Isolation and cloning of a C-type lectin from the hexactinellid sponge Aphrocallistes vastus: a putative aggregation factor
Dietmar Gundacker, Sally P. Leys2, Heinz C. Schröder, Isabel M. Müller and Werner E.G. Müller1
Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany, and 2Department of Biology, University of Victoria, British Columbia V8W 3N5, Canada
Among the sponges (Porifera), the oldest group of metazoans in phylogenetic terms, the Hexactinellida is considered to have diverged earliest from the two other sponge classes, the Demospongiae and Calcarea. The Hexactinellida are unusual among all Metazoa in possessing mostly syncytial rather than cellular tissues. Here we describe the purification of a cell adhesion molecule with a size of 34 kDa (in its native form; 24 kDa after deglycosylation) from the hexactinellid sponge Aphrocallistes vastus. This adhesion molecule was previously found to agglutinate preserved cells and membranes in a non–species-specific manner (Müller, W. E. G., Zahn, R. K, Conrad, J., Kurelec, B., and Uhlenbruck, G. [1984] Cell adhesion molecules in the haxactinellid Aphrocallistes vastus: species-unspecific aggregationfactor. Differentiation, 26, 30–35). The fact that the aggregation process required Ca2+ and was inhibited by bird’s nest glycoprotein and D-galactose but not by D-mannose or N-acetyl-D-galactosamine suggests that this cell adhesion molecule is a C-type lectin. To test this assumption, two highly similar C-type lectins were cloned from A.vastus. The deduced polypeptides of the two cDNA species isolated classified these molecules as C-type lectins. The calculated Mr of the 191 aa long sequences were 22,022 and 22,064, respectively. The C-type lectins showed highest similarity to C-type lectins (type-II membrane proteins) from higher metazoan phyla; these molecules are absent in non-Metazoa. The two sponge C-type lectins contain the conserved domains known from other C-type lectins (e.g., disulfide bonds, the amino acids known to be involved in Ca2+-binding, as well as the amino acids involved in the specificity of binding to D-galactose) and a hydrophobic N-terminal region. The N-terminal part of the purified C-type lectin was identical with the corresponding region of the deduced polypeptide from the cDNA. It is proposed that the A.vastus lectins might bind to the cell membrane by their hydrophobic segment and might interact with carbohydrate units on the surface of the other cells/syncytia.
Key words: sponges/Hexactinellida/Aphrocallistes vastus/aggregation factor/lectin/C-type lectin/evolution/cell adhesion
Introduction
Multicellularity has arisen several times in evolution and in all major kingdoms (prokaryotes, plants, fungi, and animals; Schopf, 1993). It is generally agreed that multicellular plants, the red algae, the brown algae, the land plants and the fungi arose separately from unicellular ancestors (Devereux et al., 1990; Kirk, 1997). Molecular (Müller, 1995) and morphological (Müller, 1997; Wimmer et al., 1999) data indicate that the transition from the Protozoa to the Metazoa occurred only once in evolution. Sequence data from a variety of proteins involved in cell–cell interactions indicate that all animals, including sponges, are of monophyletic origin (Müller, 1995).
The phylum Porifera, the oldest group of multicellular animals, includes three classes: the Demospongiae, the Calcarea, and the Hexactinellida. The Hexactinellida differ substantially from the other two sponge classes in having largely syncytial tissues (Reiswig, 1979; Mackie and Singla, 1983; Leys, 1995, 1999). The majority (75%) of the sponge constitutes a multinucleated syncytium; the remaining portions of the sponge function independently as cells but are still connected to the whole by open or plugged cytoplasmic bridges (Leys, 1999). Knowledge of hexactinellid embryology is still very limited, as for the most part these sponges inhabit deep waters accessible only by submersible or dredge. Early descriptions of hexactinellid embryos obtained from dredged specimens (Ijima, 1901; Okada, 1928) and an examination of embryos from a population of hexactinellids recently discovered (Boury-Esnault et al., 1999) suggest that the embryo is “cellular” but that the larva is clearly syncytial. How the syncytial tissue arises remains unclear.
Two alternative hypotheses have been proposed to explain relationships between the major sponge classes. One suggests that the Demospongiae are more closely related to Hexactinellida based on presumed larval similarities (Böger, 1988). The other divides the Porifera into the adelphotaxa of the Hexactinellida and the Demospongiae/Calcarea based on the gross difference in tissue structure (Reiswig and Mackie, 1983; Leys, 1999). Recent molecular evidence supports the latter view (Koziol et al., 1997; Kruse et al., 1998; Skorokhod et al., 1999) suggesting that Calcarea are most closely related to other diploblasts and form a clade with the Demospongiae. The Hexactinellida are thought to have diverged first from a common ancestor of the Metazoa (Müller et al., 1998; Müller and Müller, 1999).
There is now a wealth of cellular and molecular evidence to suggest that the Demospongiae have many of the features characteristic of much evolutionarily younger metazoan taxa (e.g., the receptor tyrosine kinases (Müller and Schäcke, 1996), integrins (Pancer et al., 1997; Wimmer et al., 1999), collagen (Exposito et al., 1991), and the metabotropic glutamate/GABA-like receptor (Perovic et al., 1999)).
The role of the aggregation factor (AF) in sponges has been studied in detail (reviewed in Müller, 1982, and Fernà ndez-Busquets and Burger, 1999). The AF isolated from demosponges has been shown to function in a species-specific manner (Moscona, 1968; Müller et al., 1979a). Furthermore, in the Demospongiae a lectin belonging to the S-type lectins (Pfeifer et al., 1993) has been found to be involved in a species-specific aggregation complex (Wagner-Hülsmann et al., 1996). A similar “factor” isolated from the hexactinellid Aphrocallistes vastus aggregated cells in the presence of Ca2+ non-species-specifically (Müller et al., 1984). The preparation contained several fractions with one dominant protein species of 34,000 kDa. Sugar analysis revealed that the A. vastus AF was a glycoprotein consisting of 55% (w/w) protein, 40% neutral carbohydrate and 2% hexuronic acid (Müller et al., 1984). The experiments also showed that the A. vastus AF “agglutinated” the cells by interacting in a homo- or heterophilic manner of the second order.
Until now no cell adhesion molecules have been cloned from hexactinellid sponges. However, the syncytial nature of hexactinellid tissue, namely, its ability to fuse to form a syncytium after dissociation through fine mesh, and the evidence that hexactinellid sponges may have been the earliest multicellular animals to have evolved on earth, suggest that a study of cell adhesion molecules in hexactinellid sponges may reveal new mechanisms of cell–cell recognition within the Metazoa.
In the present study, the previously isolated AF was purified and shown to have a size of 34 kDa (24 kDa after deglycosylation). The factor caused aggregation in the presence of Ca2+ and this function was inhibited by D-galactose. These properties—Ca2+-dependency and sugar specificity—suggest that the previously termed AF is in fact a Ca2+-dependent lectin. To test this assumption, two highly similar Ca2+-dependent lectins (C-type lectin) have been cloned from A.vastus. Phylogenetic analysis revealed that the cloned putative C-type lectin represents the oldest (phylogenetic) member of this class within the Metazoa.
The sequences reported here are deposited in the EMBL/GenBank data base under the accession no. AJ276450 (APHRLECC1) and AJ276451 (APHRLECC2) as Aphrocallistes vastus C-type lectins.
Results
Aggregation-promoting activity of an extract from A.vastus
It was previously found that the partially enriched fraction of the A.vastus extract (formerly termed AF) caused aggregation of preserved cells and syncytia at Ca2+ concentrations greater than 1 mM (Müller et al., 1984). By applying the enrichment procedure described previously, aggregates larger than 500 µm diameter (Figure 1B,C) were obtained from single cells/membranes (Figure 1A). The size of the aggregates increased with increasing concentration of the extract: at 5 units of extract the diameter of aggregates was approximately 500 µm (Figure 1B); at 20 units of extract they were larger than 4 mm (Figure 1 C).
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