating in media without SPARC. SPARC caused rounding of bovine endothelial cells, fibroblasts, and smooth muscle cells; however, the cell lines F9, PYS-2, and 3T3 were not affected. The activity of native SPARC was inhibited by heat denaturation and prior incubation with anti-SPARC IgG. The effect of SPARC on endothelial cells appeared to be independent of the rounding phenomenon produced by the peptide GRGDSP. Immunofluorescence localization of SPARC on endothelial cells showed preferential distribution at the leading edges of membranous extensions. SPARC bound Ca+2 in both amino- and carboxyl-terminal (EF-hand) domains and required this cation for maintenance of native structure. Solid-phase binding assays indicated a preferential affinity of native SPARC for several proteins comprising the extracellular matrix, including types III and V collagen, and thrombospondin. This binding was saturable, Ca+2 dependent, and inhibited by anti-SPARC IgG. Endothelial cells also failed to spread on a substrate of native type III collagen complexed with SPARC. We propose that SPARC is an extracellular modulator of Ca+2 and cation-sensitive proteins or proteinases, which facilitates changes in cellular shape and disengagement of cells from the extracellular matrix.

Animals↗
PubMed1989

The calcium-binding protein SPARC is secreted by Leydig and Sertoli cells of the adult mouse testis.

In mammals, polypeptides secreted by cells of the testis are believed to influence spermatogenesis and to affect the behavior of the resident somatic cell populations. The 43,000-MW, secreted, calcium-binding glycoprotein SPARC (Secreted Protein, Acidic and Rich in Cysteine) is synthesized by a number of embryonic, fetal, and adult somatic cells and is associated with areas of cellular differentiation, proliferation, and morphological reorganization. Here, we report on the expression of SPARC in the testes of adult mice. By immunohistochemistry, SPARC was observed in the cytoplasm of Leydig cells and of Sertoli cells bearing late-stage, elongate spermatids. Testicular mRNA, translated in vitro, yielded a polypeptide of approximately 42,000 MW that bound anti-SPARC antibodies. Northern blot analysis revealed 2.3 kilobase (kb) SPARC mRNA in the testis, a size comparable to that of SPARC mRNA in nongonadal cells. Western blot assays of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an immunoreactive polypeptide of 43,000 MW in purified mouse Sertoli cells and their culture supernatants. Similar assays of testis interstitial fluid revealed 43,000 MW and 30,000 MW immunoreactive polypeptides. By indirect immunofluorescence, purified mouse Leydig cells cultured 24-48 h expressed SPARC in cytoplasmic granules. Cultured Leydig cells incorporated [35S]methionine into a secreted polypeptide of 43,000 MW that was recognized by anti-SPARC antibodies. In metal binding assays, purified SPARC bound Ca2+, Fe2+ and Cu2+. The function of SPARC in testes may be to sequester or transport certain metallic cations. Our recent discovery that SPARC induces changes in shape of certain nongonadal cell types also suggests that this glycoprotein may influence the functions of both Leydig and Sertoli cells by affecting their morphology.

Animals↗
PubMed1989

Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice.

SPARC (Secreted Protein that is Acidic and Rich in Cysteine), a Ca++-binding glycoprotein also known as osteonectin, is produced in significant amounts by injured or proliferating cells in vitro. To elucidate the possible function of SPARC in growth and remodeling, we examined its distribution in embryonic and adult murine tissues. Immunohistochemistry on adult mouse tissues revealed a preferential association of SPARC protein with epithelia exhibiting high rates of turnover (gut, skin, and glandular tissue). Fetal tissues containing high levels of SPARC included heart, thymus, lung, and gut. In the 14-18-day developing fetus, SPARC expression was particularly enhanced in areas undergoing chondrogenesis, osteogenesis, and somitogenesis, whereas 10-day embryos exhibited selective staining for this protein in Reichert's membrane, maternal sinuses, and trophoblastic giant cells. SPARC displayed a Ca++-dependent affinity for hydrophobic surfaces and was not incorporated into the extracellular matrix produced by cells in vitro. We propose that in some tissues SPARC associates with cell surfaces to facilitate proliferation during embryonic morphogenesis and normal cell turnover in the adult.

Animals↗
PubMed1987

Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues.

SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.

Amino Acid Sequence↗
PubMed1987

Distinct patterns of glycosylation of colligin, a collagen-binding glycoprotein, and SPARC (osteonectin), a secreted Ca2+-binding glycoprotein. Evidence for the localisation of colligin in the endoplasmic reticulum.

Mouse parietal endoderm PYS cells were labelled with [2-3H]mannose for 16-24 h. Colligin, an Mr-47000 collagen-binding protein, and SPARC, a Mr-43000 protein, highly homologous to the Ca2+-binding protein osteonectin, were isolated from labelled cell extracts and culture medium respectively. Glycopeptides obtained by exhaustive digestion with pronase were analysed by lectin-affinity, ion-exchange, and gel-filtration chromatography and by paper chromatography of high-mannose oligosaccharides after endo H release. The results show that the N-linked carbohydrate chains of colligin are exclusively the high-mannose type, of which (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2 make up 77%. This carbohydrate structure provides strong evidence that colligin is a component of the endoplasmic reticulum, and argues against a role in cell-surface interactions. By contrast to colligin, SPARC secreted by PYS cells contains predominantly a diantennary complex type of chain containing a variable number of sialic acid and core-substituted fucose residues. Similar glycosylation patterns to those discussed above were seen in colligin isolated from primary mouse embryonic parietal endoderm cells and the murine 3T3 cell line, and in SPARC secreted by bovine corneal endothelial cells. Unlike the type-IV-collagen-binding glycoprotein studied by Dennis, J., Waller, C. and Schirrmacher, V. [J. Cell Biol. 99, 1416-1423 (1984)], removal of N-linked oligosaccharides from colligin had no effect on its binding to native type IV collagen.

Animals↗
PubMed1987

Extracellular matrix-induced synthesis of a low molecular weight collagen by fetal calf ligament fibroblasts.

Fetal calf ligamentum nuchae fibroblasts, cultured from animals of different gestational age, synthesize a unique, low molecular weight collagen termed FCL-1 (Sage, H., Mecham, R., Johnson, C., and Bornstein, P., 1983, J. Cell Biol. 97:1933-1938). Previous studies on the elastogenic differentiation of these cells in vitro demonstrated that the extracellular matrix (ECM) protein elastin was specifically induced in undifferentiated fibroblasts when they were grown on ligament ECM isolated from animals at later stages of development (Mecham, R.P., Madaras, J.G., and Senior, R.M., 1984. J. Cell Biol. 98:1804-1812). To investigate the expression of FCL-1 as a function of developmental age, we grew fetal calf ligament fibroblasts from an 85 d (first trimester) animal (FCL 85d) on three different substrata: ligament from a 120 d (second trimester) animal, ligament from a 270 d (term) animal, and unmodified plastic tissue culture dishes. FCL 270d fibroblasts were grown on plastic substrata and served as a differentiated cellular control. Analysis of metabolically radiolabeled proteins from both the culture media and the cell layers showed that the synthesis of FCL-1 was selectively increased in those cells cultured on ligament ECM. For FCL 85d fibroblasts grown on 120 d and 270 d ligaments, FCL-1 comprised 17% and 22%, respectively, of the culture medium proteins that precipitated at concentrations of ammonium sulfate from 20-50%. FCL 85d and 270d fibroblasts grown on plastic substrata yielded values of 2.5% and 1.0%, respectively. This effect appeared to be specific for this collagen and did not reflect a general increase in the synthesis of connective tissue proteins of the ECM (e.g., types I and III procollagen). As percent of total newly-synthesized cellular protein, the output of FCL-1 was 10-fold higher by FCL 85d cells grown on 270d ligament ECM (5.8%) as compared to that of the same cellular population grown on a plastic surface (0.56%). The presence of the ligament ECM also altered the levels and distribution of secreted proteins between the culture medium and the cell layer. These studies provide evidence for differential expression of the novel collagen FCL-1 by FCL fibroblasts during development and suggest that such expression is affected, at least in part, by interaction of the cell with a ligament ECM.

Animals↗
PubMed1986

Structural characteristics and intermolecular organization of human pulmonary-surfactant-associated proteins.

The structural relationships and intermolecular organization among the proteins associated with pulmonary surfactant are largely unknown. We studied the pulmonary-surfactant-associated proteins in the bronchoalveolar lavage fluid obtained from a patient with the clinical syndrome of alveolar proteinosis. The major proteins with Mr values of 32,000-36,000 and 62,000 formed thiol-dependent complexes (Mr greater than 400,000) with intermolecular disulphide bonds present in the collgenase-sensitive domains of these proteins. In contrast, other proteins, which were collagenase-insensitive, formed thiol-dependent oligomers that were not covalently linked to the major proteins. The associations of these proteins in the surfactant of a normal individual were similar. By amino acid analysis, two-dimensional peptide mapping and bacterial-collagenase digestion the 32,000-36,000-Mr and 62,000-Mr proteins were nearly identical. Differences in CNBr cleavage products suggested that the larger of the proteins was formed by non-disulphide, covalent, cross-links in the collagenase-sensitive domains of the 32,000-36,000-Mr proteins. Thus the evidence suggested that the lipid-associated proteins of Mr 32,000-36, 000 contained both disulphide and non-disulphide cross-links in the collagen-like N-terminal region of the proteins and form higher-Mr complexes. This organization may support the three-dimensional conformation of surfactant in the alveolar space.

Amino Acids↗
PubMed1986

Interaction of platelets and purified collagens in a laminar flow model.

Damage to the endothelial surface of the vessel wall can result in exposure of circulating blood components to collagen and other subendothelial structures. Collagen types I, III, IV, and V have been demonstrated in the vessel wall by chemical and immunohistological methods; type V is thrombin-sensitive, and is present on the endothelial cell surface. In an earlier study using a rocking model, both unstimulated and ADP-induced platelet adherence was reduced on wells coated with type V collagen in comparison to uncoated wells; and increased on plastic surfaces coated with types III and IV collagen in comparison to those coated with type V collagen. The present study was designed to determine the effect of erythrocytes and shear rate on platelet adherence to these purified collagen types in a laminar flow system. With platelet-rich plasma, adherence of labeled platelets was much lower in the laminar flow system compared with the rocking model. Erythrocytes significantly enhanced platelet adherence to surfaces that were untreated or absorbed with collagen types I, III, and IV. However, this enhancement was not seen in the presence of type V collagen. These studies provide additional evidence for the selectively nonthrombogenic nature of type V collagen.

Animals↗