Species-specific Aggregation Factor Research Articles

Synergy between phorbol esters, 1-oleyl-2-acetylglycerol, urushiol, and calcium ionophore in eliciting aggregation of marine sponge cells.

Aggregation of marine sponge cells (Microciona prolifera) resembles stimulus-response coupling of higher organisms in which activation of protein kinase C and movements of intracellular Ca provide twin signals. We now report that activators of protein kinase C (phorbol esters) and ionomycin act synergistically to aggregate sponge cells. Surprisingly--since extracellular Ca is required for integrity of the species-specific aggregation factor--synergistic aggregation proceeded in the complete absence of added extracellular Ca (2.5-20 mM EDTA). The order of activity of phorbol esters and related compounds was that of their effect on protein kinase C (phorbol myristate acetate, phorbol dibutyrate greater than phorbol diacetate much greater than phorbol, 4 alpha-phorbol). 1-Oleyl, 2-acetylglycerol a synthetic activator of protein kinase C, also showed synergy with ionomycin. Phorbol esters and 1-oleyl, 2-acetylglycerol acted in synergy with ionomycin to liberate membrane Ca as detected by decreased fluorescence of chlortetracycline in prelabeled cells. Moreover, urushiol, the toxic principle of poison ivy, but not pentadecanylcatechol, its inert analogue, showed synergy with ionomycin. Synergistic aggregation was inhibited by calmidazolium (10 microM), piroxicam (20-100 microM), and pertussis toxin (20 micrograms/ml). The data not only confirm that marine sponge cell aggregation follows the general sequence of stimulus-response coupling in the cells of higher organisms but also support, in this most ancient of multicellular creatures, the hypothesis that mobilization of intracellular Ca and activation of protein kinase C provide the twin signals for cell activation in the absence of added extracellular Ca.

Aggregation of marine sponge cells induced by Ca pulses, Ca ionophores, and phorbol esters proceeds in the absence of external Ca

Cells of the sponge Microciona prolifera dissociated in Ca,Mg-free sea water reaggregate upon addition of Ca, an observation classically attributed to the requirement for Ca of a species-specific aggregation factor. We now report that, unexpectedly, extracellular Ca is not required during aggregation; brief Ca pulses (1–3 sec) terminated by excess EDTA suffice to prepare the cells for aggregation by Ca ionophores (e.g. A23187). We also show that phorbol myristate acetate (PMA) promotes aggregation of pulse-prepared cells. Since PMA and A23187 act synergistically in Microciona , the “twin signal” hypothesis, signalling by Ca and protein kinase C in parallel, is validated in this primitive animal.

Calcium dependent aggregation of marine sponge cells is provoked by leukotriene B 4 and inhibited by inhibitors of arachidonic acid oxidation

Dissociated cells of the marine sponge, Microciona prolifera , aggregate in response to a species-specific aggregation factor (MAF) and Ca ionophores. We now report that leukotriene B 4, a 5-lipoxygenase product, also causes aggregation of sponge cells. No other lipoxygenase products provoked aggregation. However, nordihydroguaiaretic acid (NDGA), colchicine, indomethacin, piroxicam and ibuprofen inhibited MAF-induced aggregation; the latter three agents inhibited Ca movements. Inhibition of prostaglandin release cannot be the mechanism whereby these agents inhibit aggregation of sponge cells, since the cells do not respond to exogenous cyclooxygenase products.

Sponge aggregation factor and sponge hemagglutinin: Possible relationships between two different molecules

A lectin from the marine sponge GEODIA CYDONIUM was isolated and characterized. GEODIA lectin (GL) agglutinates human red blood cells irrespective of the ABO blood group and precipitates with a variety of D-galactose containing glycosubstances, i.e. certain snail galactans, bovine erythrocyte glycoprotein and PNEUMOCOCCUS type XIV polysaccharide. The only simple sugars inhibiting the GL-mediated hemagglutination were lactose and n-acetyl-D-galactosamine.GL was purified by affinity chromatography on Sepharose 4B almost to homogeneity as tested by polyacrylamide disc gel electrophoresis. Positive staining of the lectin band with Coomassie brilliant blue and PAS suggest that GL is a glycoprotein. Under dissociating conditions GL exhibits an apparent mol. wt. of 12,000 daltons. The elution behaviour of the lectin on Biogel suggests that GL exists as an oligomeric molecule in its native state.Our data obtained with GL demonstrate that the species-specific aggregation factor (AF) from GEODIA is not identical with this lectin. Two major hypotheses for a biological role of GL are discussed: first, the cooperation of GL with AF in sorting-out processes of GEODIA cells and second, the possibility that GL is involved in defense mechanisms.

Species-Specific Aggregation Factor in Sponges: Isolation, Purification and Characterization of the Aggregation Factor from Suberites domuncula

An aggregation factor (AF) from the siliceous sponge Suberites domuncula has been isolated and purified by the following steps: Sepharose 2 B gel chromatography, sucrose gradient, Nonidet treatment, Sephadex G-100 gel chromatography and DEAE-Sephadex ion-exchange chromatography. By this procedure the AF was purified 1340-fold with a 63% yield nearly to homogeneity. The AF is originally associated with large particles, characterized by a sedimentation of 2200 S. These particles have been visualized electron microscopically; they are characterized by a filament-like shape of a length of 3400 A and a cross-sectional diameter of 230 A. The purified, low-molecular weight AF has a buoyant density of 1.38 g/cm 3 and an absorbance maximum at 282 nm. The isoelectric pH is approximately 5.75. The molecular weight of the AF has been determined to be 55,000. Chemical analysis revealed that AF consists mainly of protein. The reaggregation process of Suberites cells to large aggregates (>1000 μm), mediated by Suberites AF, is strongly dependent on pH, ionic strength and the presence of calcium ions, and independent of incubation temperature between 0°C and 20°C. Aggregation can be inhibited by trypsin, D-glucosamine and dodecyl sulphate. The Suberites AF is species-specific if tested in a system containing cells from the siliceous sponge Geodia cydonium .

Species-Specific Aggregation Factor in Sponges: Transfer of a Species-Specific Aggregation Receptor from Suberites domuncula to Cells from Geodia cydonium

An aggregation receptor (AR) from the siliceous sponge Suberites domuncula has been isolated and purified by chromatography to about 55% purity. The AR consists primarily of neutral carbohydrate and is characterized by a buoyant density of 1.59 g/ml and by an apparent molecular weight of 42,500. The average density of the AR on Suberite cells is about 3.8 × 105 per μm2. The AR contains considerable amounts of hexuronic acid. The isolated AR can bind not only to receptor-depleted Suberites cells but also to receptor depleted cells from another siliceous species (Geodia cydontum). After being charged with Suberites ARs, Geodia cells form aggregates in the presence of the species-specific Suberites aggregation factor.

Species-specific aggregation factor in sponges : VIII. Nature and alteration of cell surface charge.

Isolated cells from the siliceous spongeGeodia cydonium have been studied with respect to their partition behaviour in a two-phase aqueous polymer system. With this method it is possible to determine subtle changes in the cell surface charge. Addition of a homologous aggregation factor to the isolated cells lowers the partition rate, a finding which indicates that after binding of the aggregation factor to the cells their surface charge is reduced. The partition rate of the cells is strongly correlated with their content of membranebound sialic acid.Sixty-nine percent of the total, membrane-bound hexuronic acid is associated with the aggregation receptor; 1.8×107 aggregation receptor molecules are present on the surface of one cell which means that the average surface density amounts to 2.8×105 molecules per μm2.Removal of the aggregation receptor molecules from the cell surface results in a decrease of the partition rate in the two-phase system. After charging the receptor-depleted cells with soluble aggregation receptor, the partition behaviour of these cells can be reconstituted.

Species-specific aggregation factor in sponges XIII. Entire and core structure of the large circular proteid particle from Geodia cydonium

High-molecular weight particles have been isolated from the sponge Geodica cydonium. In the ‘native’ form these particles consist of a spherical center and have 25–30 filaments attached to it. The core structure of the particles is assembled of a central circle and 25 radially-arranged filaments. The core structure is obtained from the entire structure by incubation in a medium, containing a non-ionic detergent and EDTA. The molecular weight of the entire structure was in the range of 1.4 × 10 9 daltons or more and of the core structure 6.1 × 10 8 daltons. Two functional proteins are released from the ‘native’ particles: the aggregation factor and the sialyltransferase.

Species-specific aggregation factor in sponges. Sialyltransferase associated with aggregation factor.

The sialyltransferase (= glycoprotein-sialic acid transferase) was studied in the sponge Geodia cydonium, a mesozoan organism. The experiments were performed both in intact cellular and in isolated enzyme systems. It is shown, that desialylated cells show a lower aggregation potency than the controls. During aggregation enzymic sialylation of desialylated sponge cells occurs in the presence of an aggregation factor, which is associated with a high molecular weight particle. The sialylation process is temperature-dependent and can be inhibited by N-ethylmaleimide. Sialylation occurs predominantly at a distinct cell surface component, the aggregation receptor. The sialyltransferase was isolated and purified by the following steps: Sepharose 4B, CM-cellulose, Nonidet treatment, and Sephadex G-100. By this procedure the enzyme was purified 680-fold with a 31% yield. The sialyltransferase is originally associated with the high molecular weight particle also carrying the aggregation factor. In the last step the aggregation factor was separated from the sialyltransferase. The enzyme catalyzes the transfer of sialic acid from CMP-sialic acid to the desialylated aggregation receptor. The molecular weight of the sialyltransferase has been determined to be 52,000. Kinetic studies revealed no lag phase and a dependence on enzyme concentration. The purified transferase has a pH optimum of 7.75 and requires 200 mM NaCl for activity. No requirement for Mg2+ or Ca2+ could be observed. The reaction is inhibited by 10 micronM N-ethylmaleimide.

Species-specific aggregation factor in sponges. I. Characterization of the large circular proteid particle

We have isolated circular proteid particles (CPP) from the sponge Geodia cydonium, which carry the aggregation factor, and have freed them from concomitant impurities. The CPP have been visualized by electron microscopy as annular particles with a circular contour length of 3530 Å. The circles are surrounded by 25 filaments 610 Å in length. The CPP are characterized by a sedimentation coefficient ( S° 20, w ) of 3000, by a buoyant density of 1.32 g/cm 3 and by a particle weight of about 2–5 · 10 9 daltons. They consist primarily of protein. It is suggested that the fibrous complex found electron microscopically is only the core structure of a more complex particle.

Species-specific aggregation factor in sponges: VI. aggregation receptor from the cell surface

An aggregation receptor from the siliceous sponge Geodia cydonium has been isolated and purified in an almost pure form. It sediments at about 2-6s, has a buoyant density of 1-51 g/ml in CsCl and elutes from Sephadex G-50 at a Ve/V0 value of 1-311. Chemical analysis revealed that the receptor consists of 81% neutral carbohydrate and 7-5% protein. The activity of the receptor is rapidly destroyed by Na-periodate. The receptor is released from the cell surface after removal of Ca2+ from the medium or after incubation of the cells with trypsin. The depleted cells can be charged again with isolated receptor molecules. The binding of the receptor molecules on the cell surface is prevented in the presence of trypsin. For optimal binding, physiological salt concentrations with respect to NaCl (540 mM NaCl) and Ca2+ ions are necessary. The receptor whose isolation is described in this report, is involved in secondary aggregation processes, which are initiated by a soluble aggregation factor. The primary aggregation of the cells is not influenced by the receptor. Time-course studies with receptor-depleted cells revealed that new aggregation receptor molecules are formed during the aggregation process. By competition experiments it could be shown that high concentrations of soluble aggregation receptor molecules inhibit secondary aggregation. The soluble receptor molecules can complete with surface-bound receptor molecules only if these are not linked with the aggregation factor.

Species-specific aggregation factor in sponges: IV. Inactivation of the aggregation factor by mucoid cells from another species

The isolated and purified aggregation factor of the siliceous sponge Geodia cydonium has been shown to act species-specifically in combination with three horny sponges (one of them: Ircinia muscarum), but unspecifically towards a variety of other sponge species to be classed with Calcispongiae, Demospongiae and Keratosa. The property of the Geodia aggregation factor to cause aggregates only with isolated Geodia cells and not with Ircinia cells is not determined by the aggregation factor itself, but by substances which are located on the cell surfaces of the mucoid cells from Ircinia. The Ircinia inactivator, capable to inactivate the Geodia aggregation factor, has a molecular weight of 9800 D and behaves like a protein: first it can be inactivated by heat, extreme pH conditions and by pronase and second it has a buoyant density of 1.39± 0.04 g/cm 3. The inactivation of the Geodia aggregation factor by Ircinia cells can be prevented by preincubation of the aggregation factor with reagents for thiol groups. The bonding of the Geodia aggregation factor to Ircinia cells can be split by adding excess of the thiol compound dithiothreitol. From the results presented it is supposed that the Geodia aggregation factor is bound to Ircinia mucoid cells via disulfate linkages. The biological relevance of the finding that the capacity of the Geodia aggregation factor is limited by alien cells can be seen in a defence mechanism of the Ircinia organism against gemmules from Geodia.

Species-specific aggregation factor in sponges V. Influence on programmed syntheses

Isolated cells from the siliceous sponge Geodia cydonium as well as small primary aggregates (diameter: 70 μm) consisting of them show no increase in rates of programmed syntheses and mitotic activity with time. After addition of a highly purified aggregation factor to a culture with primary aggregates which subsequently form secondary aggregates (diameter: larger than 1000 μm), a dramatic increase of DNA, RNA and protein synthesis occurs. Together with this increase, the cells show a high mitotic activity. The values for the mitotic coefficient reach a first maximum 8 h after the beginning of the secondary aggregation process. The stimulation of the mitotic activity of cells during the aggregation factor induced secondary aggregation process can be suppressed by inhibitors of RNA and protein synthesis as well as by a blocker of DNA synthesis. This finding may indicate that cells from the G 0 -population enter the proliferating cell pool via the G 1 -phase.

Purification and characterization of a species-specific aggregation factor in sponges

The events during re-aggregation of sponge cells, dissociated in Ca 2+- and Mg 2+-free artificial sea water, containing trypsin, can be subdivided into three phases. The first event is the formation of primary aggregates with a diameter of 82 μm. It is Ca 2+- and Mg 2+-dependent and insensitive towards trypsin and puromycin even at high concentrations. The formation of secondary aggregates with a diameter greater than 1 000 μm, is initiated by an aggregation factor. This factor can be separated from the formed elements with calcium- and magnesium-free artificial sea water. Its action is Ca 2+- and Mg 2+-dependent, temperature-independent and insensitive to puromycin. The last event is the reconstitution of functional aquiferous systems in the aggregates. The aggregation factor which was found to be species-specific could be purified 158-fold. Its functional group seems to be a protein probably with polar amino acids in critical positions. Secondary aggregates generated with the aggregation factor show high viability. Some evidence is presented that the aggregation factor may be an annular particle with a circular contour length of 3 500 Å with about 25 filaments attached to it.