Molecular cloning of the mountain cedar (Juniperus ashei) pollen major allergen, Jun a 1.
BACKGROUND
Cedar pollens cause allergic disease in diverse geographic areas. We have recently purified and characterized the major mountain cedar (Juniperus ashei) pollen allergen, Jun a 1.


OBJECTIVE
A full-length complementary DNA for Jun a 1 was cloned and sequenced, and the recombinant protein was expressed.


METHODS
Messenger RNA from mountain cedar pollen was purified and Jun a 1 sequences were established with use of reverse transcriptase-PCR and primers based on the N-terminal amino acid sequence of Jun a 1 and the homologous protein Cry j 1. Portions of the nucleotide sequence were confirmed by comparison with N-terminal amino acid sequencing of the intact tryptic fragments of the purified native protein. Recombinant Jun a 1 was cloned into pET 30, expressed in BL21, and purified by HPLC, and its allergenicity was analyzed by Western blotting with patient sera.


RESULTS
Jun a 1 possesses a high level of amino acid sequence homology with Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar. The amino acid sequence of a region with putative pectate lyase activity was identical to that of Cry j 1 and Cha o 1. Jun a 1 contained 2 potential N-glycosylation sites that were distinct from those found in Cry j 1. The IgE from patient sera bound recombinant Jun a 1 in Western blot analysis.


CONCLUSIONS
The high degree of homology of Jun a 1 with Cha o 1 and Cry j 1 may explain the cross-reactivity of conifer pollens. Differences in N-glycosylation suggest little overlap of glycopeptide epitopes.
Midoro-Horiuti
T
T
Department of Pediatrics, Child Health Research Center, University of Texas Medical Branch at Galveston, 77555-0366, USA.
Goldblum
R M
RM
Kurosky
A
A
Wood
T G
TG
Schein
C H
CH
Brooks
E G
EG
D000485:Allergens / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant; D018653:Juniperus; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011058:Pollen; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000485:Allergens; D052179:Antigens, Plant; D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C121951:jun a 1 protein, Juniperus ashei; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin