Proteomic analysis of the sponge Aggregation Factor implicates an ancient toolkit for allorecognition and adhesion in animals

Abstract

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.

Keywords: Porifera; adhesion; allorecognition; evolution; proteomics.

Fig. 1.

The Aggregation factor (AF) is composed of glycoproteins that form linear or circular structures with radiating arms. (A) Phylogenetic placement of the four major lineages of sponges (blue). The AF is believed to be unique to demosponges. (B) Atomic force microscopy (AFM) images of linear vs. ring-like AF purified from different demosponge species. AF purification and AFM imaging were performed as described in the Materials and Methods Section (red arrowhead = MAFp4 arms, yellow arrowhead = MAFp3 ring). (C) Cartoon depiction of Clathria prolifera AF. Wreath domain-containing MAFp3 makes up the central ring and is decorated with g-200 glycans responsible for Ca²⁺-dependent AF–AF interactions. Calx-beta domain-containing MAFp4 arms are decorated with g-6 glycan responsible for Ca²⁺-independent AF–cell interactions, but it is unclear how the AF attaches to the surface of cells.

Fig. 2.

Known and predicted AF components were abundant in all replicates. (A) Venn diagram of proteins detected in each of the Crude, Filtered, and SEC samples. Sample complexity was high, irrespective of the preparatory method, potentially indicating low purity. However, (B) a violin plot showing protein proportion in the SEC sample of the 319 proteins common to all samples (plotted on a log10 scale) illustrates that most proteins were actually very low in abundance. The 25 most abundant proteins are highlighted in orange. (C) These included the known AF components, MAFp3/p4, and predicted AF components that contained wreath and calx-beta domains (sorted by proportion in the SEC sample).

Fig. 3.

Wreath domain-containing proteins detected in AF proteomic samples have structural similarity to the vWFD domain. (A) Wreath domain-containing proteins in order of their relative proportion in AF proteomic datasets. Wreath domain (W) is highlighted in magenta. Proteins are highlighted in green in Fig. 2C. (B and B’) AlphaFold3 prediction and secondary structure diagram of MAFp3 wreath domain, with beta-strands highlighted to reflect their position in the tertiary structure. Beta sheet switches between strands 3/4 and 8/9 are highlighted in magenta. Putative cleavage site residues Asp-Pro between strands 1 and 2 are highlighted in orange. Disordered N and C termini were manually trimmed for visibility. (C) Superposition of the central MAFp3 wreath domain beta-sandwich (green) with vWFD domain of A. millepora mucin-like (UniprotID: B3EWY9, aa 705 to 858; gray). rmsd = 4.15 over 200 atoms. Secondary structure diagram of the vWFD domain shown in C’. Conserved Asp-Pro autocleavage motif in the vWFD domain is highlighted in orange. Conserved beta sheet switches between strands 3/4 and 8/9 are highlighted in magenta (39). (D) Multiple sequence alignment of domains structurally similar to the MAFp3 wreath domain. Only the N terminus of the domains is shown, highlighting the conserved cleavage motif Asp-Pro between beta strands 1 and 2 (orange) (40). The cysteine residue needed for covalent attachment after cleavage is shown in red.

Fig. 4.

Top MAFAP1 hits in the predicted proteome are unrelated but share a highly conserved, candidate AF-interaction domain. (A) Domain architecture of C. prolifera proteins that contain a conserved AF-interacting region (pink box) in common with MAFAP1. Also like MAFAP1, three of these also contain a disordered repeat region (green oval) adjacent to the AF-interacting region. All but S168704 were detected in proteomics results for the AF. (B) Multiple sequence alignment of the AF-interacting regions from proteins depicted in panel A. (C) AlphaFold3 prediction of the C-terminal AF-interacting region of MAFAP1, highlighting a divergent Ig-like domain (region 1) and a divergent Fn3-like domain (region 2).

Fig. 5.

MAFAP1 C-terminal domains resemble Ig and Fn3 domains. (A) Sequence alignment of region 1 of MAFAP1 C-terminal domain to Ig domains (C2-type) from Foldseek hits. Conserved cysteine residues, typical for Ig domains, are highlighted in red. (B) Sequence alignment of region 2 of MAFAP1 C-terminal domain to Fn3 domains from Foldseek hits and NCAM sequences shown in A. (A’) Structural superposition of AlphaFold3 prediction of region 1 of MAFAP1 C-terminal domain (green, aa722 to 853) to M. musculus NCAM2 Igdomain (UniprotID: O35136, aa21 to 108, gray) (rmsd: 3.14 over 350 atoms). Conserved cysteine residues that form a stabilizing, intramolecular disulfide bridge are highlighted in magenta (for MAFAP1) and yellow (for NCAM2). (B’) Structural superposition of AlphaFold3 prediction of region 2 of MAFAP1 C-terminal domain (green, aa854 to 947) to NCAM2 Fn3-domain (UniprotID: O35136, aa495 to 591, gray) (rmsd: 3.52 over 435 atoms).