HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marchot, P. Articles by Taylor, P. Search for Related Content PubMed PubMed Citation Articles by Marchot, P. Articles by Taylor, P. Social Bookmarking                      What's this?

Volume 272, Number 6, Issue of February 7, 1997 pp. 3502-3510
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Expression and Activity of Mutants of Fasciculin, a Peptidic Acetylcholinesterase Inhibitor from Mamba Venom

(Received for publication, July 15, 1996, and in revised form, October 8, 1996)

Pascale Marchot ^§ , Claudine N. Prowse , Joan Kanter , Shelley Camp , Elizabeth J. Ackermann , Zoran Radi , Pierre E. Bougis ^§ and Palmer Taylor

From the  Department of Pharmacology, University of California at San Diego, La Jolla, California 92093-0636 and ^§ CNRS, Unité de Recherche Associée 1455, Institut Fédératif de Recherche Jean Roche, Université de la Méditerranée, Faculté de Médecine Secteur Nord, 13916 Marseille Cedex 20, France

Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, Pro³⁰, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg²⁷, Pro³⁰, and Pro³¹ mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg²⁴, Lys³², and Met³³ mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys²⁵, Arg²⁸, Val³⁴-Leu³⁵, Asp⁴⁵, and Lys⁵¹ mutants appeared as active as Fas2. The Thr⁸-Thr⁹, Arg¹¹, and His²⁹ mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. Kukkonen, M. Perakyla, K. E. O. Akerman, and J. Nasman Muscarinic Toxin 7 Selectivity Is Dictated by Extracellular Receptor Loops J. Biol. Chem., December 3, 2004; 279(49): 50923 - 50929. [Abstract] [Full Text] [PDF]

				K. Jeyaseelan, D. Ma, and A. Armugam Real-time detection of gene promoter activity: quantitation of toxin gene transcription Nucleic Acids Res., June 15, 2001; 29(12): e58 - e58. [Abstract] [Full Text] [PDF]