The putative sponge aggregation receptor. Isolation and characterization of a molecule composed of scavenger receptor cysteine-rich domains and short consensus repeats.

Porifera (sponges) are the oldest extant metazoan phylum. Dissociated sponge cells serve as a classic system to study processes of cell reaggregation. The reaggregation of dissociated cells is mediated by an extracellularly localized aggregation factor (AF), based on heterophilic interactions of the third order; the AF bridges two cells by ligating a cell-surface-bound aggregation receptor (AR). In the present study we report cloning, expression and immunohistochemical localization of a polypeptide from the marine sponge Geodia cydonium, which very likely represents the AR. The presumed AR gene gives rise to at least three forms of alternatively spliced transcripts of 6.5, 4.9 and 3.9 kb, as detected by northern blotting. Two cDNA clones corresponding to the shorter forms were already reported earlier; here we present an analysis of the largest. All three putative polypeptides feature scavenger receptor cysteine-rich (SRCR) domains. The largest form, SRCR-SCR-Car, is a cell-surface receptor of molecular mass 220 kDa, which is assumed to be the cell-adhesion receptor AR; the second form, SRCR-Re, is also a putative receptor of 166 kDa, while the third form, SRCR-Mo, is a soluble molecule of 129 kDa. The SRCR-SCR-Car molecule consists of fourteen SRCR domains, six short consensus repeats (SCRs), a C-terminal transmembrane domain and a cytoplasmic tail; its fourteenth SRCR domain features an Arg-Gly-Asp tripeptide. To obtain monoclonal antibodies, a 170-amino-acid-long polypeptide that is found in all three forms of the SRCR-containing proteins was expressed in E. coli. In a western blot of sponge cells lysate the monoclonal antibody raised against the recombinant polypeptide recognized two major immuno-reacting polypeptides (220 and 117 kDa) and two minor bands (36 and 32 kDa). The antibody was found to react with antigen(s) predominantly localized on the plasma membranes of cells, especially those of spherulous cells. In a functional assay Fab' fragments of the antibodies suppressed AF-mediated cell-cell reaggregation. Additionally, a recombinant SRCR-soluble fragment effectively inhibited AF-mediated cell-cell reaggregation. We conclude that the 220 kDa SRCR-containing protein of the sponge G. cydonium is very likely the AR.

A novel member of an ancient superfamily: sponge (Geodia cydonium, Porifera) putative protein that features scavenger receptor cysteine-rich repeats.

Proteins featuring scavenger receptor cysteine-rich (SRCR) domains are prominent receptors known from vertebrates and from one phylum of invertebrates, the echinoderms. In the present study we report the first putative SRCR protein from the marine sponge Geodia cydonium (Porifera), a member of the lowest phylum of contemporary Metazoans. Two forms of SRCR molecules were characterized, which apparently represent alternative splicing of the same transcript. The long putative SRCR protein, of 1536 aa, features twelve SRCR repeats, a C-terminal transmembrane domain and a cytoplasmic tail. The sequence of the short form is identical with the long form except that it lacks a coding region near the C terminus, thus the 1195 aa deduced protein consists of only the first ten SRCR domains and the last 26 C-terminal aa residues, without the transmembrane domain. Homology searches revealed that the sponge putative SRCR protein shares with bovine T-cell antigen WC1 29.2% identity in 1054 aa overlap, 33.9% identity in 475 aa overlap with sea urchin speract and 56% identity in 110 aa overlap with macrophage scavenger receptor type I. Based upon the number and location of the conserved Cys residues, the sponge SRCR domain repeats were classified as belonging to group A of the SRCR superfamily. With twelve SRCR repeats, one more than those in any of the previously described SRCR proteins, and several membrane-bound and soluble forms, it seems that the most primitive known member of this family may be the structurally most complex one among SRCR containing proteins.

Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis.

Age-dependent studies show that the amount of inorganic polyphosphate in rat brain strongly increases after birth. Maximal levels were found in 12-months old animals. Thereafter, the concentration of total polyphosphate decreases to about 50%. This decrease in the concentration of total polyphosphate is due to a decrease in the amount of insoluble, long-chain polyphosphates. The amount of soluble, long-chain polyphosphates does not change significantly in the course of ageing. In rat embryos and newborns, mainly soluble polyphosphates could be detected. In rat liver, the age-dependent changes are less pronounced. The changes in polyphosphate level are accompanied by changes in exopolyphosphatase activity, which degrades the polymers to orthophosphate; highest enzyme activities were found when the polyphosphate level was low. Induction of apoptosis in the human leukemic cell line HL-60 by actinomycin D results in degradation of long polyphosphate chains. The total polyphosphate content does not change significantly in apoptotic cells.

A novel method for determination of inorganic polyphosphates using the fluorescent dye fura-2.

A method for determining inorganic polyphosphate, which is based on the Mn(2+)-induced quenching of the fluorescence of the calcium indicator fura-2, is described. The effect of Mn2+ ions on fura-2 fluorescence is gradually abolished in the presence of increasing concentrations of polyphosphate; this allows the quantification both of synthetic polyphosphates and of the naturally occurring polymer isolated from tissues or cells. The described method has some advantages compared to conventional procedures for detection of polyphosphates based on the metachromatic effect on toluidine blue. It can be applied for the determination of pyrophosphate, tripolyphosphate and other short-chain polyphosphates not detectable by toluidine blue and it can be used for measurement both of pyrophosphatase and exopolyphosphatase activity.