Carbohydrate-carbohydrate interactions of a novel acidic glycan can mediate sponge cell adhesion.

G.N Misevic,M.M Burger

doi:10.1016/s0021-9258(18)53484-0

Abstract

Cell recognition and adhesion were first demonstrated in marine sponges. These phenomena were later shown in Microciona prolifera sponge to be mediated by a Ca(2+)-dependent self-association of adhesion proteoglycans (APs) attached in a species-specific manner to cell-surface receptors. Using the same experimental system we now provide three lines of evidence that highly polyvalent Ca(2+)-dependent carbohydrate-carbohydrate interactions of a novel AP glycan represent the basis of AP-AP self-binding and thus of cell adhesion. 1) A specific monoclonal antibody which blocks cell aggregation and AP bead adhesion identified a highly repetitive novel carbohydrate epitope (2500 sites) in an acidic glycan of M(r) = 200 x 10(3) (g200) from AP. 2) Reconstitution of the Ca(2+)-dependent self-interaction activity of AP was achieved by cross-linking the purified protein-free g200 glycan into polymers of similar valency as the native AP. 3) Beads coated with the protein-free g200 glycan showed a Ca(2+)-dependent aggregation equivalent to that of AP beads. Carbohydrate and amino acid analyses of the g200 glycan purified by gel electrophoresis, high performance liquid chromatography gel filtration, and ion exchange chromatography yielded six components in the following proportions; 68 fucose, 32 glucuronic acid, 2 mannose, 18 galactose, 19 N-acetylglucosamine, and 1 asparagine residue. These unique chemical features together with immunological and enzymological analyses suggest that the g200 glycan is a large highly fucosylated, acidic, N-linked polysaccharide with a novel structure distinct from that of other known glycosaminoglycans.

Highlights

(2500 sites) in an acidic glycan of M, = 200 X lo3 such highly polyvalent, but very low single site affinity bind
Jumblatt et al reported in 1980 that thecell-binding function of AP alone was not sufficient to promote cell adhesion, but adhesion did occur when AP-AP self-interaction was activated by the addition of physiological seawater containing 10 mM Ca2+.It was shown in a cell-free system that the AP-AP binding can mediate aggregation of AP-coated beads
Mg/ml Pronase type B (Boehringer Mannheim) which have been The self-binding assay with lZ5I-AP,3H-g200,and 3H-xg200was preincubated for 30 min at 60 "C was added threetimes at 24-h performed in 100 p1 of CSW containing 0.5% BSA and with inclusion intervals, during which digestion was carried out at 60"C.Amino of 2 or 10 mM Ca2+.After incubation for 20 min at room temperature, acids and small peptides were removed from total AP glycans by gel samples were centrifuged for 10 min at 10,000X g and theradioactivfiltration and ion exchange chromatography (Misevic et al, 1987). ity in supernatants and redissolved pellets was determined

Summary

MATERIALS AND METHODS

Sponges-M. prolifera sponges were collected by the Marine Biomarked “advertisement” in accordance with 18 U.S.C. Mg/ml Pronase type B (Boehringer Mannheim) which have been The self-binding assay with lZ5I-AP,3H-g200,and 3H-xg200was preincubated for 30 min at 60 "C was added threetimes at 24-h performed in 100 p1 of CSW containing 0.5% BSA and with inclusion intervals, during which digestion was carried out at 60"C.Amino of 2 or 10 mM Ca2+.After incubation for 20 min at room temperature, acids and small peptides were removed from total AP glycans by gel samples were centrifuged for 10 min at 10,000X g and theradioactivfiltration and ion exchange chromatography (Misevic et al, 1987). Monoclonal Antibodies-BALB/c mice were immunized with AP Analytical Methods-Carbohydrate analysis of AP glycans was as described previously (Misevic et al, 1987).Monoclonal antibodies performed according to Chaplin (1982) after methanolysis and triwere prepared from culture supernatants of AP-positive clones ob- methylsilylation either in a Hewlett-Packardgas chromatograph 7620 tained afterfusion of mice spleen cells with the FO myelomaline and A using a glass column (2 mm X 2 m) packed with 4% SE-30 on 100/.

RESULTS

A P and g200 binding to cells

I i II IIIii

Findings

DISCUSSION