Cloning and expression of the putative aggregation factor from the marine sponge Geodia cydonium.
Sponges (phylum Porifera) have extensively been used as a model system to study cell-cell interaction on molecular level. Recently, we identified and cloned the putative aggregation receptor (AR) of the sponge Geodia cydonium, which interacts in a heterophilic way with the aggregation factor (AF) complex. In the present study, antibodies against this complex have been raised that abolish the adhesion function of the enriched sponge AF, the AF-Fraction 6B. Using this antibody as a tool, a complete 1.7 kb long cDNA, GEOCYAF, could be isolated from a cDNA library that encodes the putative AF. Its deduced aa sequence in the N-terminal section comprises high similarity to amphiphysin/BIN1 sequences found in Protostomia and Deuterostomia. However, the C-terminal portion of the sponge sequence lacks the SH3 domain characteristic for amphiphysin/BIN1. The polypeptide with a calculated size of 47 kDa was expressed in Escherichia coli. The recombinant, soluble 36 kDa putative AF was prepared and found to compete with the AF complex-associated adhesion protein of the AF-Fraction 6B for the binding sites at the cell surface. Furthermore, the recombinant putative AF was recognized by the antibody used to screen the cDNA library by western blotting. In addition, there is evidence that the recombinant putative AF binds to the G. cydonium galectin. It is concluded that the putative G. cydonium AF--a further autapomorphic molecule characteristic for Metazoa--binds to the AR present on the cell surface in association with the homologous galectin.
Schütze
J
J
Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany.
Krasko
A
A
Diehl-Seifert
B
B
Müller
W E
WE
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002449:Cell Aggregation; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D015723:Gene Library; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010802:Phylogeny; D011161:Porifera / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
D000906:Antibodies; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D010455:Peptides; D011994:Recombinant Proteins; C013065:cell aggregation factors