Proteoglycan Mechanics Studied by Single-molecule Force Spectroscopy of Allotypic Cell Adhesion Glycans

Abstract

Early Metazoans had to evolve the first cell adhesion system addressed to maintaining stable interactions between cells constituting different individuals. As the oldest extant multicellular animals, sponges are good candidates to have remnants of the molecules responsible for that crucial innovation. Sponge cells associate in a species-specific process through multivalent calcium-dependent interactions of carbohydrate structures on an extracellular membrane-bound proteoglycan termed aggregation factor. Singlemolecule force spectroscopy studies of the mechanics of aggregation factor self-binding indicate the existence of intermolecular carbohydrate adhesion domains. A 200-kDa aggregation factor glycan (g200) involved in cell adhesion exhibits interindividual differences in size and epitope content which suggest the existence of allelic variants. We have purified two of these g200 distinct forms from two individuals of the same sponge species. Comparison of allotypic versus isotypic g200 binding forces reveals significant differences. Surface plasmon resonance measurements show that g200 self-adhesion is much stronger than its binding to other unrelated glycans such as chondroitin sulfate. This adhesive specificity through multiple carbohydrate binding domains is a type of cooperative interaction that can contribute to explain some functions of modular proteoglycans in general. From our results it can be deduced that the binding strength/surface area between two aggregation factor molecules is comparable with that of focal contacts in vertebrate cells, indicating that strong carbohydrate-based cell adhesions evolved at the very start of Metazoan history.Specific carbohydrate-carbohydrate interactions are rarely reported in biologically relevant situations such as cell recognition (1). However, carbohydrate structures have immense structural diversity (2), a ubiquitous distribution in vertebrate and invertebrate tissues (3), and are associated with the cell surface (4, 5), as required of cell recognition molecules. Carbohydrate-carbohydrate interactions are characterized by relatively weak forces that, when multimerized, can be easily potentiated by orders of magnitude, representing a highly versatile form of cell adhesion given the extraordinary plasticity of their structures (6). Among the few known examples of carbohydrate self-recognition proposed to be specific in biological processes are the multivalent binding of Lewis x epitopes involved in the first steps of embryogenesis (7, 8), glycolipid-glycolipid interactions controlling cell adhesion, spreading, and motility (9 -11), and self-interactions of the glycan portion of sponge proteoglycans leading to species-specific cell adhesion (12, 13).Sponge cells associate in a species-specific process through multivalent interactions of carbohydrate structures on a type of extracellular proteoglycan termed aggregation factor (14 -16). Based on their molecular structure, aggregation factors have been related to hyalectans (15): large, extracellular aggregating modul...

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans

“…DAF self-interaction forces measured in experiments conducted in lower concentrations of calcium (2 mM) were weaker (data not shown), in agreement with previous SMFS studies using MAF (37). However, this observation requires careful interpretation because the integrity of AF protein cores depends on a residual calcium concentration (14). The data used to calculate the F max were restricted to the last peak of the force curves to ensure that only single molecule self-interaction forces were taken into consideration.…”

“…The average force (F max ) measured at a retract velocity of 2000 nm s Ϫ1 for the last DAF self-adhesion event was 273 Ϯ 6 pN (Fig. 1D) and was in the same order of magnitude as MAF self-interaction forces (14). DAF self-interaction forces measured in experiments conducted in lower concentrations of calcium (2 mM) were weaker (data not shown), in agreement with previous SMFS studies using MAF (37).…”

“…Sites-Images of DAF acquired via high resolution AFM revealed a proteoglycan remarkably similar to the AF of the sponge M. prolifera (MAF) described elsewhere (8,14,15), which enabled us to trace parallels between their structures, compositions, and chemical behaviors. DAF has a sunburst-shaped core constructed by a ring, ϳ130 nm across, and radiating arms up to 190 nm in length (Fig.…”

“…The structures of AFs resemble those of aggrecans: large, extracellular membrane-bound, aggregating, and modular proteoglycans (15). In the marine sponge Microciona prolifera the AF is constructed by a protein core attached to two different carbohydrate units: a small 6-kDa glycan that mediates the interaction of the AF with putative receptors present in the cell membrane and a larger 200-kDa sulfated polysaccharide, which binds homophilically with identical units on the AFs of adjacent cells (13)(14)(15)(16).…”

“…Proteoglycan-like molecules termed aggregation factors (AFs) 4 mediate intercellular adhesion and recognition in sponges (14). The structures of AFs resemble those of aggrecans: large, extracellular membrane-bound, aggregating, and modular proteoglycans (15).…”