Sponge cell aggregation

Summary Dissociated sponge cell system has proved to be a useful model to study the process of cell aggregation both on cellular and subcellular level. The purpose of this review is to discuss recent results obtained from experiments with the marine sponge Geodia cydonium.Dissociated cells form functional aggregates during a process which can be sub-divided into three phases: first, formation of small primary aggregates in the presence of Ca²⁺; second, formation of secondary aggregates in the presence of an aggregation factor and third, reconstitution of a functional system of watercontaining channels by rearrangement in the secondary aggregates.On subcellular level a series of macromolecules are known which are involved in the control of aggregation and separation of sponge cells: Aggregation factor, aggregation receptor, anti-aggregation receptor, glucuronidase, ß-glucuronosyltransferase, ß- ß-galactosidase and a lectin. These components might be linked in the following sequence: (a) Activation of the aggregation receptor by its enzymic glucuronylation; (b) Adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) Inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated ß-glucuronidase; (d) Cell separation due to either the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is most likely controlled by the Geodia lectin.The events leading to cell-cell recognition cause a change in the following metabolic events: Increase of oxygen uptake, decrease of cyclic AMP level, increase of cyclic GMP level and stimulation of programmed syntheses.Abbreviations AF aggregation factor - CPP circular proteid particle - AR aggregation receptor - aAR anti-aggregation receptor - CMF calcium- and magnesium-free artificial sea water - ASW calcium- and magnesium-containing artificial sea water This paper is dedicated to PROF. DR. R. K. ZAHN on the occasion of his 60^th birthday.     

Aggregation of sponge cells. Isolation and characterization of an inhibitor of aggregation receptor from the cell surface.

From the cell membranes of the sponge Geodia cydonium a component was isolated and purified which inhibits the aggregation factor isolated from the same source; the component was termed anti-aggregation receptor. This molecule was characterized as a glycoprotein (54% neutral carbohydrate) and its molecular weight is in the range of 180,000 One biological site of the anti-aggregation receptor was determined to be D-galactose. Indirect evidence presented seems to indicate that this molecule is present in an active form in aggregation-deficient cells and absent in aggregation-susceptible cells.     

Aggregation of sponge cells: 22. Species-specific reactivity of a lectin from the sponge Geodia cydonium

Single cells of the marine sponge Geodia cydonium aggregate species-specifically in the presence of a soluble aggregation factor to form large cell clumps. A lectin isolated from the same sponge species does not cause agglutination of Geodia cells but agglutinates only cells from heterologous species (e.g. Tethya lyncurium, Hemimycale columella, Pellina semitubulosa, Cacospongia scalaris, Verongia aerophoba). The process of agglutination is independent of divalent cations (they do not affect the agglutination process at concentrations up to 50 mM), occurs at 2°C, causes a reduction in the viability of the cells and results in an inhibition of programmed syntheses. The observed differences between the properties of cell agglutination (effect of a lectin in a heterologous system) and cell aggregation (effect of an aggregation factor in the homologous system) is discussed. Cell aggregation is dependent upon the presence of an aggregation factor, the presence of cations and an incubation temperature 2̃0°C; cell aggregation results in a stimulation of programmed syntheses. Cell agglutination requires a heterologous macromolecule (e.g. lectin), it is independent of divalent cations and causes inhibition of programmed syntheses in the cells.     

Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium

Summary The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The M_r of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing conditions three protein species (M_r: 16 500, 15 500 and 13 500) were identified in the pAF preparation. The pAF was precipitable by Ca⁺⁺ and did not cross-react with antisera against homologous purified secondary aggregation factor and lectin. It is mainly composed of protein (48.0%) and carbohydrate (50.2%). The isolated pAF restored the aggregation potency not only of factor-depleted Geodia cells but also of cells from other Demospongiae. However, the pAF displayed no aggregation enhancing effect on urea-treated cells from species belonging to the Calcispongiae or Hexactinellida. We hypothesize that in contrast to the secondary aggregation, the initial aggregation of Geodia cells is mediated by the one-component system, the bivalent and bifunctional pAF.     

Structure-activity relationship of clovamide and its related compounds for the inhibition of amyloid β aggregation

Alzheimer’s disease (AD), a neurodegenerative disorder, is characterized by aggregation of amyloid β-protein (Aβ). Aβ aggregates through β-sheet formation and induces cytotoxicity against neuronal cells. Inhibition of Aβ aggregation by naturally occurring compounds is thus a promising strategy for the treatment of AD. We have already reported that caffeoylquinic acids and phenylethanoid glycosides, which possess two or more catechol moieties, strongly inhibited Aβ aggregation. Clovamide (1) containing two catechol moieties, isolated from cacao beans (Theobroma cacao L.), is believed to exhibit preventive effects on Aβ aggregation. To investigate the structure-activity relationship of clovamide (1) for the inhibition of Aβ aggregation, we synthesized 1 and related compounds 2–11 through reaction between l-DOPA, d-DOPA, l-tyrosine, or l-phenylalanine and caffeic acid, p-coumaric acid, or cinnamic acid, and compounds 12 and 13 were derived from 1. Among tested compounds 1–13, those containing one or two catechol moieties exhibited potent anti-aggregation activity, whereas the non-catechol-type related compounds showed little or no activity. This suggests that at least one catechol moiety is essential for inhibition of Aβ42 aggregation, and this activity increases depending on the number of catechol moieties. Consequently, clovamide (1) and its related compounds may be a promising therapeutic option for inhibiting Aβ-mediated pathology in AD.     

Directed migration of cells from the sponge Geodia cydonium

The migration behavior of cells from the sponge Geodia cydonium was studied in vitro, applying the 'Tissue Culture Slide Chamber' technique. The homologous lectin caused a directed cell migration with a maximal locomotory rate of 1.6 mum/min. Competition experiments using the solubilized lectin receptor (= antiaggregation receptor) revealed that the chemotactic ligand (= lectin) interacts directly with the lectin receptor which-in consequence-functions as the chemotactic receptor. The ability of the lectin to promote cell migration is abolished by coincubation with purified leucine aminopeptidase. Biochemical and immunochemical data revealed that this enzyme is present also on the surface of sponge cells. Furthermore, we present evidence that the chemotactic receptor (= anti-aggregation receptor) on the cell surface is, in an hitherto unknown manner, coupled with the intracellularly present actin filaments. From these data we conclude that the directed migration of Geodia cells is mediated by the interaction between the lectin (= chemotactic ligand) and the lectin receptor (= chemotactic receptor); it is very likely that also intracellular structural elements operate simultaneously and coordinately during cell migration.     

Aggregation of sponge cells. Function of a lectin in its homologous biological system.

For the first time, the biological role of a lectin in the process of reaggregation of single cells from the same species (marine sponge: Geodia cydonium Jam.) is described. The galactose-specific lectin does not promote aggregation, but prevents the antiaggregation receptor from disaggregating cell clumps. Competition experiments showed that the lectin inactivates the antiaggregation receptor by binding to it, most likely via its terminal galactose residues. The lectin converts reversibly aggregation-deficient cells (carrying functional cell membrane-bound antiaggregation receptor molecules) to aggregation-susceptible cells.     

Further characterization of a lectin and its in vivo receptor from Geodia cydonium

From the results of two-dimensional isoelectric focusing, SDS-gel electrophoresis and from immunochemical data it became evident that lectin I and lectin II (corresponding to fractions Geodia I and Geodia II isolated on immobilized lactose) from the sponge Geodia cydonium are apparently identical mixtures of several isolectins, the pI values of their subunits ranging, in contrast to our previous report, from 4.8–7.5. The hypothetical concept of sugar-mediated, specific lectin-lectin interactions (self-recognition) could not be verified by binding of FITC-labelled isolectins (Geodia I) to the lectin subunits, which had been purified by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The concept should also be dismissed on the basis of carbohydrate analyses revealing in contradiction with previous results the exclusive presence of alkali-labile bound tetraglucose on the purified isolectins (1 mol/mol lectin protein). The combining site of the isolectins was shown by a quantitative microprecipitation inhibition assay to be most complementar to oligosaccharides of the β-galactoside series and to interact specifically with particular structural elements of the subterminal sugar(s). Carbohydrates of the anti aggregation receptor, which are assumed to represent the functional ligand of the Geodia-isolectins in vivo, could be demonstrated to have a high affinity for the lectin combining site, exceeding that of the best disaccharide inhibitor, lactose, by five orders of magnitude. A preliminary chemical characterization of the receptor carbohydrate revealed that D-galactose and D-glucose (each approx. 200 mol/mol receptor) are organized in an oligosaccharide, which could be cleaved from the protein by trifluoroacetolysis.     

Aggregation of sponge cells: 23. Interaction of Geodia lectin with Tethya cell-surface glycoprotein

A macromolecule has been isolated from the cell membranes of the sponge Tethyalyncurium. This macromolecule was purified and found to bind to a d-galactose specific lectin from the sponge Geodia cydonium. The lectin receptor was characterized as a glycoprotein with a molecular weight in the region of 155 000. Evidence is presented indicating that the binding of lectin with the lectin receptor is caused by hydrophobic interactions.     

The Role of Cell-Surface Lectins in the Aggregation of Azospirilla

The mutant strain Azospirillum brasilenseSp7.2.3 with impaired lectin activity exhibited poorer cell aggregation than its parent strain A. brasilenseSp7(S) both in the exponential and stationary growth phases. The pretreatment of bacterial cells with the specific haptens (L-fucose and D-galactose) of a lectin located at the cell surface of the mutant strain was found to inhibit the aggregation of azospirilla. The specific binding of the A. brasilenseSp7(S) lectin to the extracellular polysaccharide-containing complexes of this strain was revealed by dot immunoblotting on nitrocellulose membrane filters. The interaction of the lectins of A. brasilense75, A. brasilenseSp7, and A. lipoferum59b with the polysaccharide-containing complexes that were isolated from these strains was not specific. No interstrain cross-interaction between the exopolysaccharides and lectins of azospirilla was found. A coflocculation of A. brasilenseSp7 cells with Bacillus polymyxa1460 cells was shown. The involvement of autogenous lectins in the aggregation of bacterial cells is discussed.     

Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous aggregation factor and insulin. Insulin-mediated phosphorylation of the ras protein resulted in a decrease in its Kd with GTP from 2 microM to 80 nM. The activated ras protein displayed high GTPase activity if the partially purified protein was incubated with homologous lectin and lectin receptor molecules. These results suggest that in the sponge, ras is activated by the insulin/insulin(insulin-like)-receptor system. This transition enables the ras protein to interact with the lectin-receptor/lectin complex, a process which may ultimately lead to an initiation of an intracellular signal-transduction chain.     

Protein kinase C phosphorylates the sponge aggregation receptor after its binding to the homologous aggregation factor

The aggregation factor from the sponge Geodia cydonium functions also as a growth factor after binding to the aggregation receptor (= growth factor receptor) on the plasma membrane of homologous cells. We have recently shown that protein kinase C is involved in the pathway transducing the growth factor signal. Here we report that the aggregation receptor (a polypeptide with an Mr of 43,500) is phosphorylated by protein kinase C. Using a plasma membrane fraction only this phosphoprotein (pp) 43.5 became phosphorylated by kinase C. The phosphorylation of pp43.5 in intact cells in response to the binding of the aggregation factor to this polypeptide was a late event and occurred 10 to 15 h after addition of the aggregation factor. Based on studies with phorbol esters it appears to be very likely that protein kinase C also phosphorylates pp43.5 in vitro. The degree of phosphorylation of pp43.5 paralleled with both the extent of DNA synthesis and ras oncogene expression. The latter process resulted in a switch of the responsiveness of the cells to growth factors signals: 10 to 15 h after addition of the aggregation factor to dissociated cells, this factor lost its growth factor function while the homologous lectin gained the ability to stimulate cell proliferation (to be published). These results support the idea that phosphorylation of pp43.5 (= aggregation receptor) results in an inhibition of its function, i.e., the transduction of the growth factor (= aggregation factor) signal.     

Amyloid inhibitors enhance survival of cultured human islets

Background

Amyloid fibrils created by misfolding and aggregation of proteins are a major pathological feature in a variety of degenerative diseases. Therapeutic approaches including amyloid vaccines and anti-aggregation compounds in models of amyloidosis point to an important role for amyloid in disease pathogenesis. Amyloid deposits derived from the β-cell peptide islet amyloid polypeptide (IAPP or amylin) are a characteristic of type 2 diabetes and may contribute to loss of β-cells in this disease.

Methods

We developed a cellular model of rapid amyloid deposition using cultured human islets and observed a correlation between fibril accumulation and β-cell death. A series of overlapping peptides derived from IAPP was generated.

Results

A potent inhibitor (ANFLVH) of human IAPP aggregation was identified. This inhibitory peptide prevented IAPP fibril formation in vitro and in human islet cultures leading to a striking increase in islet cell viability.

Conclusions

These findings indicate an important contribution of IAPP aggregation to β-cell death in situ and point to therapeutic applications for inhibitors of IAPP aggregation in enhancing β-cell survival.

General significance

Anti-amyloid compounds could potentially reduce the loss of β-cell mass in type 2 diabetes and maintain healthy human islet cultures for β-cell replacement therapies.     

Cytophilic binding of IgE to the macrophage. III. Involvement of cyclic GMP and calcium in macrophage activation by dimeric or aggregated rat myeloma IgE

Discharge of lysosomal enzymes, measured by release of β-glucuronidase, was studied in uninduced rat macrophages stimulated in vitro with rat monoclonal IgE (IR 162) in different states of aggregation. Monomeric IgE showed negligible activity, while dimeric and aggregated IgE were shown to induce a rapid and selective release of β-glucuronidase as well as new synthesis of the enzyme, without change in the cytoplasmic marker, leucine aminopeptidase. Lysosomal enzyme release is related to the dose of dimeric IgE, becoming maximal above 2.5 μg/ml. β-Glucuronidase release from macrophages by dimers is competitively inhibited by monomeric IgE but only at high ratios, approximately 100-fold greater than those needed to block mast cell release of the same enzyme. The difference in inhibitability is consistent with the difference in binding affinity of macrophages and mast cells for monomeric IgE. This observation rules out the participation of the few remaining mast cells contained in the macrophage monolayer in β-glucuronidase release. Dimeric or aggregated IgE produced a rise in cyclic GMP coincident with the peak fixation of IgE by macrophages. Elevation of cyclic GMP by pharmacological means also stimulated β-glucuronidase release and new synthesis, as well as enhancing the effect on these of aggregated IgE. Enzyme release by IgE did not occur in the absence of extracellular calcium. We conclude that IgE, which has been cross-linked to form dimers before binding to specific macrophage receptors, triggers the cell and that cyclic GMP (and perhaps calcium) modulates the early step of macrophage activation.     

Formation of stable platelet aggregates by lectin from Solanum tuberosum

The GlcNAc-specific lectin from Solanum tuberosum is shown to induce haptenic-sugar-resistant contacts in platelet aggregation but not to induce stable neutrophil and lymphocyte aggregation. The formation of such contacts in platelets was significantly hindered by the inhibitors of cAMP phosphodiesterase (papaverine) or arachidonic acid metabolism (indomethacin, aristolochic acid, or MK-886) and by a sulfhydryl reagent (N-ethylmaleimide). This lectin can be useful in studying the mechanisms of stable platelet aggregation, drug screening for antithrombotic activity, and developing the cell engineering techniques.     

Selective separation of microorganisms by lectins: yeast and concanavalin A as a model system

Specific aggregation and separation of microorganisms was investigated using yeasts and concanavalin A as a model system. Cells of Saccharomyces cerevisiae were specifically aggregated and so separated from those of Schizosaccharomyces pombe. Optimum aggregation with over 99% of cells aggregated was achieved by adjustment to pH value and applied agitation. Dimeric lectin structure caused a far higher degree of aggregation than did tetrameric. Degree of aggregation was also strongly influenced by the ratio of lectin/cell concentrations, optimum aggregation occurring in the middle range of ratios. A high ratio of lectin to cells inhibited aggregation, occupation of most of the available receptors preventing intercellular bonding by divalent lectins. Detachment and reuse of concanavalin A was demonstrated using switching from moderate to low pH value. Potential uses for species-specific-separation of microorganisms are discussed. (c) 1992 John Wiley & Sons, Inc.     

Isolation and characterization of a cyanobacterium-binding protein and its cell wall receptor in the lichen Peltigera canina

Peltigera canina, a cyanolichen containing Nostoc as cyanobiont, produces and secretes arginase to a medium containing arginine. Secreted arginase acts as a lectin by binding to the surface of Nostoc cells through a specific receptor which develops urease activity. The enzyme urease has been located in the cell wall of recently isolated cyanobionts. Cytochemical detection of urease is achieved by producing a black, electron-dense precipitate of cobalt sulfide proceeding from CO₂ evolved from urea hydrolysis in the presence of cobalt chloride. This urease has been pre-purified by affinity chromatography on a bead of active agarose to which arginase was attached. Urease was eluted from the beads by 50 mM α-D-galactose. The experimentally probed fact that a fungal lectin developing subsidiary arginase activity acts as a recognition factor of compatible algal cells in chlorolichens can now been expanded to cyanolichens.Key words: arginase, lectin, Peltigera canina, recognition, urease     

Species-Specific Association of the Cell-Aggregation Molecule Mediates Recognition in Marine Sponges

Reaggrcgation of dissociated cells of marine sponges, resulting in reformation of functional sponges, is a calcium-dependent process mediated by large, proteoglycan-like molecules termed aggregation factors (AF). During aggregation, species-specific sorting of cells is often observed. We purified and characterized AFs from three different sponge species and investigated their role in species-specific aggregation using novel approaches. The calcium-dependent association between purified AFs is species-specific in most combinations, as was shown in overlay assays and bead-sorting assays with AFs immobilized onto colored beads. Species-specific interactions of living cells and AF-beads resulted in incorporation of only homospecific AF-beads into reforming cell aggregates. Sequences from peptides obtained from the AF core proteins could all be aligned to the sequence of one species, the Microciona prolifera AFp3 core protein. In contrast to this similarity, major species-specific differences were seen in carbohydrate composition and in the response of AFs to specific carbohydrate-recognizing antibodies. In summary, our data point to a prominent role for the calcium-dependent association of AFs in recognition processes during aggregation. As this association of AFs occurs via carbohydrate -carbohydrate interactions, we speculate that the specificity of those interactions may be fundamental to recognition mechanisms required for regeneration of individuals from dissociated cells and for rejection of foreign material by sponge individuals.