Publications

The discovery that sponges (Porifera) can fully regenerate from aggregates of dissociated cells launched them as one of the earliest experimental models to study the evolution of cell adhesion and allorecognition in animals. This process depends on an extracellular glycoprotein complex called the Aggregation Factor (AF), which is composed of proteins thought to be unique to sponges. We used quantitative proteomics to identify additional AF components and interacting proteins in the classical model, Clathria prolifera, and compared them to proteins involved in cell interactions in Bilateria. Our results confirm MAFp3/p4 proteins as the primary components of the AF but implicate related proteins with calx-beta and wreath domains as additional components. Using AlphaFold, we unveiled close structural similarities of AF components to protein domains in other animals, previously masked by the mutational decay of sequence similarity. The wreath domain, believed to be unique to the AF, was predicted to contain a central beta- sandwich of the same organization as the vWFD domain (also found in extracellular, gel- forming glycoproteins in other animals). Additionally, many copurified proteins share a conserved C- terminus, containing divergent immunoglobulin (Ig) and Fn3 domains predicted to serve as an AF-interaction interface. One of these proteins, MAF- associated protein 1, resembles Ig superfamily cell adhesion molecules and we hypothesize that it may function to link the AF to the surface of cells. Our results highlight the existence of an ancient toolkit of conserved protein domains regulating cell-cell and cell-extracellular matrix protein interactions in all animals, and likely reflect a common origin of cell adhesion and allorecognition.

JTD Keywords: Adhesion, Allorecognitio, Binding, Calcium, Carbohydrate-carbohydrate interactions, Cell-cell adhesion, Evolution, Marine sponge, Microciona-prolifera, Molecule, Porifera, Protein, Proteomics, Recepto, Recognition

Carbohydrate-carbohydrate interactions in cell adhesion are being increasingly explored as important players in cell-cell and cell-extracellular matrix interactions that are characterized by finelytuned on-off rates. The emerging field of glycomics requires the application of new methodologies to the study of the generally weak and multivalent carbohydrate binding sites. Here we use the quartz crystal microbalance (QCM) for the analysis of the self-binding activity of the g200 glycan, a molecule of marine sponge origin that is responsible for Ca2+-dependent species-specific cell adhesion. The QCM has the advantages over other highly sensitive techniques of having only one of the interacting partners bound to a surface, and of lacking microfluidics circuits prone to be clogged by self-aggregating glycans. Our results show that g200 self-interaction is negligible in the absence of Ca2+. Different association kinetics at low and high Ca2+ concentrations suggest the existence of two different Ca2+ binding sites in g200. Finally, the observation of a non-saturable binding indicates that g200 has more than one self-adhesion site per molecule. This work represents the first report to date using the QCM to study carbohydrate-carbohydrate interactions involved in cell adhesion.

JTD Keywords: Ca2+-dependent binding, Carbohydrate-carbohydrate interaction, Cell adhesion, Proteoglycan, Quartz crystal microbalance, Sponges