Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a 1.
The family Cupressaceae is a relevant source of allergens that causes winter respiratory allergies. Cloning and sequencing the major antigen of Cupressus arizonica is important for a better diagnosis and treatment of sensitized patients. To obtain a full-length complementary DNA for Cup a 1, the major allergen of Cupressus arizonica pollen. It was cloned and sequenced and the recombinant protein was expressed. Messenger RNA from Cupressus arizonica pollen was obtained and the Cup a 1 sequence was established using a 3'-RACE system and primers based on the N-terminal amino acid sequence. Recombinant Cup a 1 was cloned in pBluescript and expressed in a glycosylated form in rabbit reticulocytes. The cDNA was subcloned in pGEX-5X-1 and expressed in Escherichia coli as a fusion protein with GST. Recombinant Cup a 1 is highly homologous with the major allergens of mountain cedar (Jun a 1), Japanese cypress (Cha o 1) and Japanese cedar (Cry j 1). Cup a 1 contains three potential N-glycosylation sites that are different from those found in Jun a 1 and Cry j 1. The cloned protein contains a pectate lyase active site identical to those of Cry j 1 and Jun a 1. The IgE from patients' sera recognizes recombinant Cup a 1, and this reactivity is higher with the glycosylated protein. Cup a 1 has been cloned and sequenced. As expected, the high degree of homology with Cha o 1, Jun a 1 and Cry j 1 explains the cross-reactivity of conifer pollens. Different IgE reactivity with the glycosylated and non-glycosylated protein suggests the importance of carbohydrate moieties in the IgE binding site.
Aceituno
E
E
Immunology Department, Fundación Jiménez Díaz, Madrid, Spain.
Del Pozo
V
V
Mínguez
A
A
Arrieta
I
I
Cortegano
I
I
Cárdaba
B
B
Gallardo
S
S
Rojo
M
M
Palomino
P
P
Lahoz
C
C
D000485:Allergens / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001665:Binding Sites; D003001:Cloning, Molecular; D018745:Genome, Plant; D006031:Glycosylation; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics*; D011058:Pollen / Q000737:chemistry*; D011133:Polysaccharide-Lyases; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000737:chemistry; D012130:Respiratory Hypersensitivity / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D014197:Trees
D000485:Allergens; D052179:Antigens, Plant; C419643:Cup a 1 protein, Cupressus arizonica; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007073:Immunoglobulin E; D011133:Polysaccharide-Lyases; C023911:pectate lyase