title: Structural, immunological and functional properties of natural recombinant Pen a 1, the major allergen of Brown Shrimp, Penaeus aztecus.
abstract: BACKGROUND
  Recombinant allergens are considered the basis for new diagnostic approaches and development of novel strategies of allergen-specific immunotherapy. As Pen a 1 from brown shrimp Penaeus aztecus is the only major allergen of shrimp and binds up to 75% of all shrimp-specific IgE antibodies this molecule may be an excellent model for the usage of allergens with reduced IgE antibody-binding capacity for specific immunotherapy.
  
  
  OBJECTIVE
  The aim was to clone, express and characterize a full-length recombinant Pen a 1 molecule and compare it with natural Pen a 1 in regard to structural and immunological parameters such as IgE antibody capacity and ability to induce IgE-mediated mediator release.
  
  
  METHODS
  Total RNA was isolated from P. aztecus and a rapid amplification of cDNA ends (5' RACE) was performed to obtain full-length cDNA coding for Pen a 1. Using a gene-specific primer, PCR was performed and full-length cDNA was cloned and sequenced. Recombinant His-tagged Pen a 1 was isolated from Escherichia coli under native conditions by immobilized metal affinity chromatography. Secondary structure of natural and recombinant Pen a 1 was compared by circular dichroism (CD) spectroscopy, and the IgE antibody-binding capacity evaluated by RAST. The allergenic potency was tested by the capability of natural and recombinant Pen a 1 to induce mediator release in a murine and human in vitro model of IgE-mediated type I allergy.
  
  
  RESULTS
  The deduced amino-acid sequence was 284 residues long and amino-acid sequence identities with allergenic and non-allergenic tropomyosins ranged from 80% to 99% and 51% to 58%, respectively. The analysis of the secondary structure of natural and recombinant Pen a 1 by CD spectroscopic analysis showed that both nPen a 1 and rPen a 1 had alpha-helical conformation that is typical for tropomyosin. The IgE antibody binding capacities of nPen a 1 and r Pen a1 were found to be essentially identical by RAST. The mediator release experiments using both wild-type and humanized rat basophilic leukaemia 30/25 cells showed that rPen a 1 and nPen a 1 induced a similar level of mast cell activation.
  
  
  CONCLUSIONS
  Recombinant Pen a 1 and natural Pen a 1 are structurally and immunologically identical and rPen a 1 may be used as the basis for component-resolved diagnosis and the generation of modified shrimp tropomyosin for allergen-specific immunotherapy. The results of the animal studies indicate that C3H/HeJ mice that were sensitized with shrimp extract in combination with cholera toxin as adjuvant may be a suitable model to study shrimp allergy.
authors.affiliation: Paul-Ehrlich-Institut, Department of Allergology, Langen, Germany. reege@pei.pe
authors.forename: G
authors.initials: G
authors.lastname: Reese
authors.forename: S
authors.initials: S
authors.lastname: Schicktanz
authors.forename: I
authors.initials: I
authors.lastname: Lauer
authors.forename: S
authors.initials: S
authors.lastname: Randow
authors.forename: D
authors.initials: D
authors.lastname: Lüttkopf
authors.forename: L
authors.initials: L
authors.lastname: Vogel
authors.forename: S B
authors.initials: SB
authors.lastname: Lehrer
authors.forename: S
authors.initials: S
authors.lastname: Vieths
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001491:Basophils / Q000276:immunology; D002478:Cells, Cultured; D002942:Circular Dichroism / Q000379:methods; D004270:DNA, Circular / Q000737:chemistry; D005260:Female; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007938:Leukemia / Q000276:immunology; D051379:Mice; D008809:Mice, Inbred C3H; D008954:Models, Biological; D033561:Penaeidae / Q000276:immunology*; D011487:Protein Conformation; D011852:Radioallergosorbent Test / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology*; D014162:Transfection / Q000379:methods; D014335:Tropomyosin / Q000276:immunology
chemical_list: D000485:Allergens; D004270:DNA, Circular; C513564:Pen a 1 allergen, Penaeus aztecus; D011994:Recombinant Proteins; D014335:Tropomyosin; D007073:Immunoglobulin E