Báo cáo khoa học: Aplysia temptin ) the ‘glue’ in the water-borne attractin pheromone complex pdf

Aplysia temptin ) the ‘glue’ in the water-borne attractin pheromone complex Scott F. Cummins 1,2 , Fang Xie 3 , Melissa R. de Vries 1 , Suresh P. Annangudi 3 , Milind Misra 4 , Bernard M. Degnan 2 , Jonathan V. Sweedler 3 , Gregg T. Nagle 1 and Catherine H. Schein 4,5 1 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA 2 School of Integrative Biology, University of Queensland, St Lucia, Australia 3 Department of Chemistry and the Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA 4 Sealy Center for Structural Biology, Department of Biochemistry and Molecular Biology, Sealy Center for Vaccine Design, University of Texas Medical Branch, Galveston, TX, USA 5 Department of Microbiology and Immunology, Sealy Center for Vaccine Design, University of Texas Medical Branch, Galveston, TX, USA Pheromones play an important role in coordinating male and female reproductive behavior in many aqua- tic 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 Keywords enticin; fibrillin; epidermal growth factor-like domains; Marfan’s syndrome; signaling network Correspondence C. H. Schein, 218 Clay Hall, Department of Biochemistry and Molecular Biology, UTMB, Galveston, TX 77555-0857, USA Fax: +1 409 747 6000 Tel: +1 409 747 6843 E-mail: chschein@utmb.edu (Received 29 June 2007, revised 15 August 2007, accepted 28 August 2007) doi:10.1111/j.1742-4658.2007.06070.x Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aply- sia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein–cell surface contact dur- ing 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 pre- dicted 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 pro- posed metal-binding loop. The water-borne pheromone attractin in egg cor- don 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 attr- actin. 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. FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS 5425 the albumen gland that play a role in water-borne pheromone signaling. The pheromone signal must radi- ate long distances (tens of meters from the secreting Aplysia organism) to attract other Aplysia organisms to fresh egg cordons [1,2]. Binary blends of attractin and either temptin, enticin, or seductin, three other characterized Aplysia protein pheromones that are released during egg laying, are sufficient to attract ani- mals in T-maze assays [3,4]. The first water-borne pro- tein pheromone characterized in invertebrates was the water-borne Aplysia pheromone attractin [5,6], a 58-residue protein released during egg laying that is involved in forming and maintaining egg-laying and mating aggregations in Aplysia [6–9]. The disulfide- bonding pattern and NMR solution structure indicated that attractin has a novel helical fold that most resembles the pheromones identified for unicellular organisms [10–12]. We have also characterized the disulfide-stabilized helical fold of enticin as being simi- lar to that of attractin [13]. In this article, we provide biophysical evidence that temptin, a third member of the complex, resembles the disulfide-linked, primarily b-strand structures of members of the epidermal growth factor-like (EGF), family and functions in pheromone signaling by binding to attractin in the protein bouquet. Native temptin, a 103-residue protein, was purified from albumen gland extracts by RP-HPLC, character- ized by N-terminal microsequence analyses of the puri- fied protein and of tryptic fragments, which defined the site of signal sequence cleavage, and cloned [3]. Temptin was 91% conserved between two geographi- cally and physically distinct species, Aplysia californica (Pacific coast) and Aplysia brasiliana (Gulf of Mexico) [3]. Sequence analysis of temptin indicated it would have a fold quite different from that of other members of the pheromone bouquet, such as enticin and attrac- tin. Fold recognition server results and a blast search indicated that temptin would have a fold similar to the Ca 2+ -binding, EGF-like domain of the extracellular matrix protein fibrillin [2]. This finding immediately suggested a function for temptin in pheromone signal- ing, as mutations in the multiple repeats of the EGF- like domains in fibrillin have been linked to Marfan’s syndrome, an autosomal dominant disease affecting connective tissues [14,15]. Whereas the disease was originally thought to be due to loss of elasticity in con- nective tissues, recent results show instead that fibrillin specifically binds to and controls the available levels of transforming growth factor-b (TGF-b), and that one treatment for Marfan’s syndrome is based on control- ling binding of TGF-b to its receptor [16]. Here, we present evidence that temptin has a similar role in the pheromone complex to that of fibrillin in the extra- cellular matrix, and mediates binding of attractin to sensory cells in the chemosensory rhinophores of target Aplysia. In the present study, we expressed and purified temptin and characterized its secondary structure with CD measurements. Recombinant full-length temptin was efficiently expressed in a soluble form using a recently developed ‘cold-shock’ method for protein expression in Escherichia coli [17,18], as demonstrated by immunoblot analysis. We determined temptin’s disulfide-bonding pattern using a combination of lim- ited proteolysis and MS. The CD spectrum of the puri- fied protein indicated that temptin was largely b-sheet in structure, consistent with the suggested homology to EGF-like domains of proteins involved in coagulation and interaction with the cell surface [2]. One of the disulfide bonds, determined by limited proteolysis and MS, was consistent with the previous alignment of the temptin sequences with EGF-like domains from other proteins. The closest related known structure, detected by fold recognition servers, was that of the type 1, Ca 2+ -binding, EGF-like repeat domains that are con- served in the fibrillins, notch proteins, and clotting factors. We generated a three-dimensional homology model for temptin, based on that of human fibrillin type I (Protein Data Bank file 1EMN), that was con- sistent with these data, and showed an extended, disul- fide-stabilized hairpin-like structure with a stabilized metal-binding loop. We also found, using protein gel shifts, that recombinant temptin formed complexes with attractin that resembled those in egg cordon elu- ates. Docking results with our model of temptin, and our previously determined NMR structure of attractin, indicated a preferred conformation for the complex that was consistent with these results. Thus, temptin may play a role during pheromone detection by organizing the interaction of attractin with the cell membrane. Further studies of temptin, e.g. with multi- dimensional NMR, should reveal how similar the structure of this protein pheromone is to those of other EGF-like proteins, and yield more information about its role in signal transduction in Aplysia. Results Recombinant expression of temptin using pCold III vector Using temptin cDNA as template, the region enco- ding the mature temptin protein was amplified by PCR and the resulting PCR product was cloned into a pCold III vector. The insert was present as deter- mined by PCR analysis and nucleotide sequence Aplysia temptin coordinates pheromone complex S. F. Cummins et al. 5426 FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS analysis. SDS ⁄ PAGE analysis demonstrated that temptin expression was induced by 0.1 mm isopropyl thio-b-d-galactoside (IPTG) after 20 h of incubation of bacteria at 15 °C, as judged by Coomassie Blue staining (data not shown). Recombinant temptin was soluble, as the protein was detected in the superna- tant following sonication and centrifugation of E. coli lysates. Following C18 Sep-Pak purification and C18 RP-HPLC purification of bacterial lysates containing temptin (see below), the presence of temptin was verified by Coomassie Blue staining (Fig. 1A; left panel) and western blot analysis using temptin anti- serum (Fig. 1A; right panel). Temptin was sequen- tially purified on C18 Sep-Pak cartridges and by C18 RP-HPLC using heptafluorobutyric acid (HFBA) as counterion. An immunoreactive temptin peak was obtained in pooled fractions 124–134 (Fig. 1B). The protein was further purified on a second RP-HPLC gradient using trifluoroacetic acid as counterion (data not shown) for CD spectra analyses and disul- fide bond determination. Temptin concentration was estimated by densitometry (integrated density value) following each stage of purification, and temptin was purified 9.0-fold relative to the bacterial cell lysate supernatant, with 23.9% recovery after the first RP-HPLC purification. CD spectra of temptin The CD spectrum of purified temptin (Fig. 2; 0.3 mgÆmL )1 ) was interpreted with the cdsstr program [19,20]. This indicated that the protein was primarily b-strand ⁄ turn, with < 10% helix. This was consistent with our alignment with the predominantly b-strand EGF-like domains of fibrillins (Fig. 3). A temptin-like expressed sequence tag cDNA that was recently isolated from the mollusk Haliotis [21] also aligns well with the temptin family. Fold recognition servers suggested 1EMN (fibrillin 1 in the sequence alignment of Fig. 3) as a potential template. However, temptin contains only four cysteine residues, and only two of these aligned with cysteine residues involved in disulfide bonds in the conserved EGF-like fold. Determination of the disulfide bond pattern in temptin The full-length amino acid sequence of mature temptin predicted by the cDNA is shown in Fig. 4; a 13-resi- due N-terminal translation-enhancing element (TEE) sequence encoded by the pCold vector is included in the final sequence. We next determined the disulfide- bonding pattern of the protein experimentally. Purified AB Fig. 1. Expression and purification of recombinant temptin. (A) SDS ⁄ PAGE and western blot analysis of recombinant temptin expression. Temptin expression was induced by 0.1 m M IPTG. After 20 h of induction, bacteria were lysed and the supernatant was fractionated in paral- lel SDS-polyacrylamide gels, one for Coomassie Blue staining (left panel) and the other for western blot analysis using temptin antiserum (right panel). The amount of soluble protein added in each lane corresponded to 30 lL of an initial 10 mL culture. The Coomassie Blue- stained gel in the left panel indicates the level of purity of temptin following C18 Sep-Pak Vac purification and two successive C18 RP-HPLC gradient purification steps using HFBA and trifluoroacetic acid, respectively, as counterions. Arrow, temptin protein. (B) RP-HPLC purification of pCold-expressed lysate containing recombinant temptin. The supernatant of a temptin-containing bacterial extract was purified on a C18 Sep-Pak Vac cartridge and fractionated by C18 RP-HPLC. The sample was eluted with a linear gradient of 0.1% HFBA and 100% CH 3 CN ⁄ 0.1% HFBA. Fractions indicated by the solid bar were pooled and lyophilized. Pooled fractions 124–134 from several RP-HPLC runs were repurified with a linear gradient of 0.1% trifluoroacetic acid and 100% CH 3 CN ⁄ 0.1% trifluoroacetic acid (data not shown) and analyzed by Coomassie Blue staining and western blot analysis (A). S. F. Cummins et al. Aplysia temptin coordinates pheromone complex FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS 5427 recombinant temptin expressed using the pCold III vector was assayed by MS and had a monoisotopic mass of 12405.7 Da, which corresponded to the pre- dicted mass of temptin with the addition of a 13-resi- due N-terminal bacterial TEE (residues ) 13 to ) 1in Fig. 4). The pCold III-expressed temptin was successfully used to determine the disulfide connectivity. Initial reduction and alkylation experiments resulted in the addition of four alkyl moieties, indicating the presence of two disulfide bonds in temptin (data not shown). To deduce the bonding pattern, the protein was ini- tially digested with trypsin, which cleaves after lysine and arginine residues. The enzymatically digested peptides were analyzed further by reduction–alkyl- ation. Figure 4 shows several MS spectra obtained fol- lowing enzymatic digestion and reduction–alkylation treatments that enabled determination of the disulfide linkages. Following trypsin digestion of temptin, four peaks were detected (1679.7 Da, 1814.7 Da, 2231.0 Da, and 6430.4 Da) that were consistent with four peptides containing free cysteines at positions 103, 57, 77, and 18, respectively. In addition, two other key peaks were detected (4043.6 Da and 8106.8 Da) that were consis- tent with fragments containing disulfide bonds between Cys57 and Cys77, and Cys18 and Cys103, respectively. This indicated that the first and fourth cysteines were disulfide-bonded and the second and third cysteines were disulfide-bonded. To confirm this pattern, trypsin digests were subjected to reduction–alkylation. Follow- ing this treatment, only four alkylated peaks (1737.5 Da, 1872.6 Da, 2289.2 Da, and 6488.7 Da) were detected, indicating that the disulfide bonds in those two peaks (4043.6 Da and 8106.8 Da) were bro- ken during reduction, and each peak was broken into the two corresponding alkylated peptides with free cysteines. These results confirmed that the disulfide- bonding pattern in recombinant temptin was Cys18- Cys103 and Cys57-Cys77 in the mature protein. Three-dimensional modeling of temptin The sequences of both A. californica and A. brasiliana temptin aligned well with the EGF-like domains of the Fig. 2. The CD spectrum of HPLC-purified recombinant temptin. The CD spectrum (+ signs) for temptin is shown overlaid with the CDSSTR2-reconstructed spectrum (rectangles), which is based on the protein sample containing 35% b-strand, 24% turn ⁄ coil, and 34% disordered structure. Fig. 3. Alignment of the central 1 region of temptin (residues 38–91 of the recombinant protein) with the central conserved Ca 2+ -binding, EGF-like domains of fibrillin sequences from human, chicken, and pufferfish. Residues that are identical in fibrillins, known temptins (A. cali- fornica, A. brasiliana) and a temptin-like expressed sequence tag sequence (Haliotis asinina) are shaded black; identical and similar residues within the temptin family are in dark gray (red in the online version), and those that are similar to conserved residues in the fibrillins are shaded light gray (green in the online version). Cysteines involved in the disulfide bond that is common to the fibrillins and temptins are marked by the top line, and the residues involved in the proposed metal-binding loop are shown by the bottom line; see Fig. 5C. The Trp52 and Trp79 residues that flank the two disulfide bonds in our model (shown in Fig. 5A) align with two conserved cysteines in the fibrillins. Aplysia temptin coordinates pheromone complex S. F. Cummins et al. 5428 FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS fibrillin family, with 11 of the residues being conserved in all five fibrillins (Fig. 3). Most significantly, the interior disulfide bond in temptin, determined experi- mentally, was also consistent with the disulfide bond between two of the EGF-like cysteines that these resi- dues aligned with. We generated a model based on the fibrillin 1 domain (Protein Data Bank structure 1EMN), which allowed us to generate the interior disulfide based on homology and insert the second by minimization (see Experimental procedures). The resulting model (Fig. 5) had several interesting fea- tures. The first was that the two tryptophan residues in temptin, which both aligned with conserved cysteine residues in fibrillins, flanked the two disulfide bonds. This suggested that they might serve as an electron- rich barrier to stabilize these disulfides from reduction. Trp79, near the interior disulfide, could also stabilize a possible negatively charged metal-binding loop (Fig. 5C). This metal-binding site is part of a ‘top face’ of temptin that could interact with the cell surface, consistent with that seen for the interaction of EGF with its receptor [22]. Fibrillins form part of the extracellular matrix; recent research has also revealed an active role for these proteins in signal transduction, in which they bind growth factors. To learn more about the possible intermediary role of temptin in the pheromone signal- ing process, we docked our previous NMR structure of attractin [10] to the temptin model. Two possible complexes (Fig. 5B,D) show a potential binding sur- face on the bottom face of temptin for attractin. In both configurations, the surface of the second attractin Relative Intensity 2231.0 1814.7 1679.7 2289.2 1872.6 1737.5 1200 1600 2000 2400 m/z 4043.6 6430.4 (Met-ox) 8106.8 6488.7 4000 6000 8000 5x 1 MNHKVHMELGTLEYPQYQAV IPNGSSVPNP 30 31 C N TSQ IAQGVGH INFQGTGPLNP FGED FKA 60 61 A G K Q W T T D L C DMDSDGDGRSNGVELGDPE C 90 91 V W S Q G E T P A R T T D L S H P G F D E A T V S C 116 A B C D T 101 -C 116 * * * * * Q 64 -R 79 S 80 -R 100 Q 64 -R 79 & S 80 -R 100 M 1 -K 59 M 1 -K 59 & T 101 -C 116 E Fig. 4. Summary of disulfide linkage determination showing diagnostic MALDI TOF MS spectra of enzymatic digests of recombinant temp- tin. Following digestion with trypsin, six key peptides were observed in (A), (C) and (E). The peak at m ⁄ z 6430.4 corresponds to the tryptic peptide Met13–Lys46 which includes the 13-residue bacterial TEE (Met13–Glu1 from the vector; dashed underline in sequence) and an oxidized methionine. Peaks at m ⁄ z 4043.6 Da and 8106.8 Da are dimers that are each composed of two disulfide-bonded peptides. After further reduction–alkylation of the whole enzymatic digest, four alkylated peptides were observed in (B) and (D) that confirmed the disulfide bond pattern (Cys18-Cys103, Cys57-Cys77) labeled on the sequence. (E) is an enlarged spectrum of the boxed area in (C). The MALDI matrix is a-cyano-4-hydroxycinnamic acid in (A), (C), and (E), and sinapinic acid in (B) and (D). The residues shown by an asterisk are the potential cleavage sites (C-terminal) for trypsin. S. F. Cummins et al. Aplysia temptin coordinates pheromone complex FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS 5429 helix, which we previously showed by mutagenesis to be essential for pheromonal attraction [8], is turned outwards, whereas the first helix forms a multitude of contacts (atom–atom distances less than 3 A ˚ ) to the bottom face of temptin. These complexes suggest how temptin could mediate binding of attractin to phero- mone signaling proteins in the rhinophores of Aplysia. Temptin complexes with attractin to form complexes similar to those seen in egg cordon eluates We used gel shifts and immunoblotting to determine whether temptin would indeed complex with attractin (Fig. 6). Essentially all the immunoreactive attractin in the extracts from the egg cordon of A. californica is complexed with other proteins, as it runs signifi- cantly above the position of the free protein. Adding recombinant temptin to attractin, at an equimolar ratio, was sufficient to shift all the immunoreactive protein to a higher position on the gel, similar to that seen for the egg cordon eluate. This indicates that temptin can form complexes with attractin, which may enhance its binding to sensory cells in the target Aplysia (or prevent degradation of the protein). Attempts to determine which face of attrac- tin was involved in the binding, using our previously described mutant proteins [8], were unsuccessful, as both of these proteins were difficult to detect with the attractin antibody. A C B D W79 W52 C18-C103 C57-C77 Backbone O- G65,D64, S61, G63 R66 OD2-D64 OD1,2-D62 W79 S67-O Fig. 5. Views of a three-dimensional model structure of temptin alone (A, C) and in docked complexes with attractin (B, D). (A) Ribbon dia- gram of the temptin model, showing the two disulfide bridges (Cys18-Cys103, Cys57-Cys77) and their flanking tryptophan residues (Trp52 and Trp79, respectively). (C) Solid view of the model, same orientation as (A), showing the proposed metal-binding loop stabilized by Trp79 (right side, labeled residues). (B, D) Ribbon diagram (B) and space-filling view (D) of possible complexes of temptin with attractin (the lowest and 10th lowest energy complexes from ZDock, respectively). Temptin is in approximately the same orientation as in (A) and (C), but is tilted slightly away from the viewer (rotation arrow to the right) to show the interacting face with the first helix of attractin; attractin residues that are in closest contact with temptin are Met15 and Cys20–Thr28. The side chains of interacting residues in (B) are colored blue for temptin and brown for attractin. In (D), attractin atoms are colored according to the Corey–Pauling–Koltun convention (O, red; C, black; N, blue; S, yellow), whereas all atoms of temptin are colored khaki. In both (B) and (D), the residues in the second helix of attractin, which constitute the conserved pheromone signaling site [8], face outwards towards the viewer. Aplysia temptin coordinates pheromone complex S. F. Cummins et al. 5430 FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS Expression of temptin in other Aplysia tissues The sequencing of the A. californica genome (http:// www.ncbi.nlm.nih.gov ⁄ ) revealed several isoforms of temptin (temptins 2–5 in Fig. 3). Northern blot analy- sis (Fig. 7) indicated these other isoforms may be expressed in other Aplysia tissues, particularly in the ovotestis, central nervous system, and buccal muscle; the sizes of the cDNAs agreed with the predicted sizes of the albumen gland transcript. This suggests that temptins may have similar, but not identical, functions in other Aplysia tissues, and may indeed bind other Aplysia growth factors in these tissues. To date, no molluskan fibrillin has been character- ized. A tblastn search of the A. californica genomic sequence database at NCBI GenBank (http://www. ncbi.nlm.nih.gov ⁄ ), using the Homo sapiens sequence of fibrillin 1 (accession number CAA45118) revealed one candidate sequence, contig AASC01127679.1. This encodes a protein similar to those of the Notch protein family, conserved throughout the Metazoa, which are membrane receptors that contain Ca 2+ -binding EGF- like repeats similar to those of fibrillins [23]. Notch proteins use these repeats to control extracellular signaling, in areas distinct from fibrillins. This lends further support to our suggestion that molluskan temptin is derived from a common ancestral gene that also yielded EGF and the EGF-like family of signaling proteins. Discussion We originally identified temptin as one of four proteins that were expressed in the exocrine albumen gland and released during egg laying, and showed, using T-maze attraction assays, that temptin was an essential compo- nent of the pheromone bouquet that attracts Aplysia to freshly laid egg cordons [3]. Here, we expressed and purified recombinant temptin (Fig. 1) to use in struc- tural and functional studies. Our results indicate that temptin has a fold and function similar to those of repeat EGF-like domains in human fibrillins [24]. The evidence for this is as follows: (a) the CD spectrum of the purified recombinant protein is predominantly b-strand (Fig. 2); (b) the sequence alignment (Fig. 3) shows the conservation of residues in both temptin sequences, and the other isoforms known, with those common to all fibrillins; (c) this alignment also predicts the correct position of a disulfide bond found for recombinant temptin (Fig. 4); (d) modeling and Fig. 6. Gel shift experiments show that recombinant temptin forms complexes with recombinant attractin that are similar in size to the com- plexed forms of attractin seen in egg cordon eluates. A western blot is shown, developed with antibody to attractin. Recombinant attractin (1 nmol) was combined with a 1 : 1 or 1 : 5 molar excess of temptin, fractionated on a native gel, and transferred as described in Experi- mental procedures. The picture is a composite of three experiments, with a repetition of the temptin–attractin equimolar complex. From left to right, attractin, temptin, and the complexes, a Coomassie Blue stain, followed by a blot of egg cordon eluates, and a repeat gel showing attractin, temptin and their 1 : 1 complex. For Coomassie Blue visualization, proteins were loaded at 5 nmol each. Arrow 1 shows the posi- tion of unshifted, native attractin, Arrow 2 indicates the middle of the shifted attractin–temptin complex (which migrates nonuniformly on the nondenaturing gel). S. F. Cummins et al. Aplysia temptin coordinates pheromone complex FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS 5431 docking results show a structure that could mediate the interaction of attractin with the cell surface of tar- get Aplysia (Fig. 5), and gel shift results show the for- mation of a stable, high molecular weight complex of temptin with attractin (Fig. 6). Although the disulfide bonds are fewer than those that stabilize other EGF- like proteins, our model suggests that those in temptin are stabilized by a novel mechanism. Individual trypto- phan side chains overlay both disulfides, suggesting that they could serve as electron-rich barriers to reduc- tion in seawater. A region between the two cysteines that form the internal disulfide (Fig. 5C) has a nega- tively charged surface ideal for binding hard metals such as Ca 2+ or Mg 2+ [25]. Temptin might thus repre- sent a homolog of the EGF family. One could propose that the mechanism of structure stabilization by over- lapping disulfides that stabilize the EGF fold [26] evolved from a simpler architecture in more oxidizing environments. Further evidence for this is that the pufferfish homolog of fibrillin (Fig. 3) has a valine in place of one of the conserved six cysteine residues characteristic of mammalian EGF repeats. Our results are particularly important, as they assign a role for temptin in the pheromone signaling pathway that may have evolutionary significance. Binary combinations of attractin and temptin are sufficient to stimulate mate attraction and are thought to act in concert with enticin and seductin, which are also released during egg laying [2,3]. The presence of an expressed temptin-like cDNA in the abalone Haliotis (Fig. 3) and several temptin-like iso- forms within the genome of the limpet Lottia (data not shown) suggests that a temptin-like protein could play a similar role in coordinating pheromone signal- ing in other mollusks. Blends of soluble pheromones have also been characterized in vertebrates, e.g. the salamander Plethodon, which contains a pheromone blend consisting of plethodontid-modulating factor, plethodontid receptivity factor, and sodefrin precur- sor-like factor [27]. Plethodontid-modulating factor is a hypervariable courtship pheromone that is a mem- ber of the three-finger protein superfamily, all of which share a relatively simple three-dimensional structure consisting of three adjacent ‘finger-like’ loops extending from a small, hydrophobic core that is crosslinked by a common disulfide bond bridging pattern. All plethodontid-modulating factor sequences contain a conserved  20-residue signal sequence and a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms, as well as xenotoxins from Xenopus [27]. Expression and biophysical characterization of recombinant temptin Temptin was efficiently expressed in a soluble form using the pCold bacterial expression system (Fig. 1A). Although temptin has been previously expressed in insect cells [3], the present study demonstrates a better method for expressing the protein, as: (a) larger amounts of temptin protein are obtained relative to the insect cell expression system (data not shown); (b) temptin comprises a significant percentage of the expressed peptides and protein, as judged by RP- HPLC (Fig. 1B); and (3) the labeling of bacterially expressed proteins in defined media for NMR struc- tural studies is relatively inexpensive as compared to using the insect cell expression system. An important question is whether the recombinant protein folded in a similar way to the native protein. The same disulfide folding was observed when using native and recombinant Aplysia enticin [13]. In the case of enticin, the additional 13-residue tag at the N-terminus of recombinant protein, which contained a bacterial TEE, did not affect the activity or pattern of Cys-Cys bonding, as the disulfide-bonding patterns were identical in native and recombinant enticin. Thus, Fig. 7. Tissue distribution of Aplysia temptin mRNAs. The blot was overexposed to detect the presence of isoforms in tissues outside the albumen gland. Total RNA (10 lg) was isolated from the albu- men gland, atrial gland, large hermaphroditic duct (LHD; combined red and white hemiduct), small hermaphroditic duct (SHD), ovotes- tis, pooled central nervous system (CNS; pooled cerebral, pleural, pedal, buccal, and abdominal ganglia) and buccal muscle, and frac- tionated on agarose ⁄ 1% formaldehyde gels, and the membranes were hybridized with radiolabeled cDNA probes for temptin. RNA size markers (Invitrogen) are indicated. Equivalent amounts of RNA were loaded in each lane and confirmed by ethidium bromide stain- ing. Autoradiography was performed for 24 h. Aplysia temptin coordinates pheromone complex S. F. Cummins et al. 5432 FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS the pattern indicated by trypsin digestions⁄ MALDI- TOF MS analysis from the recombinant temptin (I–IV and II–III) is most likely the correct one. Role of temptin as the glue in the attractin complex Our model of temptin, based on the class 1 EGF-like domain of fibrillin [24], and the gel shift experiments (Figs 5 and 6), indicate that this protein could serve to organize the pheromone complex and facilitate signal- ing, by binding to both the pheromone and a cell sur- face receptor. A crystal structure of the complex of EGF with the extracellular domain of its receptor indi- cated that EGF can bring two receptor domains together, from two different binding surfaces [19]. Our docking and gel shift results indicate a similar role for temptin in the signaling complex, whereby attractin would bind with one conserved face to temptin, while still displaying the residues on the second helix that are essential for pheromone activity [8]. Temptin could also mediate the activity of another pheromone com- plex protein, enticin [13]. This role for temptin would be consistent with a direct role for fibrillin in mediating TGF-b signaling that has direct therapeutic implications. Data accu- mulated since 1991 from Marfan’s syndrome patients have shown mutations in fibrillins, particularly those that affect the structure and number of EGF-like domains. Whereas this was taken to mean that the symptoms were due to a weakness in the extracellu- lar matrix that contains fibrillin, more recently it was shown that mutations also decreased the ability of fibrillins to bind and control the activity of TGF-b; many symptoms of Marfan’s syndrome can also be found in patients with mutations in the receptor for this factor [28]. Reagents that antagonize TGF-b binding (such as losartan) can completely reverse the symptoms of fibrillin mutants in a mouse model; these results form the basis for a clinical trial in humans with Marfan’s syndrome [16]. In an analogous fashion, we propose that temptin modulates attractin signaling during the mate attrac- tion process, and either enhances its ability to bind to cells or controls its free concentration. Alternatively, complexation with temptin may be necessary for attractin to bind to the host cell. For example, the parasitic protozoan, Eimeria tenella, synthesizes a microneme protein (EtMIC4) containing multiple extracellular Ca 2+ -binding EGF-like repeats [29] that forms a high molecular mass ([EtMIC4] 8 [EtMIC5] 4 ; > 2 MDa) complex with a soluble protein EtMIC5. This complex, secreted to the protozoan surface, mediates the binding of the parasite to mammalian cells [30]. Does temptin serve as Aplysia fibrillin? Although our primary purpose was to determine the role of temptin in pheromone signaling, the northern blot (Fig. 7) indicated expression of temptin isoforms in muscle and other tissues. We are only beginning to understand the full functions of the extracellular matrix proteins, such as fibrillins [31], in mammalian intercellular signaling and control. If temptin does indeed represent the mollusk fibrillin, then it may play a more general role in the mollusk temptin isoforms expressed in the central nervous system and buccal muscle (Fig. 7) of Aplysia, and support a comparable role in the binding of other proteins, such as growth factors or those involved in connective tissue organiza- tion. Conclusions These results indicate that temptin has a disulfide- stabilized structure that resembles that of metal-bind- ing, class 1 EGF-like domains found in mammalian factors that play a major role in formation of the extracellular matrix. As with these proteins, temptin could regulate the activity of the attractin complex by mediating the binding of the potent pheromone signaling molecule, attractin, to chemosensory cells in the sensory rhinophores of target Aplysia . Possibly, the signaling complex contains many copies of temp- tin, and further structural characterization of the Aplysia pheromone bouquet proteins should provide more details about how these molecules function in the signaling that initiates reproduction in mollusks. The recombinant protein isolation methods developed during this work will aid in determining the three- dimensional structure of temptin, as we previously did for attractin [10]. Experimental procedures Cloning of temptin in a pCold expression vector PCR was used to amplify residues 23–125 of the temptin precursor using A. californica temptin cDNA as template (GenBank Accession number AY309079; bases 89–397); this corresponds to the mature full-length temptin protein [3]. The sense primer (5¢-ACTT CTCGAGTACCCCCAAT ACCAG-3¢) was synthesized with an XhoI site (underlined). The antisense primer (5¢-TACT GAATTCTTAGCAC GATACTGTAGC-3¢) was synthesized with a stop codon S. F. Cummins et al. Aplysia temptin coordinates pheromone complex FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS 5433 followed by an EcoRI site (underlined). Samples were heated at 94 °C for 3 min and amplified for 36 cycles (94 °C, 1 min; 50 °C, 1 min; 72 °C, 1 min), and this was followed by a 7 min extension at 72 °C. PCR products were cloned into the bacterial expression vector pCold III (Takara Shuzo Co. Ltd., Shiga, Japan) and transfected into Origami (DE3) bacteria. Recombinant plasmids were sequenced to confirm that no base changes had been intro- duced during PCR amplification. Expression and purification of recombinant temptin and native temptin Expression of recombinant temptin was optimized in pCold III-transfected Origami bacteria by controlling both temperature and IPTG concentration. Single colonies of bacteria were used to inoculate 10 mL of overnight cultures and incubated at 37 °C in LB medium containing ampicil- lin, with shaking. An aliquot of the overnight culture was inoculated into fresh LB medium (1 : 100) containing ampi- cillin. Bacteria were incubated at 37 °CtoaD 600 of 0.6, chilled for 30 min at 15 °C, induced with 0.1 mm IPTG, and grown for an additional 20 h at 15 ° C. Cells were cen- trifuged (using a GLC-2B Sorvall centrifuge and HL-4 rotor at 4 °C), the pellets were frozen () 70 °C) and resus- pended (10 mm Tris ⁄ HCl, pH 8.0, 2 mm EDTA) at 4 °C, lysozyme (100 lgÆmL )1 ) and Triton X-100 (0.1% v ⁄ v) were added, and the lysates were incubated for 15 min at 30 °C. Lysates were sonicated and centrifuged (20 000 g using a J2-21 centrifuge, Beckman, rotor type JA-20 for 30 min; 4 °C), and the supernatant was heated (5 min; 95 °C) and fractionated by SDS ⁄ PAGE to examine temptin expression. Supernatants were also purified on C18 Sep-Pak Vac car- tridges (5 g; Waters Corp., Milford, MA, USA) prior to RP-HPLC. Peptides were eluted with 70% acetonitrile (CH 3 CN) ⁄ 0.1% HFBA and lyophilized, and the lyophiliz- ate was resuspended in 0.1% HFBA and purified on a semipreparative Vydac C18 RP-HPLC column (1 · 25 cm) using a two-step linear gradient (0–10% CH 3 CN ⁄ 0.1% HFBA in 5 min; 10–65% CH 3 CN ⁄ 0.1% HFBA in 195 min). Fractions were pooled, lyophilized and repurified using the same gradient conditions, except that 0.1% tri- fluoroacetic acid was the counterion. Native albumen gland temptin was purified using RP-HPLC as previously described [3]. SDS ⁄ PAGE and western blot analyses Protein was quantified using the Protein Coomassie Blue Assay Kit (Bradford method; Bio-Rad, Hercules, CA, USA). Sep-Pak- and RP-HPLC-purified samples of temp- tin were mixed with sample buffer (75 mm Tris ⁄ HCl, pH 6.8, 20% glycerol, 10% 2-mercaptoethanol, 4% SDS, 0.25% Bromophenol Blue) and fractionated on 12% SDS-polyacrylamide gels. Gels were either stained with Coomassie Brilliant Blue R-250, or were used for immu- noblot analyses, performed essentially as previously described [3]; membranes were incubated with temptin antiserum (1 : 500 dilution). Details of temptin antiserum production have been previously described [3]. As a nega- tive control, the primary antiserum was replaced with temptin antiserum preincubated with the corresponding antigen (20 lgÆmL )1 ). Detection of temptin was performed using the ECL western blotting detection system (Amer- sham Biosciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. Band density on SDS ⁄ PAGE gels was quantified using an Alpha Imager (Alpha Inno- tech Corp., San Leandro, CA, USA). Gel shift analysis of complex formation between recombinant attractin and temptin Proteins were resuspended in filtered seawater (1 nmol of attractin, 1 nmol of temptin, 1 nmol of attractin ⁄ 1 nmol of temptin, 1 nmol of attractin ⁄ 5 nmol of temptin, 20 lgof egg eluate), and then mixed with native gel sample buffer (75 mm Tris ⁄ HCl, pH 6.8, 50% glycerol, 0.25% Bromophe- nol Blue) to a final volume of 5 lL. Preparations were applied to a 12% Tris ⁄ glycine gel (375 mm Tris ⁄ HCl, pH 8.0) and run at constant voltage (120 V) for 60 min. Gels were used for immunoblot analyses, performed essen- tially as previously described [3]. Details of attractin antise- rum production have been previously described [3]. As a negative control, the primary antiserum was replaced with attractin antiserum preincubated with the corresponding antigen (20 lgÆmL )1 ). Blots were incubated with Super- Signal chemiluminescence reagent (Pierce, Rockford, IL, USA); light emission was detected with autoradiography films. For Coomassie stain visualization, proteins were pre- pared as described above and loaded onto the same gel (5 nmol). CD To characterize the secondary structure of temptin, CD analysis was performed using a 0.1 cm path length cell at 0.2 nm intervals with two scans (190–250 nm) averaged for each at 25 °C (Jasco J-715 spectropolarimeter, JASCO, Easton, MD, USA). Lyophilized temptin was slowly dissolved in 10 mm sodium phosphate buffer (pH 6.5), centrifuged to remove precipitated protein at 15 000 g using a 5415D Eppendorf centrifuge and GE 009 rotor, and then concentrated by centrifugation (7000 g using a J2-21 centri- fuge; Beckman, and rotor type JA-20) in a Centricon 3 con- centrator (Amicon, Beverly, MA, USA) to a volume of 0.5 mL. After one more addition of buffer and concentra- tion, the protein was diluted with sodium phosphate (pH 6.5) to a final concentration of 0.3 mgÆmL )1 . Far-UV CD spectra were taken at a protein concentration of 0.1 mgÆmL )1 . The resultant spectra were corrected for the Aplysia temptin coordinates pheromone complex S. F. Cummins et al. 5434 FEBS Journal 274 (2007) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]...S F Cummins et al buffer signal The CD spectrum of temptin was interpreted with the programs k2d, contin, and cdsstr, using the DICHROWEB site [32,33]; all three programs indicated a predominantly b-strand secondary structure for temptin, but cdsstr2 gave the best spectral reconstruction (Fig 2) Determination of the disulfide bond pattern in temptin Portions of recombinant HPLC-purified temptin (and... with the two different MALDI matrix solutions onto the target plate as described above for MALDI-TOF MS analysis Aplysia temptin coordinates pheromone complex Protein modeling The temptin sequences from A californica and A brasiliana were aligned with proteins suggested by the fold recognition server results (Fig 3) The 1EMN template of two Ca2+binding, EGF-like domains from fibrillin [24] using the. .. used The final model had an overall backbone rmsd to the ˚ template of 3.66 A, and for the N-terminal 65 residues, ˚ Most of the deviations were in the C-terminus of the 2.5 A molecule, especially those necessitated by insertion of the Cys18-C103 linkage Molecular docking of the temptin model with the NMR structure of attractin and our model of enticin were done with the zdock program [37] Northern... native temptin 1–103 with 1EMN [11,34,35] To fix the two disulfide bonds, the Diamod model from mpack (before FANTOM minimization) was first minimized using the Biopolymer module of sybyl (version 7.1; Tripos, St Louis, MO, USA) using the Amber02 force field with Amber charges after specifying only the internal disulfide The second disulfide (Cys18Cys10 3) was then specified, and the model was minimized with the. .. DK070285 to J V Sweedler); protein modeling FEBS Journal 274 (200 7) 5425–5437 ª 2007 The Authors Journal compilation ª 2007 FEBS 5435 Aplysia temptin coordinates pheromone complex S F Cummins et al and analysis of interacting surfaces were supported by grants from the NIH (R01AI064913-0 1) and the EPA to Werner Braun and C H Schein We acknowledge the technical assistance of the UTMB Protein Chemistry Laboratory... References 13 1 Cummins SE, Schein CH, Xu Y, Braun W & Nagle GT (200 5) Molluscan attractins, a family of water-borne protein pheromones with interspecific attractiveness Peptides 26, 121–129 2 Cummins SF, Nichols AE, Schein CH & Nagle GT (200 6) Newly identified water-borne protein pheromones interact with attractin to stimulate mate attraction in Aplysia Peptides 27, 597–606 3 Cummins SF, Nichols AE,... Hummon AB, Sweedler JV & Nagle GT (200 4) Characterization of Aplysia enticin and temptin, two novel water-borne protein pheromones that act in concert with attractin to stimulate mate attraction J Biol Chem 279, 25614– 25622 4 Cummins SF, Nichols AE, Warso CJ & Nagle GT (200 5) Aplysia seductin is a water-borne protein pheromone that acts in concert with attractin to stimulate mate attraction Peptides... GT & Painter SD (199 7) Molecular cloning of a cDNA encoding a potential water-borne pheromonal attractant released during Aplysia egg laying Mol Brain Res 48, 167–170 6 Painter SD, Clough B, Garden RW, Sweedler JV & Nagle GT (199 8) Characterization of Aplysia attractin, the first water-borne peptide pheromone in invertebrates Biol Bull 194, 120–131 7 Painter SD, Clough B, Black S & Nagle GT (200 3) Behavioral... scoring matrix (PSSM), had a low TITO score and significant identity (after gapping) Whereas the alignment with 1EMN gave good matching of the cysteine residues Cys57 and Cys77, and was consistent with the bond between these residues being the same as that in the template, the bond between Cys18 and Cys103 was not in the template Homology modeling was carried out with the mpack program suite, using... characterization of attractin, a water-borne peptide pheromone in the genus Aplysia Biol Bull 205, 16–25 8 Painter SD, Cummins SF, Nichols AE, Akalal DB, Schein CH, Braun W, Smith JS, Susswein AJ, Levy M, de Boer PA et al (200 4) Structural and functional analysis of Aplysia attractins, a family of water-borne protein pheromones with interspecific attractiveness Proc Natl Acad Sci USA 101, 6929–6933 9 Susswein AJ . could mediate binding of attractin to phero- mone signaling proteins in the rhinophores of Aplysia. Temptin complexes with attractin to form complexes similar. proteins, temptin could regulate the activity of the attractin complex by mediating the binding of the potent pheromone signaling molecule, attractin, to