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

Abstract

Aggregation factors are the molecules responsible for species-specific cell adhesion in sponges. Here, we present the structure of the aggregation factor from the marine sponge Microciona prolifera, which constitutes the first description of a circular proteoglycan. We have analyzed chemically dissociated and enzymatically digested aggregation factor with atomic force microscopy, agarose gel electrophoresis, and Western blots using antibodies against the protein and carbohydrate moieties. Twenty units from each of two N-glycosylated proteins, MAFp3 and MAFp4, form the central ring and radiating arms, respectively, stabilized by a hyaluronidase-sensitive component. MAFp3 carries a 200-kDa glycan involved in homologous self-interactions between aggregation factor molecules, whereas MAFp4 carries a 6-kDa glycan that binds cell surface receptors. A 68-kDa lectin found in cell membranes of several sponge species binds the aggregation factor and its protein-free glycans, as well as chondroitin sulfate and hyaluronan. Here, we show that despite their lack of clear sequence homologies with other known proteoglycan structures, the protein and carbohydrate components of sponge aggregation factors assemble to form a supramolecular complex remarkably similar to classical proteoglycans.

Molecular Recognition between Glyconectins as an Adhesion Self-assembly Pathway to Multicellularity

“…4). Staining of gels by alcian blue revealed that GN1 has two major species of acidic glycans with molar masses of 200 and 6 kDa as reported previously (14,15,18,29). GN2 had one major large acidic glycan type with a molar mass of 180 kDa, representing more then 60% of the total carbohydrate content, and heterogeneous populations of acidic oligosaccharides with sizes below 10 kDa very faintly stained by alcian blue because of the larger area spread (Fig.…”

“…Whether these peptides are identical would have to be verified by direct sequencing. Furthermore, the cloning and sequencing of GN2 and GN3 genes for their respective core proteins will show the exact differences to the already cloned gene of GN1 (16,18).…”

“…Hydrazinolysis of intact GNs under standard conditions released glycans; however, these conditions are now known to cleave both N-linked and O-linked glycans. Gas chromatography coupled to mass spectroscopy analyses and alkali treatment of GNs indicate that the large glycans of GN2 and -3 are N-linked to the protein core as in the case of both GN1 glycans (16,18,29). To confirm the nature of the glycan-protein linkage, a more quantitative analysis of the isolated linkage region must be made.…”

“…However, these and subsequent experiments from 1960 to 1980 used semi-purified and chemically ill-defined extracts, termed aggregation factors, and thus lacked quantitative and biochemical data about the underlying molecular mechanisms (9 -12). In the last two decades, the first structure-to-function-related studies have been performed on M. prolifera glyconectins (1,(13)(14)(15)(16)(17)(18). Here, we have isolated glyconectins (GNs) from three marine sponge species, M. prolifera (GN1), Halichondria panicea (GN2), and Cliona celata (GN3).…”

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge Microciona prolifera

“…Later studies revealed that the speciesspecific cell aggregation of the marine sponge Microciona prolifera is mediated by a proteoglycan-like aggregation factor (MAF). Hereby, the N-glycans of two functional domains of MAF, MAFp4 and MAFp3, play an essential role: (i) the MAFp4-bound 6-kDa glycan (known as g-6), which in a Ca 2+ -independent way adheres to cell surface receptors, and (ii) the MAFp3-bound 200-kDa glycan (known as g-200), which promotes cell adhesion via a Ca 2+ -dependent self-association process [2,3]. Specific monoclonal antibodies against purified MAF, called Block 1 and Block 2, could inhibit the Ca 2+ -dependent self-association process without interfering with the cell-binding activity.…”