C.B. Daul

Identification of the Major Brown Shrimp (Penaeus aztecus) Allergen as the Muscle Protein Tropomyosin

Shrimp, a major seafood allergen, was investigated as a model food allergen. Extracts from both shrimp (Penaeus aztecus) meat and cooking fluid contain a substantial and similar amount of allergenic activity. A 36-kD allergen, demonstrated in both extracts by SDS-PAGE/Western blot analysis, reacted with 28/34 (82%) sera from shrimp-sensitive, skin test and RAST-positive, individuals. This allergen, named Pen a I, was isolated by SDS-PAGE; its amino acid composition was rich in aspartic and glutamic acids. A 21-residue peptide, obtained from endoproteinase Lys-C digested Pen a I by high-performance liquid chromatography, demonstrated significant homology (60-87%) with the muscle protein tropomyosin from various species and origins. The greatest homology (87%) was noted with tropomyosin of the fruit fly (Drosophila melanogaster) reflecting the phylogenic relationship between these two arthropods. These studies demonstrate that tropomyosin is the major shrimp allergen. Although the amino acid sequence of this shrimp muscle protein shares considerable homology with tropomyosins of other species including man, significant differences remain in allergenic activity.

Identification of the first major allergen of a squid (Todarodes pacificus)

BACKGROUND In Japan, squid is an important seafood, and some patients with food allergies are sensitive to squid. There has been no report, however, describing the major allergens of squid. OBJECTIVE To characterize squid allergens, we isolated a major allergen from the Pacific flying squid (Todarodes pacificus) and compared it with a major allergen from a shrimp (Penaeus orientalis). METHODS The major squid and shrimp allergens were isolated by column chromatography on diethylaminoethyl-Sepharose (Pharmacia, Uppsala, Sweden), hydroxylapatite, and Sephacryl S-300 (Pharmacia). The IgE reactivity of the isolated allergens was assessed by immunoblotting. The cross-reactivity between the squid and shrimp allergens was examined by use of mouse polyclonal and monoclonal antibodies to the major allergens. Amino acid sequence analyses of the isolated allergens were done. RESULTS The isolated squid allergen is a 38 kd, heat-stable protein. IgE antibody binding to the purified squid allergen was demonstrated by immunoblotting. Cross-reactivity between major squid and shrimp allergens was demonstrated with sera from patients allergic to squid or shrimp or with allergen-specific monoclonal antibodies. The amino acid sequence analysis of the major squid allergen showed a marked homology with tropomyosin from blood fluke planorbid (Biomphalaria glabrata), which is a common vector snail of Schistosoma mansoni. CONCLUSION This 38 kd protein is a major allergen of the squid, Todarodes pacificus, and is believed to be squid muscle protein tropomyosin. We named it Tod p 1 according to International Union of Immunological Societies allergen nomenclature regulation.

Provocation-challenge studies in shrimp-sensitive individuals☆

Thirty individuals with history of immediate, objective, adverse reactions after shrimp ingestion underwent double-blind, placebo-controlled shrimp-food challenges. All individuals who did not exhibit a positive response (reproduction of objective symptoms) were administered an open challenge of 16 whole cooked shrimp. Positive challenge responses occurred in 9/30 subjects (30%); six of these subjects experienced a positive response during the double-blind phase. Of the 21 remaining subjects, 12 experienced generalized pruritus as their only symptom, whereas nine subjects had completely negative challenge responses. All placebo challenges were negative. Although a positive skin test was strongly associated with challenge symptoms (p less than 0.001), the shrimp prick skin test titration end points were not different among the challenge groups. The serum shrimp RAST percent was significantly higher in the positive challenge group (p less than 0.02). Mean levels of shrimp-specific serum IgG, IgA, and IgM levels were not different among the challenge groups. Although no single immunologic variable could be consistently used to identify subjects more likely to exhibit a positive challenge response, the composite of a positive shrimp prick skin test and elevated serum shrimp-specific IgE (RAST percent label bound greater than 11%) demonstrated a correct predictive value of 87% in this group of shrimp-sensitive subjects.

Quantification of the major brown shrimp allergen Pen a 1 (tropomyosin) by a monoclonal antibody-based sandwich ELISA

BACKGROUND Among 13 allergens found in extracts of cooked brown shrimp (Penaeus aztecus) the 36 kd muscle protein tropomyosin has been identified as the only major shrimp allergen (Pen a 1). Cross-reacting molecules with similar molecular weights were detected in other crustacea species such as crab, lobster, and crawfish. Because Pen a 1 and Pen a 1-like allergens are important in crustacea allergy, the aim of this study was to develop a monoclonal antibody (mAb)-based sandwich ELISA to quantify Pen a 1 and to evaluate Pen a 1 levels in four commercial shrimp, crab, and lobster extracts. METHODS Two Pen a 1-specific mAbs with different epitope specificities were selected. ELISA plates coated with captured mAb 3.2 were incubated with samples containing Pen a 1. Bound Pen a 1 was detected by a combination of biotinylated mAb 4.9.5 and alkaline phosphatase-labeled streptavidin. RESULTS The optimized sandwich ELISA could detect Pen a 1 concentrations ranging from 4 to 125 ng/ml. Four commercial shrimp extracts demonstrated a 40-fold difference in Pen a 1 levels (24 to 920 microg/ml). Crab and lobster extracts contained detectable levels of Pen a 1-like proteins. No reactivity to cockroach, house dust mite, oyster, codfish, or peanut extracts was detected, which indicates that the developed assay is crustacea-specific. CONCLUSION A sensitive sandwich assay was developed to quantify Pen a 1. This assay will be helpful to standardize shrimp extracts in regard to the content of the major allergen, Pen a 1, and to study cross-reactivities among and evaluate occupational exposure to different crustacea species.

Characterization of Recombinant Shrimp Allergen Pen a 1 (Tropomyosin)

Tropomyosin (Pen a 1) from brown shrimp, Penaeus aztecus, has been identified as the only major shrimp allergen. Since beef, pork and chicken are other tropomyosin-containing foods that are not very allergenic, tropomyosins can serve to investigate the contribution of the structural properties of a protein to its allergenicity. The aim of this study was to determine the primary structure of Pen a 1 and to identify IgE-binding epitopes. The screening of a unidirectional expression cDNA library from shrimp tail muscle with the Pen-a-1-specific monoclonal antibody 4.9.5 resulted in 4 positive Escherichia coli clones. Immunoblot analysis with human sera from shrimp-allergic subjects demonstrated IgE binding of all 4 recombinant shrimp proteins. Three of 4 expressed recombinant proteins have a molecular weight of approximately 36 kD, consistent with the molecular weight of natural Pen a 1. The DNA sequence analysis identified these recombinant shrimp proteins as tropomyosin and could be aligned with the sequence of greasyback shrimp (Metapenaeus ensis) tropomyosin (Met e 1). In order to characterize contiguous IgE-binding epitopes of Pen a 1, a peptide library (Novagen epitope mapping system) expressing 10-30 amino-acid-residue-long recombinant Pen a 1 peptides was constructed and screened with human IgE. Four recombinant, IgE-reactive Pen a 1 peptides were selected and sequenced. They show various degrees of sequence identity with tropomyosins of other arthropods, such as fruitfly and house dust mite, helminths and vertebrates.

Hypersensitivity reactions to ingested crustacea: Clinical evaluation and diagnostic studies in shrimp-sensitive individuals☆

Adverse reactions to ingested crustacea are common and may be life-threatening. We studied 14 individuals with histories of such reactions to shrimp by immediate skin tests and RAST with extracts of shrimp, crab, crayfish, and lobster. Nine of these subjects (8/8 atopics and 1/6 nonatopics) had positive immediate skin tests (wheal greater than or equal to 2 mm) and RAST (ratios greater than 3.0) to shrimp. Their skin tests and RAST ratios to the other crustacea were also frequently positive even, in several cases, in the absence of prior exposure. In contrast, only 1/10 volunteers with no history of intolerance to crustacea had a weak positive skin test to raw shrimp. These studies suggest that both skin tests and RAST are useful in the confirmation of hypersensitivity to shrimp in atopic individuals and that cross-reactivity among crustacea may exist.

Angiotensin-forming enzyme in human brain.

Abstract The subcellular and regional distribution of angiotensin-forming enzyme was examined in human brain. A relatively purified form of this enzyme was obtained by discontinuous sucrose density gradient subfractionation of the crude mitochondrial fraction. Of the six brain regions examined, the cerebellar cortex crude mitochondrial fraction had the highest angiotensin-forming enzyme content. Kinetic characteristics, with respect to hog plasma renin and synthetic tetradecapeptide substrates and optimum pH range (4·5–5·5), were similar for both the particulate and soluble forms of the enzyme. Both forms also had a lower affinity for human plasma renin substrate. Different substrate kinetics and optimum pH range were obtained for the brain angiotensin-forming enzyme and kidney renin. Because of its potential clinical implication, a brain renin-angiotensin-aldosterone system, which is independent of kidney and adrenals, merits further study.

The natural history of shrimp hypersensitivity

Sera collected sequentially during a 24-month interval from 11 individuals with shrimp hypersensitivity and 10 nonhypersensitive control subjects were evaluated for shrimp-specific IgE, IgG, IgM, and IgA reactivity. Shrimp-hypersensitive subjects underwent double-blind, placebo-controlled shrimp challenges; seven exhibited positive challenges, and four subjects reported the subjective symptom of oropharyngeal pruritus. Shrimp-specific IgE levels in all subjects were relatively constant during the 24 months of this study and not affected by shrimp challenge, although some fluctuation in the shrimp-specific IgG, IgM, and IgA reactivity were noted, apparently unrelated to shrimp challenge. Shrimp-specific IgE and IgG, but not IgM and IgA, were significantly higher in the group with shrimp hypersensitivity as compared to the control subjects. Moreover, the challenge-positive subjects had higher levels of both shrimp-specific IgE and IgG than subjects reporting pruritus. The levels of shrimp-specific IgG correlated directly with shrimp-specific IgE reactivity. These studies indicate that serum levels of shrimp-specific IgE are significantly elevated in shrimp-hypersensitive subjects who exhibit positive food challenges, and these baseline levels did not appear to be altered long term by isolated shrimp challenge. Furthermore, baseline shrimp-specific antibody (IgG, IgM, and IgA) levels noted in normal subjects were not markedly affected by frequent ingestion of shrimp.

Immunologic evaluation of shrimp-allergic individuals

Thirty-three individuals with a history of immediate hypersensitivity reactions after shrimp ingestion and 29 nonshrimp-sensitive control subjects were evaluated for evidence of crustacea-specific immunity by skin prick test titration end point, RAST, and ELISA, with extracts of shrimp, crab, crayfish, and lobster. Individuals were categorized as either atopic or nonatopic on the basis of history and skin test reactivity to common inhalant allergens. Most (28/33) shrimp-sensitive subjects had positive skin prick tests to shrimp extract, whereas skin tests were negative in 27/29 control subjects. Eighty-one percent of atopic and 41% of nonatopic shrimp-sensitive subjects had elevated shrimp-RAST ratios. The RAST ratios of atopic individuals were significantly higher than ratios of nonatopic individuals, and there was a significant correlation between shrimp-RAST ratios and historical clinical symptom scores. RAST determinations of all control subjects were negative. Shrimp-sensitive subjects also had significantly elevated serum levels of shrimp-specific IgG and IgA as compared to control individuals. Both IgG and IgA shrimp-specific reactivity demonstrated a significant positive correlation with shrimp-RAST ratios. These studies indicate that IgE-mediated, type I mechanisms, detected by positive shrimp skin tests and RASTs, appear to be operative in crustacea-sensitive individuals, particularly those with concurrent respiratory allergy. Although the role of shrimp-specific IgG and IgA antibodies in the immunopathogenesis of crustacea allergy remains unclear, such antibodies appear to represent increased immunologic recognition of shrimp allergens/antigens in shrimp-sensitive subjects.

The relationships among shrimp-specific IgG subclass antibodies and immediate adverse reactions to shrimp challenge†

High levels of shrimp-specific IgE, in association with a positive prick test, are not always predictive of a positive, immediate response to double-blind, placebo-controlled, food challenge (DBPCFC) with shrimp. The observation that shrimp-sensitive individuals in general have increased levels of circulating shrimp-specific IgG is of interest because antigen/allergen-specific IgG subclasses have been associated with adverse reactions to foods. Therefore, this current study measured shrimp-specific IgG subclass and IgE antibodies in 31 individuals with histories of immediate, adverse reactions to shrimp immediately before DBPCFC and 20 shrimp-tolerant subjects. Individuals with a history of shrimp sensitivity had significantly raised shrimp-specific IgG2 and IgG4 compared to shrimp-tolerant individuals. Challenge-positive subjects were distinguished from subjects with negative or equivocal responses by an increased IgG2 (p less than or equal to 0.001). Specific IgG4 was not raised (p less than or equal to 0.065). These studies indicate that some shrimp-specific IgG subclass levels are increased in shrimp-sensitive subjects. However, none of the subclass responses were significantly predictive of a positive response to DBPCFC and therefore were not diagnostic of shrimp intolerance.