A protein of the basal lamina of the sea urchin embryo

The purification, biochemical characterization and functional features of a novel extracellular matrix protein are described. This protein is a component of the basal lamina found in embryos from the sea urchin species Paracentrotus lividus and Hemicentrotus pulcherrimus . The protein has been named PI-200 K or Hp-200 K, respectively, because of the species from which it was isolated and its apparent molecular weight in SDS-PAGE under reducing conditions. It has been purified from unfertilized eggs where it is found packed within cytoplasmic granules, and has different binding affinities to type I collagen and heparin, as assessed by affinity chromatography columns. By indirect immunofluorescence experiments it was shown that, upon fertilization, the protein becomes extracellular, polarized at the basal surface of ectoderm cells, and on the surface of primary mesenchyme cells at the blastula and gastrula stages. The protein serves as an adhesive substrate, as shown by an in vitro binding assay where cells dissociated from blastula embryos were settled on 200K protein-coated substrates. To examine the involvement of the protein in morphogenesis of sea urchin embryo, early blastula embryos were microinjected with anti-200K Fab fragments and further development was followed. When control embryos reached the pluteus stage, microinjected embryos showed severe abnormalities in arms and skeleton elongation and patterning. On the basis of current results, it was proposed that 200K protein is involved in the regulation of sea urchin embryo skeletogenesis.     

Spicule matrix protein LSM34 is essential for biomineralization of the sea urchin spicule.

Biomineralized skeletal structures are composite materials containing mineral and matrix protein(s). The cell biological mechanisms that underlie the formation, secretion, and organization of the biomineralized materials are not well understood. Although the matrix proteins influence physical properties of the structures, little is known of the role of these matrix proteins in the actual formation of the biomineralized structure. We present here results using an antisense oligonucleotide directed against a spicule matrix protein, LSM34, present in spicules of embryos of Lytechinus pictus. After injection of anti-LSM34 into the blastocoel of a sea urchin embryo, LSM34 protein in the primary mesenchyme cells decreases and biomineralization ceases, demonstrating that LSM34 function is essential for the formation of the calcareous endoskeletal spicule of the embryo. Since LSM34 is found primarily in a specialized extracellular matrix surrounding the spicule, it is probable that this matrix is important for the biomineralization process.     

Localization and functional role of a 41 kDa collagenase/gelatinase activity expressed in the sea urchin embryo

The egg storage compartment of the sea urchin embryo was investigated for a protein destined for export to the extracellular matrices. Using an antiserum prepared against a 41 kDa collagenase/gelatinase localized to the extraembryonic matrices (the hyaline layer and basal lamina), the egg storage compartment was mapped for this antigen. Indirect immunofluorescence analysis revealed the 41 kDa collagenase/gelatinase in the cortical granules as well as a second compartment which was dispersed throughout the egg cytoplasm. High resolution immunogold labeling defined this cytoplasmic compartment as the yolk granule organelle. Gelatin substrate gel zymography revealed the presence of a 41 kDa gelatin cleavage activity in purified yolk granules. These results suggest a role for yolk granules in regulated protein export and challenge the traditional view of this organelle as a benign storage compartment for nutrients. In additional experiments, embryos grown in the presence of the 41 kDa cleavage activity or the anti-41 kDa antiserum had severely delayed gut formation and spicule elongation. These results demonstrate a requirement for defined levels of the 41 kDa activity in the extracellular matrices of the developing embryo.     

On the ultrastructure of hyalin, a cell adhesion protein of the sea urchin embryo extracellular matrix

Hyalin is a large (ca. 350 x 10(3) kD by gel electrophoresis) molecule that contributes to the hyalin layer surrounding the sea urchin embryo. In previous work a mAb (McA Tg-HYL), specific for hyalin, was found to inhibit cell-hyalin adhesion and block morphogenesis of whole embryos (Adelson, D. L., and T. D. Humphreys. 1988. Development. 104:391-402). In this report, hyalin ultrastructure was examined via rotary shadowing. Hyalin appeared to be a filamentous molecule approximately 75-nm long with a globular "head" about 12 nm in diameter that tended to form aggregates by associating head to head. Hyalin molecules tended to associate with a distinct high molecular weight globular particle ("core"). In fractions containing the core particle often more than one hyalin molecule were seen to be associated with the core. The core particle maintained a tenacious association with hyalin throughout purification procedures. The site(s) of McA Tg-HYL binding to the hyalin molecule were visualized by decorating purified hyalin with the antibody and then rotary shadowing the complex. In these experiments, McA Tg-HYL attached to the hyalin filament near the head region in a pattern suggesting that more than one antibody binding site exists on the hyalin filament. From the ultrastructural data and from the cell adhesion data presented earlier we conclude that hyalin is a filamentous molecule that binds to other hyalin molecules and contains multiple cell binding sites. Attempts were made to demonstrate the existence of lower molecular weight hyalin precursors. Whilst no such precursors could be identified by immunoprecipitation of in vivo labeled embryo lysates, immunoprecipitation of in vitro translation products suggested such precursors (ca 40 x 10(3) kD) might exist.     

Nogo-B mediates HeLa cell adhesion and motility through binding of Fibulin-5

Nogo-B is a known regulator of neural functions and plays an important role in cell adhesion and migration. To our knowledge, the molecular mechanism behind its regulation of cell motility is still unknown. Here, we identified Fibulin-5, a secreted extracellular matrix protein, as a binding partner of Nogo-B. Using HeLa cells as a model, we found that Nogo-B and Fibulin-5 co-localize in the cytoplasm and plasma membrane. Furthermore, in HeLa cells that overexpress Nogo-B, cell migration and invasion was promoted by the elevated secretion of Fibulin-5. Thus, identification of the Nogo-B binding protein Fibulin-5 may contribute to uncover the pathway in which Nogo-B regulates tumor cell movement.     

Proteolytic processing of a sea urchin, ECM-localized protein into lower mol mass species possessing collagen-cleavage activity

The hyaline layer is an apically located extraembryonic matrix, which blankets the sea urchin embryo. Using gelatin substrate gel zymography, we have identified a number of gelatin-cleaving activities within the hyaline layer and defined a precursor-product processing pathway which leads to the appearance of 40- and 38-kDa activities coincident with the loss of a 50-kDa species. Proteolytic processing of the precursor required the presence of both CaCl2 and NaCl at concentrations similar to those found in sea water. The cleavage activities utilized both sea urchin and rat tail tendon gelatins as substrates but demonstrated a species-specific cleavage activity towards sea urchin collagen. The gelatin-cleaving activities were refractory to inhibition by 1,10-phenanthroline but were inhibited by benzamidine. This latter result defines the serine protease nature of the cleavage activities. Both the 40- and 38-kDa activities were found to comigrate with gelatin-cleaving activities present in the sea urchin embryo.     

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus

Summary Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, -methyl-d-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by -methyl-d-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.     

Fibronectin,not laminin,mediates heparin-dependent heparin-binding growth factor type I binding to substrata and stimulation of endothelial cell growth

Summary Fibronectin and heparin-binding growth factors (HBGF) are essential for growth of cultured endothelial cells. The stimulation of endothelial cell growth by HBGF type one (HBGF-1) in particular requires heparin or a similar glycosaminoglycan. The requirement for fibronectin and heparin for HBGF-1-stimulated endothelial cell growth may be related. HBGF-1 absorbed to the natural subcellular matrix of endothelial cells supports cell growth. [¹²⁵I]HBGF-1 specifically associates with a sequentially reconstituted matrix of collagen-fibronectin-heparin, and HBGF-1 absorbed to the reconstituted matrix supports growth of the endothelial cells. A reconstituted matrix of collagen-laminin-heparin neither supported binding of [¹²⁵I]HBGF-1 nor HBGF-1-stimulated endothelial cell growth. Association kinetics of [¹²⁵I]HBGF-1 to heparinlike sites and membrane receptor sites on endothelial cell monolayers suggest that fibronectin-heparinlike binding sites in the subcellular matrix may be an obligatory reservoir of active HBGF-1 that binds to specific cell membrane receptors. This work was carried out in the laboratory of Dr. W. L. McKeehan and supported in part by grants CA37589, DK35310 and DK38639 from the Public Health Service, Department of Health and Human Services, Washington, DC.     

Initial analysis of the molecular image of pamlin, a sea urchin cell adhesion protein, by transmission electron microscopy

Pamlin, an important extracellular protein required early for sea urchin embryogenesis, is readily isolated from the embryos of Hemicentrotus pulcherrimus . A molecular image analysis of pamlin was conducted using immuno-electron microscopy, rotary shadowing and negative staining technique-applied electron microscopy. The electron microscopy showed that a monoclonal antibody to the pamlin α-subunit bound to a position 13.5 nm from one end of a purified 255 kDa pamlin molecule, which is a 132 nm long and 6.8 nm wide linear structure. The pamlin structure is composed of three subunits, a 47 nm long 52 kDa α-subunit that attaches to one end of a 105 nm long 180 kDa β-subunit, and a 15.6 nm diameter globular 23 kDa γ-subunit that binds to the middle of the β-subunit. The α- and β-subunits together form a 125–140 nm linear structure. Intermolecular aggregation frequently occurred between the free end of two β-subunits of the αβγ pamlin molecule, leaving the entire α-subunit surface free. Occasionally associations between the ends of α-subunits, or between an α-subunit and the middle of a β-subunit also occurred, but no aggregations of pamlin formed through the γ-subunit. These homophilic molecular aggregations of pamlin formed a large supramolecular network. In addition, the single pamlin molecule rounded at one end under high calcium ion concentration to form a 'loop', suggesting the presence of a calcium sensitive region in the molecule.     

A new extracellular matrix protein of the sea urchin embryo with properties of a substrate adhesion molecule

Summary A new embryonic extracellular matrix protein has been purified from eggs of the sea urchin Paracentrotus lividus. The molecule is a 210 kD dimer consisting of two 105 kD subunits that are held together by S-S bridges. In the unfertilized egg, the protein is found within granules uniformly distributed throughout the cytoplasm. After the egg is fertilized, the antigen is polarized to the apical surface of ectodermal and endodermal cells during all of the developmental stages examined, until the pluteus larva is formed. The protein promotes the adhesion of blastula cells to the substrate and is antigenically distinct from echinonectin, a well characterized substrate adhesion molecule. This report adds a new candidate to the list of known extracellular matrix molecules for the regulation of differentiation and morphogenesis in the sea urchin embryo. Offprint requests to: V. Matranga     

Isolation of Arabidopsis extracellular ATP binding proteins by affinity proteomics and identification of PHOSPHOLIPASE C-LIKE 1 as an extracellular protein essential for fumonisin B1 toxicity

ATP is secreted to the extracellular matrix, where it activates plasma membrane receptors for controlling plant growth and stress-adaptive processes. DOES NOT RESPOND TO NUCLEOTIDES 1 (DORN1), was the first plant ATP receptor to be identified but key downstream proteins remain sought after. Here, we identified 120 proteins secreted by Arabidopsis cell cultures and screened them for putative stress-responsive proteins using ATP-affinity purification. We report three Arabidopsis proteins isolated by ATP-affinity: PEROXIDASE 52, SUBTILASE-LIKE SERINE PROTEASE 1.7 and PHOSPHOLIPASE C-LIKE 1. In wild-type Arabidopsis, the expression of genes encoding all three proteins responded to fumonisin B1, a cell death-activating mycotoxin. The expression of PEROXIDASE 52 and PHOSPHOLIPASE C-LIKE 1 was altered in fumonisin B1-resistant salicylic acid induction-deficient (sid2) mutants. Exposure to fumonisin B1 suppressed PHOSPHOLIPASE C-LIKE 1 expression in sid2 mutants, suggesting that the inactivation of this gene might provide mycotoxin tolerance. Accordingly, gene knockout mutants of PHOSPHOLIPASE C-LIKE 1 were resistant to fumonisin B1-induced death. The activation of PHOSPHOLIPASE C-LIKE 1 gene expression by exogenous ATP was not blocked in dorn1 loss-of-function mutants, indicating that DORN1 is not required. Furthermore, exogenous ATP rescued both the wild type and the dorn1 mutants from fumonisin-B1 toxicity, suggesting that different ATP receptor(s) are operational in this process. Our results point to the existence of additional plant ATP receptor(s) and provide crucial downstream targets for use in designing screens to identify these receptors. Finally, PHOSPHOLIPASE C-LIKE 1 serves as a convergence point for fumonisin B1 and extracellular ATP signalling, and functions in the Arabidopsis stress response to fumonisin B1.     

Endocytosis of beta1 integrins is an early event in migration promoted by the cell adhesion molecule L1

Directional cell motility is a complex process requiring orchestration of signals from diverse cell adhesion receptors for proper organization of neuronal groups in the brain. The L1 cell adhesion molecule potentiates integrin-dependent migration of neuronal cells and stimulates integrin endocytosis but its mechanism of action is unclear. The hypothesis was investigated that L1 stimulates cell motility by modulating surface levels of integrins through intracellular trafficking using a model cell system. Antibody-induced clustering of L1, which mimics ligand binding, induced formation of cell surface complexes of L1 and beta1 integrins in L1-expressing HEK293 cells. L1 formed cell surface complexes with integrin beta1 and alpha3 subunits but not with integrin alpha1. Following cell surface clustering, beta1 integrins and L1 became rapidly internalized into Rab5+ early endosomes. Internalization of L1 and beta1 integrins was prevented by treatment with monodansyl cadaverine (MDC), an inhibitor of clathrin-dependent endocytosis, and by deletion of the AP2/clathrin binding motif (RSLE) from the L1 cytoplasmic domain. MDC treatment coordinately inhibited L1-potentiated haptotactic migration of HEK293 cells to fibronectin in Transwell assays. These results suggested that downregulation of adhesive complexes of L1 and beta1 integrin at the plasma membrane by clathrin-mediated endocytosis is a potential mechanism for enhancing cell motility.     

Characteristics of the binding of human C-reactive protein (CRP) to laminin

Human CRP binds to the basement membrane protein laminin in vitro in a Ca2+-dependent manner via the phosphorylcholine (PC) binding site of C-reactive protein (CRP). The binding was saturable at a molar ratio of 4 (CRP/laminin). The specificity of the binding was shown by inhibition of binding of labeled CRP to laminin by unlabeled CRP, but not by human IgG. Specific binding was optimal in the presence of 5 mM Ca2+, but did not occur in the absence of Ca2+ or in the presence of EDTA. The binding of Ca2+ to CRP causes a conformational change in the molecule, which is required for binding to PC and to laminin. The PC binding site of CRP was implicated in the binding to laminin on the basis of inhibition by both soluble PC and anti-idiotypic mAbs directed to the TEPC-15 PC-binding idiotype found on mouse antibodies to PC. In addition, mouse mAbs specific for the CRP PC binding site displayed decreased reactivity with CRP already bound to laminin. The binding of CRP to laminin provides a possible explanation for selective deposition of CRP at inflamed sites. The CRP-laminin interaction may serve as a means of concentrating CRP at sites of tissue damage so that the CRP might function as a ligand for leukocytes, an event that will result in removal of necrotic tissue and cell debris.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Effect of nitrogen limitation and nature of the feed upon Oenococcus oeni metabolism and extracellular protein production

AIMS: The aim of the study was to characterize the effect of various nitrogen sources on Oenococcus oeni growth, carbon source utilization, extracellular protease activity and extracellular proteins. More generally, the goal is to understand how nitrogen-based additives might act to enhance malolactic fermentation in wine. METHODS AND RESULTS: Five yeast extracts were used. As the amino acid and nitrogen analyses revealed, they were similar in global amino acid composition, except for arginine level. Nevertheless the ratio of amino acids between free/bound, and low/high molecular weight fractions were highly different. One of the yeast extracts led to a significant protease activity in the supernatant and to a poor final biomass of the IOB84.13 strain compared to the other ones. For the IOB84.13 strain specifically, arginine addition to the arginine poor yeast extract did not restore growth. 35S-methionine-labelled extracellular proteins were separated by SDS-PAGE. Signals were detected in all media early in the growth phase and were maintained during 48 h of culture. CONCLUSIONS: A significant protease activity was detected for O. oeni supernatants during growth under nitrogen limitation but only for certain nitrogen sources. Moreover, the activity was strain dependent. Peptides (0.5-10 kDa) seemed to be more favourable for growth of wine bacteria than <0.5 kDa nitrogen sources. The extracellular protein signal patterns differed more greatly between the bacterial strains tested than between the nitrogen molecules in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study extensively considering the role of the nitrogen source composition and level upon O. oeni growth and metabolism.