Red soft coral (RSC; Dendronephthya nipponica, a marine coelenterate) causes spiny lobster fishermen living along the Pacific coast of Miyazaki Prefecture in Japan to develop occupational allergies, such as conjunctivitis, rhinitis, dermatitis and bronchial asthma. The aim of this study was to purify and to characterize RSC allergen, which causes occupational asthma in spiny lobster fishermen. The allergic responsiveness of spiny lobster fishermen to RSC was examined. The examinations included specific IgE production, skin test responses, lymphocyte stimulation tests and specific IgG production. We found that RSC has a strong sensitizing activity in humans at a molecular weight of 10 kD or more, while it has no IgE-producing activity at a molecular weight of less than 10 kD. Neither the nonatopic controls nor the atopic non-coral-allergic controls exhibited any RAST-binding activity to any fraction. For the purification and the identification of this new allergen component, repeated gel filtration of the RSC extract was performed on a Sephacryl S-200 column, followed by gel filtration on a Superose-6 column. The purified major allergen component Den n 1, which is separated on a Mono-Q column, showed intradermal responses, lymphocyte stimulating activity and specific IgG-producing activity in RSC-induced bronchial asthma patients. The 53-kD component was electroblotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence of this new allergen component (Den n 1) was determined as Asp-Asp-Ile-Asn-Arg-Tyr-Ala-Phe-Asp-Asn-Lys-Ile-Asn- Asp-Lys-Leu-Phe-Asp-His-Trp-Gln-Ser.

The red soft coral that is found around the spiny lobster represents a major allergen and many fishermen, especially those that catch the spiny lobster, suffer from allergic symptoms. Therefore, in the present study, one of the major fractions of this protein was isolated and purified from the red soft coral (Alcyonium gracillimum). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblot analyses showed that the molecular weight of the allergen is approximately 14.4–20.1 kDa. Continuous purification of the protein resulted in a peptide with a molecular weight of 17 kDa. Immunoblotting and ELISA tests showed that the isolated peptide (17 kDa) strongly cross‐reacted with the IgE of patients, thus classifying it as a significant allergen.

Spiny lobster fishermen on the Pacific coast of Miyazaki Prefecture in Japan develop bronchial asthma due to occupational sensitization to red soft coral (Dendronephthya nipponica). To assess the role of sensitization in the development of bronchial hyperresponsiveness and the relationship between bronchial responsiveness and bronchial inflammation, we established a guinea pig model of red soft coral induced asthma. Twenty-four guinea pigs were intramuscularly immunized with a priming dose of red soft coral, 5 OD₂₈₀ (15 mg protein) per 0.5 ml, and 0.5 ml complete Freund’s adjuvant on day 1. Booster doses were repeated on day 15. By day 43 all sensitized animals showed high hemagglutination titers against red soft coral conjugated sheep erythrocytes and high IgG1 titers by passive cutaneous anaphylaxis. On day 43, the animals were challenged by inhalation of red soft coral extracts, 20 OD₂₈₀ for 30 min. The respiratory resistance was monitored by the oscillation method. The respiratory resistance increased immediately upon inhalation in all sensitized animals and returned to baseline within 4 h. The bronchial reactivity to acetylcholine, measured 6 h after red soft coral inhalation in the sensitized animals when the respiratory resistance was returned to baseline, increased significantly (p < 0.05) as compared with the values measured 24 h before inhalation. The acetylcholine response measured 30 h later did not differ from preinhalation levels. There was a significant difference in the number of eosinophils (p < 0.001) in lamina propria and epithelium and of lymphocytes (p < 0.05) in the lamina propria 6 h after inhalation in the sensitized animals. There was also a significant difference (p < 0.05) in the number of eosinophils in the epithelium 30 h after inhalation. The increased airway responsiveness to acetylcholine in the sensitized animals correlated with an increase in the number of eosinophils in lamina propria and epithelium.

Certain marine organisms have been known to cause allergic reactions among occupational fishermen. We have previously reported that bronchial asthma among the workers engaged in spiny lobster fishing in Japan was caused by octocorals such as Dendronephthya sp. and Scleronephthya gracillima (previously named Alcyonium gracillimum). Now we have found another octocoral, Scleronephthya gracillima (Kuekenthal), which causes the allergic disease in fishermen. The octocoral was characterized as a new green fluorescent protein (GFP)-like family. The new allergen has a molecular mass of 27 kDa in 1D and 2D SDS-PAGE under reduced conditions. The 27 kDa component was determined to be an allergen by western blotting, ECL immune staining method and absorption of patient sera with the antigen. Furthermore, the combination of analysis with LC-ESI-MS/MS and MASCOT search in the NCBInr database concluded the 27 kDa component had the sequence YPADI/LPDYFK, and that the 22 kDa component had the sequence QSFPEGFSWER, which both matched a GFP-like protein in Acropora aculeus and in Montastraea annularis. Further analysis by MALDI-TOF/MS/MS and MASCOT search in the NCBInr database of all 27 kDa eight spot components from 2D SDS-PAGE indicated that the sequence QSFPEGFSWER also matched as GFP-like protein in Lobophyllia hemprichii and Scleractinia sp. To our knowledge, this is the first report of the new allergenic protein that corresponds to a new GFP-like protein named Akane, and which has fluorescent emissions in the red and green part of the spectra at 628 nm and 508 nm, respectively.

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