Carbohydrates at the cell surface have been proposed as mediators in cell-cell
recognition events involved in embryogenesis, metastasis, and other proliferation
processes by calcium-dependent carbohydrate to carbohydrate interactions. They are
the most prominently exposed structures on the surface of living cells, and with
flexible chains and many binding sites are ideal to serve as the major players in
initiating these cellular events. However, biological relevance of these type
interactions is often questioned because of the very low affinity binding of single
carbohydrate molecules and that they manifest themselves only through the contact
of a large number of molecules tightly arranged in the membrane. Weak interactions
are considerably more difficult to study and only a few biologically significant
examples of direct carbohydrate-carbohydrate interactions have been reported, e.g.
pioneering work showing glycosphingolipid self-interactions through multivalent
interaction of Lewis X epitopes. However, there are no reports on the existence of
specific proteoglycan self-interactions through carbohydrate-carbohydrate
interactions in cellular recognition system, as it has been done with
glycosphingolipids.
Here, we used sponges, organisms on which the first proteoglycan-mediated cell-cell
recognition in the animal kingdom was demonstrated, as a model system to study
carbohydrate-mediated cellular recognition. We show that the interaction between
single oligosaccharides from surface proteoglycans is relatively strong and
comparable to protein-carbohydrate interactions, highly specific, and dependent on
Ca2+-ions.
200 kDa glycans from the core protein of Microciona prolifera cell surface
proteoglycans have been previously shown to mediate homotypic Microciona proteoglycan-proteoglycan interactions. Here, 200 kDa glycans from four different
sponge species: Microciona prolifera, Halichondria panicea, Suberites fuscus and
Cliona celata were purified and investigated for species-specific interactions.
Selective recognition of glycans by live cells was studied to confirm the existence of
glycan-glycan recognition system in biologically relevant situations. Mature sponge
cells have the ability to reaggregate species-specifically and form homogenous
aggregates on a shaker at the right shear forces in the presence of physiological 10
mM Ca2+. Live cells were allowed to aggregate with glycan-coated beads similar in
size to small sponge cells in the presence of calcium. They specifically recognized
beads coated with their own glycans and did not mix but separated from beads
coated with glycans isolated from different species.
The glycan-glycan recognition assay was developed to mimic species-specific cellcell
recognition in sponges. 200 kDa glycans immobilized onto beads similar in size
to small sponge cells assembled species-specifically in the presence of physiological
calcium, at the same shear forces as in cell-cell aggregation. Glycans coated on
beads aggregated with glycans from the same species coated on beads, and separated
from glycans from other species. The glycan density necessary for specific live cellcell
recognition in sponges is 828 molecules/μm2. In our studies, the glycan density
necessary for specific glycan-coated bead was very similar: ~810 molecules/μm2.
Mature live cells demonstrated specific recognition of 200 kDa glycans during
selective-binding to glycans coated on surfaces in the presence of calcium. They
strongly adhered to glycans from their own surface proteoglycans coated onto a solid
polystyrene phase, while the binding to glycans from different proteoglycans was 3 -
5 times lower. Moreover, homotypic adhesion to glycan-coated plates enhanced
sponge cell differentiation and formation of mineral skeleton (spicules).
Larval cells, after settlement and spreading of larvae, can fuse species-specifically in
nature. In our studies, live larval cells recognized and adhered specifically to glycans purified from adhesion proteoglycans from their "mother sponge". They showed
almost no interaction with glycans from other species.
As in cell-glycan adhesion assays, highly species-specific adhesion of 200 kDa
glycans to glycan-coated surfaces could be observed in the presence of
physiological calcium. Tested glycans bound strongly to glycans from the same
species and showed up to a six fold reduction in binding to glycans from other
species.
Atomic force microscopy (AFM) was performed to measure for the first time
adhesion forces between single glycan molecules obtained from different surface
proteoglycans. Measurements revealed equally strong adhesion forces in the range of
several hundred piconewtons (pN) between glycan molecules as between proteins
and glycans measured in another recognition system. Moreover, statistically
significant differences (p value < 0.01) were seen between homotypic (glycans from
the same species) and heterotypic (glycans from different species) interactions.
Moreover, the polyvalent character of binding characterized mainly interactions
between glycans from the same species. This indicates that not only the higher
adhesion force per binding site as such but also the higher amount of multiple
interactions between glycans from the same species versus mixture of glycans from
different species guaranteed the specificity of the glycan-mediated recognition.
These findings confirm for the first time the existence of specific glycan-glycan
recognition system between cell surface proteoglycans. We propose that these cell's
outermost surface structures serve as important players in initiating the very first
contacts between cells through highly species-specific and flexible carbohydratecarbohydrate
interactions
