5.2.1 Molecular Biological Properties
 

Tropomyosins	References
Allergen Nomenclature Met e 1 (Metapenaeus ensis) Pen a 1 (Penaeus aztecus) Pen i 1 (Penaeus indicus) Pen o 1* (Penaeus orientalis) * not listed in the official WHO/IUIS list of allergens	(1) Larsen & Lowenstein 2000
Molecular Mass   Met e 1 Pen a 1 Pen i 1 Pen o 1 SDS-PAGE   36 kDa (2) 34 kDa (1) 39 kDa (4) calculated 34 kDa (3)	(1) Shanti et al. 1993 (2) Daul et al. 1994 (3) Leung et al. 1994 (4) Miyazawa et al. 1996
Isoelectric Point   Met e 1 Pen a 1 Pen i 1 IEF-PAGE   pI 5.2 (2) pI 4.8-5.4 (1)	(1) Shanti et al. 1993 (2) Daul et al. 1994
Amino Acid Sequence, mRNA, and cDNA   Met e 1 Pen a 1 Pen o 1 GenBank: U08008 AAB31957 (1)*   SWISS-PROT: Q25456     Amino Acids 281 (2) 265 and 229 (4) 284 (5) ps** (3) mRNA 843 bp (partial)   cDNA       *21aa peptide from endoproteinase Lys-C digest (1) ** partial sequence, 3 peptides from Acromobacter protease digest (3)	(1) Daul et al. 1994 (2) Leung et al. 1994 (3) Miyazawa et al. 1996 (4) Reese et al. 1997 (5) Reese et al. 1999b
Recombinant Proteins Expression cDNA library: cDNA library from the shrimp Metapenaeus ensis in lambda gt 11 screened with patient's sera, identified IgE-reactive clone (cDNA) purified and expressed in plasmid pGEX (1) Screening of cDNA library from shrimp tail muscle (Penaeus aztecus) with a Pen a 1 specific mAb, expression of 4 recombinant proteins in Escherichia coli (2)	(1) Leung et al. 1994 (2) Reese et al. 1997
Posttranslational Modification Acetylation: blocked N-terminus (Pen o 1) (2) Glycosylation: Carbohydrate moieties (Pen a 1): 2.9% (1)	(1) Daul et al. 1994 (2) Miyazawa et al. 1996
Biological Function Tropomyosin belongs to a highly conserved family of proteins in both muscle and non-muscle cells of all species of vertebrates and invertebrates (2); tropomyosin is associated with the troponin complex which plays a central role in calcium dependent regulation of muscle contraction (1)	(1) SWISS-PROT (2) Reese et al. 1999b
Sequence Homology Amino acid sequence identities of Met a 1 and Pen a 1 to tropomyosins from 22 sources (1):  Met e 1 Pen a 1 Met a 1 - 99% Hom a 1 (Atlantic lobster) 98% 98% Pan s 1 (spiny lobster) 98% 98% Per a 7 (American cockroach) 82% 82% Der p 10 (house dust mite) 81% 81% DromTM (fruit fly Drosophila melanogaster) 77% 77% ChlnTM (Japanese scallop) 57% 57%	(1) Reese et al. 1999b

 5.2.2 Allergenic Properties
 

Tropomyosins	References
Frequency of Sensitization IgE-binding to tropomyosins in 82% of patients (1)	(1) see 5.1 Sensitization to Shrimp Allergens
Allergenicity of Natural Proteins IgE-binding to nPen a 1: Pen a 1 inhibited about 85% of IgE binding to whole shrimp protein extract (1)	(1) Reese et al. 1999b
Allergenicity of Recombinant Proteins IgE-binding to rMet e 1: demonstrated for recombinant shrimp protein with human sera from shrimp-allergic subjects (SDS-PAGE immunoblot) (1) complete inhibition of IgE binding by rMet e 1 to recombinant crab tropomyosin (sera from crustaceae allergic patients) (3) complete inhibition of IgE binding by rMet e 1 to lobster muscle extract (sera from crustaceae allergic patients) (4) IgE-binding to rPen a 1: demonstrated for all 4 recombinant shrimp proteins with human sera from shrimp-allergic subjects (2)	(1) Leung et al. 1994 (2) Reese et al. 1997 (3) Leung et al. 1998a (4) Leung et al. 1998b
B-Cell Epitopes IgE-binding regions of Met e 1: aa 47-63 (5) aa 150-158 (5) IgE-binding regions of Pen a 1: CNBr cleavage and enzymic digestion  (Lys-C, Glu-C, trypsin, Arg-C, chymotrypsin) (c) 4-20 kDa fragments (2, 3) recombinant peptides (d): aa 136-148 (4, 6)* aa 157-169 (4, 6)* aa 167-179 (4, 6)* aa 262-282 (4, 6)* synthetic peptides (a, e): aa 1-15 (6) aa 79-93 (6) aa 109-123 (6) aa 121-135 (6) aa 133-147 (6) aa 187-201 (6) aa 217-231 (6) aa 241-255 (6) aa 253-267 (6) IgE-binding regions of Pen i 1: aa 47-63 (trypsin digest) (a) (1,5)* aa 150-158 (trypsin digest) (a) (1,5)*	(1) Shanti et al. 1993 (2) Reeese et al. 1995 (3) Reeese et al. 1996 (4) Reese et al. 1997 (5) Subba Rao et al. 1998 (6) Reese et al. 1999a     (a) Dot-immunoblot (b) ELISA inhibition (c) SDS-PAGE immunoblot (d) coding plasmid randomly cleaved by DNase 1, library screened with serum pool (e) SPOTs membrane technique *note differences in aa sequence designation in references
IgE-Binding: Critical Amino Acids  Phe-150 in Pen i 1 assumed to be essential for IgE binding since peptide aa 150-158 showed IgE binding while peptide aa 151-171 did not (1)	(1) Subba Rao et al. 1998
T-Cell Epitopes Putative T-cell epitopes deduced from Pen i 1 sequence by computer algorithm, in vitro proliferative activity of 6 synthetic peptides tested with splenocytes from mice; none of the peptides bound patients' IgE (1)	(1) Subba Rao et al. 1998