In the course of analyzing the partial amino acid sequences of Cry j I, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, we found a peptide fragment which has a significant homology to some pectate lyase isozymes secreted by plant pathogenic bacteria. Therefore, we investigated whether Cry j I has pectate lyase activity. Cry j I reacted with polygalacturonic acid, resulting in the release of unsaturated uronide products. The optimum temperature and pH for the reaction were 60-70 degrees C and pH 10. The enzymatic reaction had an absolute Ca2+ ion requirement. These characteristics were very compatible with the character of the pectate lyase isozymes reported previously. These results clearly show that Cry j I has pectate lyase activity.
-S433). According to a homology analysis, no amino acid sequence homology was observed between Cryj II and Cryj I, another major allergen. But Cryj II showed homology with polygalacturonase (PG) derived from tomato (40% identity) at the amino acid level. The sequence information can potentially be used to devise an effective course of immunotherapy for Japanese cedar pollinosis.
A purified preparation of a major allergen of Japanese cedar pollen, sugi basic protein (SBP, Cry jI), was separated into 5 subtractions of 50‐45 kDa. All of the SBP subtractions were confirmed to be reactive to IgE antibodies from patients with Japanese cedar pollinosis, and also to mouse anti‐SBP monoclonal antibodies. The sequences of 20 N‐terminal amino acids of these 5 subtractions were found to be identical. Peptide mapping analyses of the SBP subtractions showed similar patterns, with some differences which might in part be due to the existence of an N‐linked carbohydrate chain. The N‐terminal amino acid sequence of SBP was identical to the reported sequence of an allergen of mountain cedar which vegetated in North America.
We examined Cry j 2, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, for polygalacturonase enzyme activity, since a nucleotide sequence of cDNA of Cry j 2 showed a significant homology with that of tomato polygalacturonase. Polygalacturonase is well known to depolymerize preferentially polygalacturonic acid (PGA) by hydrolysis. However, Cry j 2 did not act on PGA, but was found to depolymerize pectin and methylesterified PGA in a dose-dependent manner. The substrate specificity of Cry j 2 was different from that of polygalacturonase derived from Aspergillus niger. The depolymerizing activity of Cry j 2 reached a maximum at 50%-60% of methylesterification of PGA. In contrast, polygalacturonase showed its maximum activity of PGA, and the activity decreased as the degree of methylesterification increased. Interestingly, the pectin-depolymerizing activity of Cry j 2 was due to a hydrolysis, but not a lyase, activity which splits the glycosidic bonds by beta-elimination, since no unsaturated uronides were found by measurement of absorbance at 235 nm in the reaction mixture. The enzyme activity was markedly inhibited by anti-Cry j 2 antibodies. These results indicate that Cry j 2 probably has polymethylgalacturonase enzyme activity, as postulated by von Neukom in 1963, although existence of this activity has not yet been proven.
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