Purification and Characterization of <i>Par·</i> I, Major Allergen of Parietaria officinalis Pollen

Abstract

Par·I, a major allergen of Parietaria officinalis, was purified from the pollen extract. The purified allergen was obtained by ultrafiltration, Sephadex gel filtration and DE-52 ion exchange chromatography: the purified preparation yields a single band in polyacrylamide gel isoelectric focusing (PAG-IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, a single immunoprecipitation arc in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a single peak in size exclusion high-performance liquid chromatography (HPLC). Par·I is a glycoprotein with a protein to carbohydrate ratio of 100:21. The molecular weight, determined by SDS-PAGE, Sephadex G-50 gel filtration and size exclusion HPLC, varied between 13.5 and 14.5 kDa according to the method employed. The isoelectric point was 4.6. The amino acid composition and the sequence of the first twelve N-terminal residues were determined. The allergenicity was assayed in vivo and in vitro. 29/29 Parietaria-allergic patients were skin positive to Par·I and possessed high level of specific serum IgE antibody as it determined by radioallergosorbent test (RAST). Par·I contained dominant epitopes for human IgE as inhibited to 85% the pollen extract RAST performed with a pool of sera of allergic patients. The RAST inhibitory activity was not abolished by deglycosylation.

Immunochemical Characterization of Rapid and Slowly Released Allergens from the Pollen of <i>Parthenium hysterophorus</i>

“…With respect to the contribution o f the carbohydrate moiety towards allergenicity, the allergens described so far can be broadly classified into four categories: (1) where the covalently linked sugars directly bind lgE antibodies, e.g., glycoprotein 2 from ryegrass pollen [30], H antigen from sea-squirt [31], phospholipase A2from honeybee venom [32], the high molecular weight glycoprotein/polysaccharide allergens from Pityrosporum ovale and Candida albi cans [33] and the differentially glycosylated isoforms of the major allergen (Ole e 1) of olive pollen [34]; (2) where the protein-linked carbohydrates are necessary only for the maintenance of correct conformation of the glycoprotein, and thus influence the affinity of IgE binding, e.g., Cry j 1 and Parj 1, the major allergens from pollen of Cryptomeria japonica [35] and Parietaria judaica [36], respectively; (3) where the binding of serum IgE to the glycoprotein allergen is indifferent to the presence or absence of carbohydrates, e.g., major glycoprotein allergens from Parietaria officina lis, mugwort, timothy and the cat allergen [37][38][39][40], and (4) where the deglycosylation of the allergen actually results in stronger allergenic activity than the native molecule, e.g., the fungal allergen Cla h 2 from Cladosporium herbarum [41]. Based on periodate oxidation and pronase digestion data, it appears that the glycoprotein allergens from the pol len of P. hysterophorus belong to category 1.…”

Characterization of Major Allergens of <i>Parietaria officinalis</i>

“…Recent ly, Parj 1 was described as consisting o f two isoforms with different molecular weights o f 13 and 10.5 kD and similar, but not identical, N-terminal amino acid sequences [12]. To a certain extent, these sequences coincide with the N-termi nal sequence described for Par o 1 [6]. A cDNA clone en coding Parj 1 has been isolated and its DN A sequence deter mined.…”

“…Par o 1 was identified as a glycoprotein o f about 15 kD [5] or 14.5 kD with a protcinxarbohydrate ratio of 100:21 [6], The sequence of 12 N-terminal amino acids for a purified Paro 1 protein has been described, but it is still an open question whether there are other closely related major allergens in the P officinalis extracts sharing dominant epi-topes with this protein [6]. Several publications have dealt with the major allergen Parj I from the closely related spe cies P.judaica.…”

Parietaria pollinosis: a review

“…The major allergens of P. judaica and P. ojficinalis have been purified from pollen extracts (2,17,21,51,54,60,61,64). Par o I (54) and Parj I are glycoproteins: the first has an apparent mol.…”

“…Purified Parj I and Par o I preparations contain closely related molecular species different for charge and electrophoretic mobility (53,54,61). Polo et al (61) report that charge heterogeneity of Par j I is decreased by deglycosilation.…”