Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2001 Jul;81(1):463–472. doi: 10.1016/S0006-3495(01)75714-1

Aplysia attractin: biophysical characterization and modeling of a water-borne pheromone.

C H Schein 1, G T Nagle 1, J S Page 1, J V Sweedler 1, Y Xu 1, S D Painter 1, W Braun 1
PMCID: PMC1301526  PMID: 11423429

Abstract

Attractin, a 58-residue protein secreted by the mollusk Aplysia californica, stimulates sexually mature animals to approach egg cordons. Attractin from five different Aplysia species are approximately 40% identical in sequence. Recombinant attractin, expressed in insect cells and purified by reverse-phase high-performance liquid chromatography (RP-HPLC), is active in a bioassay using A. brasiliana; its circular dichroism (CD) spectrum indicates a predominantly alpha-helical structure. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) characterization of proteolytic fragments identified disulfide bonds between the six conserved cysteines (I-VI, II-V, III-IV, where the Roman numeral indicates the order of occurrence in the primary sequence). Attractin has no significant similarity to any other sequence in the database. The protozoan Euplotes pheromones were selected by fold recognition as possible templates. These diverse proteins have three alpha-helices, with six cysteine residues disulfide-bonded in a different pattern from attractin. Model structures with good stereochemical parameters were prepared using the EXDIS/DIAMOD/FANTOM program suite and constraints based on sequence alignments with the Euplotes templates and the attractin disulfide bonds. A potential receptor-binding site is suggested based on these data. Future structural characterization of attractin will be needed to confirm these models.

Full Text

The Full Text of this article is available as a PDF (358.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adler M., Carter P., Lazarus R. A., Wagner G. Cysteine pairing in the glycoprotein IIbIIIa antagonist kistrin using NMR, chemical analysis, and structure calculations. Biochemistry. 1993 Jan 12;32(1):282–289. doi: 10.1021/bi00052a036. [DOI] [PubMed] [Google Scholar]
  2. Akada R., Minomi K., Kai J., Yamashita I., Miyakawa T., Fukui S. Multiple genes coding for precursors of rhodotorucine A, a farnesyl peptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides. Mol Cell Biol. 1989 Aug;9(8):3491–3498. doi: 10.1128/mcb.9.8.3491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brown L. R., Mronga S., Bradshaw R. A., Ortenzi C., Luporini P., Wüthrich K. Nuclear magnetic resonance solution structure of the pheromone Er-10 from the ciliated protozoan Euplotes raikovi. J Mol Biol. 1993 Jun 5;231(3):800–816. doi: 10.1006/jmbi.1993.1327. [DOI] [PubMed] [Google Scholar]
  5. Chang J. Y. The disulfide folding pathway of tick anticoagulant peptide (TAP), a Kunitz-type inhibitor structurally homologous to BPTI. Biochemistry. 1996 Sep 10;35(36):11702–11709. doi: 10.1021/bi9606915. [DOI] [PubMed] [Google Scholar]
  6. Coggeshall R. E. The structure of the accessory genital mass in Aplysia californica. Tissue Cell. 1972;4(1):105–127. doi: 10.1016/s0040-8166(72)80011-9. [DOI] [PubMed] [Google Scholar]
  7. Cuff J. A., Barton G. J. Evaluation and improvement of multiple sequence methods for protein secondary structure prediction. Proteins. 1999 Mar 1;34(4):508–519. doi: 10.1002/(sici)1097-0134(19990301)34:4<508::aid-prot10>3.0.co;2-4. [DOI] [PubMed] [Google Scholar]
  8. Fan X., Wu B., Nagle G. T., Painter S. D. Molecular cloning of a cDNA encoding a potential water-borne pheromonal attractant released during Aplysia egg laying. Brain Res Mol Brain Res. 1997 Aug;48(1):167–170. doi: 10.1016/s0169-328x(97)00154-x. [DOI] [PubMed] [Google Scholar]
  9. Frishman D., Argos P. Seventy-five percent accuracy in protein secondary structure prediction. Proteins. 1997 Mar;27(3):329–335. doi: 10.1002/(sici)1097-0134(199703)27:3<329::aid-prot1>3.0.co;2-8. [DOI] [PubMed] [Google Scholar]
  10. Higgins D. G., Bleasby A. J., Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992 Apr;8(2):189–191. doi: 10.1093/bioinformatics/8.2.189. [DOI] [PubMed] [Google Scholar]
  11. Hänggi G., Braun W. Pattern recognition and self-correcting distance geometry calculations applied to myohemerythrin. FEBS Lett. 1994 May 16;344(2-3):147–153. doi: 10.1016/0014-5793(94)00366-1. [DOI] [PubMed] [Google Scholar]
  12. Iwata T., Umezawa K., Toyoda F., Takahashi N., Matsukawa H., Yamamoto K., Miura S., Hayashi H., Kikuyama S. Molecular cloning of newt sex pheromone precursor cDNAs: evidence for the existence of species-specific forms of pheromones. FEBS Lett. 1999 Sep 3;457(3):400–404. doi: 10.1016/s0014-5793(99)01062-5. [DOI] [PubMed] [Google Scholar]
  13. Jia L. G., Wang X. M., Shannon J. D., Bjarnason J. B., Fox J. W. Function of disintegrin-like/cysteine-rich domains of atrolysin A. Inhibition of platelet aggregation by recombinant protein and peptide antagonists. J Biol Chem. 1997 May 16;272(20):13094–13102. doi: 10.1074/jbc.272.20.13094. [DOI] [PubMed] [Google Scholar]
  14. Jiménez C. R., van Veelen P. A., Li K. W., Wildering W. C., Geraerts W. P., Tjaden U. R., van der Greef J. Neuropeptide expression and processing as revealed by direct matrix-assisted laser desorption ionization mass spectrometry of single neurons. J Neurochem. 1994 Jan;62(1):404–407. doi: 10.1046/j.1471-4159.1994.62010404.x. [DOI] [PubMed] [Google Scholar]
  15. Jones D. T. GenTHREADER: an efficient and reliable protein fold recognition method for genomic sequences. J Mol Biol. 1999 Apr 9;287(4):797–815. doi: 10.1006/jmbi.1999.2583. [DOI] [PubMed] [Google Scholar]
  16. Jones D. T. Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol. 1999 Sep 17;292(2):195–202. doi: 10.1006/jmbi.1999.3091. [DOI] [PubMed] [Google Scholar]
  17. Jones M. D., Patterson S. D., Lu H. S. Determination of disulfide bonds in highly bridged disulfide-linked peptides by matrix-assisted laser desorption/ionization mass spectrometry with postsource decay. Anal Chem. 1998 Jan 1;70(1):136–143. doi: 10.1021/ac9707693. [DOI] [PubMed] [Google Scholar]
  18. Karplus K., Barrett C., Hughey R. Hidden Markov models for detecting remote protein homologies. Bioinformatics. 1998;14(10):846–856. doi: 10.1093/bioinformatics/14.10.846. [DOI] [PubMed] [Google Scholar]
  19. Kikuyama S., Toyoda F., Ohmiya Y., Matsuda K., Tanaka S., Hayashi H. Sodefrin: a female-attracting peptide pheromone in newt cloacal glands. Science. 1995 Mar 17;267(5204):1643–1645. doi: 10.1126/science.7886452. [DOI] [PubMed] [Google Scholar]
  20. Kikuyama S., Toyoda F. Sodefrin: a novel sex pheromone in a newt. Rev Reprod. 1999 Jan;4(1):1–4. doi: 10.1530/ror.0.0040001. [DOI] [PubMed] [Google Scholar]
  21. Koradi R., Billeter M., Wüthrich K. MOLMOL: a program for display and analysis of macromolecular structures. J Mol Graph. 1996 Feb;14(1):51-5, 29-32. doi: 10.1016/0263-7855(96)00009-4. [DOI] [PubMed] [Google Scholar]
  22. Li L., Moroz T. P., Garden R. W., Floyd P. D., Weiss K. R., Sweedler J. V. Mass spectrometric survey of interganglionically transported peptides in Aplysia. Peptides. 1998;19(8):1425–1433. doi: 10.1016/s0196-9781(98)00094-1. [DOI] [PubMed] [Google Scholar]
  23. Luginbühl P., Wu J., Zerbe O., Ortenzi C., Luporini P., Wüthrich K. The NMR solution structure of the pheromone Er-11 from the ciliated protozoan Euplotes raikovi. Protein Sci. 1996 Aug;5(8):1512–1522. doi: 10.1002/pro.5560050807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Luporini P., Miceli C., Ortenzi C., Vallesi A. Ciliate pheromones. Prog Mol Subcell Biol. 1996;17:80–104. doi: 10.1007/978-3-642-80106-8_5. [DOI] [PubMed] [Google Scholar]
  25. Meyer F., Schmidt H. J., Heckmann K. Pheromone 4 gene of Euplotes octocarinatus. Dev Genet. 1992;13(1):16–25. doi: 10.1002/dvg.1020130104. [DOI] [PubMed] [Google Scholar]
  26. Miceli C., La Terza A., Bradshaw R. A., Luporini P. Structural characterization of mating pheromone precursors of the ciliate protozoan Euplotes raikovi. High conservation of pre and pro regions versus high variability of secreted regions. Eur J Biochem. 1991 Dec 18;202(3):759–764. doi: 10.1111/j.1432-1033.1991.tb16430.x. [DOI] [PubMed] [Google Scholar]
  27. Mronga S., Luginbühl P., Brown L. R., Ortenzi C., Luporini P., Bradshaw R. A., Wüthrich K. The NMR solution structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi. Protein Sci. 1994 Sep;3(9):1527–1536. doi: 10.1002/pro.5560030918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mumenthaler C., Braun W. Predicting the helix packing of globular proteins by self-correcting distance geometry. Protein Sci. 1995 May;4(5):863–871. doi: 10.1002/pro.5560040506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Painter S. D., Clough B., Garden R. W., Sweedler J. V., Nagle G. T. Characterization of Aplysia attractin, the first water-borne peptide pheromone in invertebrates. Biol Bull. 1998 Apr;194(2):120–131. doi: 10.2307/1543042. [DOI] [PubMed] [Google Scholar]
  30. Rost B. PHD: predicting one-dimensional protein structure by profile-based neural networks. Methods Enzymol. 1996;266:525–539. doi: 10.1016/s0076-6879(96)66033-9. [DOI] [PubMed] [Google Scholar]
  31. Rost B. Twilight zone of protein sequence alignments. Protein Eng. 1999 Feb;12(2):85–94. doi: 10.1093/protein/12.2.85. [DOI] [PubMed] [Google Scholar]
  32. Smith J. S., Miller B. T., Knock S. L., Kurosky A. Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization. Anal Biochem. 1991 Aug 15;197(1):247–253. doi: 10.1016/0003-2697(91)90384-6. [DOI] [PubMed] [Google Scholar]
  33. Soman K. V., Midoro-Horiuti T., Ferreon J. C., Goldblum R. M., Brooks E. G., Kurosky A., Braun W., Schein C. H. Homology modeling and characterization of IgE binding epitopes of mountain cedar allergen Jun a 3. Biophys J. 2000 Sep;79(3):1601–1609. doi: 10.1016/S0006-3495(00)76410-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stewart A. E., Raffioni S., Chaudhary T., Chait B. T., Luporini P., Bradshaw R. A. The disulfide bond pairing of the pheromones Er-1 and Er-2 of the ciliated protozoan Euplotes raikovi. Protein Sci. 1992 Jun;1(6):777–785. doi: 10.1002/pro.5560010609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Takahashi N., Hirose M. Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis. Anal Biochem. 1990 Aug 1;188(2):359–365. doi: 10.1016/0003-2697(90)90621-f. [DOI] [PubMed] [Google Scholar]
  36. Thiede B., Wittmann-Liebold B., Bienert M., Krause E. MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P. FEBS Lett. 1995 Jan 2;357(1):65–69. doi: 10.1016/0014-5793(94)01323-s. [DOI] [PubMed] [Google Scholar]
  37. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Vallesi A., Giuli G., Ghiara P., Scapigliati G., Luporini P. Structure-function relationships of pheromones of the ciliate Euplotes raikovi with mammalian growth factors: cross-reactivity between Er-1 and interleukin-2 systems. Exp Cell Res. 1998 May 25;241(1):253–259. doi: 10.1006/excr.1998.4056. [DOI] [PubMed] [Google Scholar]
  39. Weiss M. S., Anderson D. H., Raffioni S., Bradshaw R. A., Ortenzi C., Luporini P., Eisenberg D. A cooperative model for receptor recognition and cell adhesion: evidence from the molecular packing in the 1.6-A crystal structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10172–10176. doi: 10.1073/pnas.92.22.10172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. White J. V., Stultz C. M., Smith T. F. Protein classification by stochastic modeling and optimal filtering of amino-acid sequences. Math Biosci. 1994 Jan;119(1):35–75. doi: 10.1016/0025-5564(94)90004-3. [DOI] [PubMed] [Google Scholar]
  41. Worster B. M., Yeoman M. S., Benjamin P. R. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the pattern of peptide expression in single neurons resulting from alternative mRNA splicing of the FMRFamide gene. Eur J Neurosci. 1998 Nov;10(11):3498–3507. doi: 10.1046/j.1460-9568.1998.00361.x. [DOI] [PubMed] [Google Scholar]
  42. Wu J., Watson J. T. A novel methodology for assignment of disulfide bond pairings in proteins. Protein Sci. 1997 Feb;6(2):391–398. doi: 10.1002/pro.5560060215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhou Z. R., Smith D. L. Assignment of disulfide bonds in proteins by partial acid hydrolysis and mass spectrometry. J Protein Chem. 1990 Oct;9(5):523–532. doi: 10.1007/BF01025005. [DOI] [PubMed] [Google Scholar]
  44. van de Poll M. L., van Vugt M. J., Lenferink A. E., van Zoelen E. J. Identification of the minimal requirements for binding to the human epidermal growth factor (EGF) receptor using chimeras of human EGF and an EGF repeat of Drosophila Notch. J Biol Chem. 1998 Jun 26;273(26):16075–16081. doi: 10.1074/jbc.273.26.16075. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES