Abstract
BACKGROUND: Humulus japonicus (HJ) is widely spread in East Asia. HJ pollen is one of the common allergens in the north of China in autumn. However, some controversy exists regarding the major allergen Hum j 1 from HJ pollen, which hamper its application in the diagnosis and treatment of HJ pollen allergy. Further studies are urgently needed to improve this aspect. METHODS: During our work in isolating potential allergens from HJ pollen, we found a protein of around 10 kDa with obvious immunoglobulin E (IgE) binding activity than other fractions. This protein was then further identified by mass spectrometry, and its gene sequence was matched from our in-house built transcripts of HJ pollen based on the identified peptides. The gene was further confirmed by polymerase chain reaction (PCR) cloning, and the recombinant protein was prepared by Escherichia coli (E. coli) expression system. The circular dichroism (CD) spectrum of natural and recombinant proteins was analyzed. The allergenicity of its natural and recombinant forms was assessed in Chinese patients and the relationship between the epitopes and its structural features was investigated under reduced, denatured and heat-treated conditions. RESULTS: The 10 kDa protein with obvious IgE binding activity was finally purified from HJ pollen with a purity of 98%. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) results identified 6 internal peptides, which covered 98.9% of the mature sequence of the 10 kDa protein with the N-terminal segment as DNCFENGMK. We surprisingly found that the 10 kDa protein was previously underrecognized Hum j 1 based on its limited sequence of only N-terminus. The sequence failed to be classified into any protein families by InterPro functional analysis, indicating the Hum j 1 could be the orphan protein. Both natural and recombinant Hum j 1 exhibited similar CD spectrum. The specific IgE binding rate against them was determined as 79% (11/14) in patients' serum with HJ pollen allergy by ELISA with a strong correlation in absorbance values (R(2) = 0.99, p < 0.0001) and decreased by 76%-87% after broking disulfide bonds, with no significant change after heat treatment or urea treatment. CONCLUSION: These findings first provided solid evidence about the major allergen Hum j 1 in HJ pollen and resolved its unclear status. The defined sequence could enhance understanding of Hum j 1 properties and aid in producing high-quality recombinant allergens for diagnosing and treating HJ pollen allergy.