Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinase.
BACKGROUND
In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue. Fruit-allergic patients represent one risk group for developing latex allergy. Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens. The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes.


OBJECTIVE
To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02).


METHODS
A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR. The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E. coli as a fusion protein to maltose binding protein. The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA. Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay.


RESULTS
The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02). The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots. Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase. The growth of F. oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture.


CONCLUSIONS
Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein.
O'Riordain
G
G
Department of Pathophysiology, University of Vienna, Vienna, Austria.
Radauer
C
C
Hoffmann-Sommergruber
K
K
Adhami
F
F
Peterbauer
C K
CK
Blanco
C
C
Godnic-Cvar
J
J
Scheiner
O
O
Ebner
C
C
Breiteneder
H
H
D018528:ATP-Binding Cassette Transporters; D000485:Allergens / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D001665:Binding Sites / Q000276:immunology; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification; D002688:Chitinases / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D029968:Escherichia coli Proteins; D005512:Food Hypersensitivity / Q000276:immunology; D005638:Fruit / Q000276:immunology; D028482:Hevea / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology; D020315:Latex Hypersensitivity / Q000276:immunology; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification
D018528:ATP-Binding Cassette Transporters; D000485:Allergens; D052179:Antigens, Plant; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D007073:Immunoglobulin E; D002688:Chitinases; C465307:Hev b 11 allergen, Hevea brasiliensis; C119825:chitinase C