Carbohydrate self-recognition mediates marine sponge cellular adhesion

Abstract

Sponges (Porifera), the simplest and earliest multicellular organisms, are thought to have evolved from their unicellular ancestors about 1 billion years ago by developing cell-recognition and adhesion mechanisms to discriminate against "non-self." Consequently, they are used as models for investigating recognition phenomena. Cellular adhesion of marine sponges is an event involving adherence of extracellular proteoglycan-like molecules, otherwise known as aggregation factors (AFs). In a calcium-independent process the AFs adhere to the cell surface, and in a calcium-dependent process they exhibit AF self-association. A mechanism which has been implied but not definitely proven to play a role in the calcium-dependent event is self-recognition of defined carbohydrate epitopes. For the red beard sponge, Microciona prolifera, two carbohydrate epitopes, a sulfated disaccharide and a pyruvylated trisaccharide, have been implicated in cellular adhesion. To investigate this phenomenon a system has been designed, by using surface plasmon resonance detection, to mimic the role of carbohydrates in cellular adhesion of M. prolifera. The results show self-recognition of the sulfated disaccharide to be a major force behind the calcium-dependent event. The interaction is not simply based on electrostatic interactions, as other sulfated carbohydrates analyzed by using this procedure did not self-associate. Furthermore, the interaction is completely eradicated on substitution of Ca(2+) ions by either Mg(2+) or Mn(2+) ions. This physiologically relevant recognition mechanism confirms the existence of true carbohydrate self-recognition, and may have significant implications for the role of carbohydrates in cellular recognition of higher organisms.

Figure 1

Structures of the sulfated disaccharide epitope (1) present on the surface of M. prolifera cells, the corresponding neoglycoconjugate (2), and the three control neoglycoconjugates (3, and 4/5).

Figure 2

Illustration of change in SPR signal (Response) with time for a typical monovalent binding event, at four different concentrations. (A) Surface bound substrate in equilibrium with buffer. (B) Initiation of flow of analyte in buffer: association. (C) Equilibrium between bound and unbound analyte: steady-state. (D) Flow of buffer restored: dissociation. (E) Further dissociation.

Figure 3

Investigation of the aggregation behavior of conjugates 2 and 4, Lewis X conjugate, and BSA, in the presence of either 10 mM CaCl₂ (A) or 10 mM MgCl₂ (B). On addition of divalent cation (1 M, 5 μl) to a solution (10 μM, 495 μl) of test molecule, the tube was mixed and the absorbance was zeroed. The absorbance was then measured at 340 nm for 6,000 s.

Figure 4

Interaction of BSA (A) and conjugate 2 (B) analyte with surface-bound conjugate 2, carboxymethylated dextran, and BSA, in the absence of Ca²⁺ ions. Concentrations of analyte: 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM.

Figure 5

Interaction of BSA with surface-bound conjugate 2, carboxymethylated dextran, and BSA, in the presence of 10 mM Ca²⁺ ions. Concentrations of analyte: 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM.

Figure 6

Interaction of conjugate 2 with surface-bound conjugate 2, carboxymethylated dextran, and BSA, in the presence of 10 mM Ca²⁺ ions. Concentrations of analyte: 10, 5, 2.5, 1.25, 0.625, and 0.3125 μM.

Figure 7

Linear fitting (21) of the self-interaction of conjugate 2 at a conjugate 2-coated surface. The accuracy of the fit is mirrored in both the size of the statistical parameter χ² (2.49, 0.37, and 0.43) and in the minor deviation of the residuals from the fit. Conc., concentration; slope, ΔResponse/ΔTime.

Figure 8

(Upper) Confirmation and illustration of the polyvalent multilayer formation of conjugate 2 (10 μM). (Lower) Binding of an antibody (291–4D10-A) to immobilized Lewis X-BSA conjugate. (A) Injection of conjugate 2 in the absence of Ca²⁺ ions. (B) Injection of conjugate 2 in the presence of 10 mM Ca²⁺ ions. (C) In comparison to B: saturation of the Lewis X-coated surface by antibody. Arrows indicate commencement of association and dissociation.