Organization and polysaccharides of sponge aggregation factor

Abstract

Aggregation factor, the macromolecular complex which mediates species-specific aggregation of dissociated sponge cells, was isolated from several species, partially characterized, and visualized by electron microscopy. All factors were large fibrous complexes with a backbone and side chains or arms. In some factors, the backbone is linear. In others it is circular and the complex appears as a sunburst with arms extending like rays from the circle. The size and location of the polysaccharide chains have been studied using purified preparations of Microciona prolifera. "Sunbursts" treated with ethylenediaminetraacetate (EDTA) for 4 weeks at 0 degrees C dissociate into 3 protein- and polysaccharide-containing components. Sodium dodecyl sulfate does not cause the sunburst to dissociate nor does it inhibit dissociation in the presence of EDTA suggesting that dissociation is not due to hydrolytic enzymes. The dissociation products were fractionated on a 977-A pore size micropore glass column. Fifteen percent of the material is excluded and appears in the electron microscope as the central circle of the sunburst. Digestion of the circles with 10(-3) M dithiothreitol (DTT) and 0.5 mg/ml proteinase K for 72 h at 37 degrees C produces 2 polysaccharide chains of 65,000 and 6,000 daltons as fractionated and sized on a 233-A pore size micropore glass column using Pharmacia dextrans as standards. The included fractions of the EDTA-treated material are subunits of the arms which contain 70% of the polysaccharide. A single polysaccharide of 6,000 daltons as measured on 233-A size glass beads and Sephadex G-75 is released from these subunits by proteinase digestion. Pharmacia dextrans are used as standard on both columns. We calculate that there would be four 65,000-dalton chains and one hundred 6,000-dalton chains per circle and fifty 6,000-dalton chains per arm. The third component of the EDTA-treated preparation is partially included on the column. It appears as linear fibrils in the electron microscope and contains polydisperse polysaccharides of several-hundred-thousand daltons. It may be an impurity since there is apparently less than 1 of the large polysaccharide chains per sunburst.

Supramolecular Structure of a New Family of Circular Proteoglycans Mediating Cell Adhesion in Sponges

“…In GuHCl dissociative density gradients, which do not cleave covalent bonds, fraction F3 contains both MAFp3 and MAFp4. Finally, SDS does not inhibit the dissociation of the arms from the ring in the presence of EDTA (Humphreys et al, 1977), suggesting that this dissociation is not due to hydrolytic enzymes. These results reinforce the hypothesis that MAFp3 and MAFp4 are two separate proteins in the native AF.…”

“…5, lanes 17 and 18), thus suggesting that it contained some g-6 glycan or fragments of it. As was already described for EDTA-dissociated MAF (Humphreys et al, 1977), GuHCl dissociation inactivates MAF irreversibly. We have tried to reconstitute the original structure through stepwise dialysis of the disassembled components back to CaCMFTSW in the presence of protease inhibitors and at 4°C.…”

“…Therefore, we propose that each of the 20 globular structures forming the MAF ring is a g-200-glycosylated MAFp3 unit, and each of the 20 arms is a g-6-glycosylated MAFp4 unit. As the ring is not sensitive to reducing conditions (Humphreys et al, 1977), its stabilization cannot be based on disulfide bonds between protein chains. Chemical deglycosylation, on the other hand, completely disrupts the ring, suggesting that interactions between glycans or between glycans and protein might be responsible for its integrity, although such treatment can affect other protein-mediated interactions contributing to the stabilization of the ring structure.…”

“…EDTA dissociation and purification of the AF fragments were done as described (Humphreys et al, 1977). Dissociative density gradients in the presence of 4 M guanidine hydrochloride (GuHCl) were performed as described (Fernà ndezBusquets et al, 1996).…”

Frequent Occurrence of HIV-Inhibitory Sulphated Polysaccharides in Marine Invertebrates

“…Many such extracts have displayed bioactivity profiles similar to dextran sulphate and other sulphated polysaccharides. Biopolymers of this class have been reported previously from diverse marine invertebrates (e.g., Katzman and Jeanloz, 1970;Humphreys et al, 1977;Junqua et aI., 1979;Albano andMourao, 1983, Coombe et aI., 1987;Valiente et aI., 1988) but have not previously been shown to have anti-HIV or other antiviral activity from such organisms.…”