Aplysia temptin − the ‘glue’ in the water‐borne attractin pheromone complex

Abstract

Pheromones play an important role in coordinating male and female reproductive behavior in many aquatic species. Strikingly, however, relatively few peptide or protein pheromones have been characterized in invertebrates or vertebrates, as they are often difficult to isolate in sufficient quantities to characterize either biochemically or behaviorally. Furthermore, as we have seen with the marine mollusk Aplysia, protein pheromones require interaction with other proteins to exert their effect. Egg laying in Aplysia, which can be reliably induced by injection of egg-laying hormone, induces secretion of at least four distinct proteins from Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly b-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca 2+ -binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-b concentration, modulates pheromone signaling by direct binding to attractin.Abbreviations EGF, epidermal growth factor; HFBA, heptafluorobutyric acid; IPTG, isopropyl thio-b-D-galactoside; TEE, translation-enhancing element; TGF-b, transforming growth factor-b.

Whole genome analysis of a schistosomiasis-transmitting freshwater snail

“…glabrata proteins from snail conditioned water (SCW) and following electrostimulation (ES), which induces rapid release of proteins. The detection by NanoHPLC-MS/MS of an orthologue of temptin, a pheromone of Aplysia 12, among these proteins (Supplementary Note 5; Supplementary Data 10) suggests an operational pheromone sensory system in B . glabrata .…”

Analyses of genome architecture and gene expression reveal novel candidate virulence factors in the secretome of Phytophthora infestans

“…Copper ion binding (GO:0005507) is another molecular function enriched in the secretome. The pheromone activity (GO:0005186) enriched in the secretome is found in proteins similar to temptins, which mediates protein-cell surface contact during fertilization in mollusks [49]. A Phospholipase D (PLD) motif (PF00614) is among the Pfam domains enriched in the P. infestans secretome.…”

An EST screen from the annelid Pomatoceros lamarckii reveals patterns of gene loss and gain in animals

“…In contrast, the N-terminal of both the Pomatoceros and Aplysia proteins are only conserved with a different Aplysia protein, the pheromone temptin [GenBank: AAS92605] [53] and with a gene predicted from the genome of the basal metazoan lineage Trichoplax [GenBank: XP_002117562]. The function and structure of Aplysia temptin have been analysed in detail, and the central 1 region of temptin (squared with red lines in Figure 4C) as well as two disulfide bonds (shown by red lines and triangles in Figure 4C) determined by a structure analysis using recombinant temptin [54] are well conserved amongst these genes. We have found that this unique combination of the temptin and mono-oxygenase sequences is also present in the Lottia genome [JGI: jgi|Lotgi1|192843|estExt_Genewise1.C_sca_480139] but is not found outside the Lophotrochozoa.…”