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The major allergens of Parietaria officinalis were characterized with a panel of nine monoclonal antibodies (mAbs). The binding of mAbs and patients’ IgE in Western blots revealed two proteins with similar molecular weights in the range of 8-10 kD. Analysis of the mAb-binding patterns in Western blots of P. officinalis extract under reducing and nonreducing conditions allows the mAbs to be divided into three different groups. mAbs of group I recognize the higher-molecular-weight component (9.4 kD), mAbs of group II recognize the lower component (8.8 kD) and mAbs of group III recognize both proteins. A comparable mAb-binding pattern was observed with Western blots of Parietaria judaica. The mAbs were used for affinity purification of the corresponding proteins from a P. officinalis extract. The purified proteins obtained with mAbs of group I–III inhibit the binding of patients’ IgE (serum pool) to a high degree, indicating that they possess the major IgE-reactive epitopes. The affinity-purified proteins were subjected to SDS-PAGE, blotted and immunologically stained by mAb binding. The results confirmed those obtained with the complete extracts. The N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of all the proteins contained highly conserved regions: GGVV (positions 4–7) and MPPLL (positions 11–15), alternating with highly variable regions (positions 1–3 for group II and 8–10 for group I). A specific group I sequence appears to be at position 1–3 with the amino acids APA and a specific group II sequence appears to be at position 8–10 with the amino acids GAL. It is possible that the two similar proteins are isoforms of Par·1.

We have investigated the importance of sequences downstream to the TATA box of an immunoglobulin promoter by transfection and in vitro transcription assays. A sequence from -11 to +10 with respect to the transcriptional start site was synthesised by a procedure allowing for random misincorporation of nucleotides. The pool of mutant oligonucleotides was cloned into the respective position of a vector carrying a fusion of a synthetic immunoglobulin heavy chain promoter with the human growth hormone gene. From 200 clones sequenced, 115 were mutants with at least one nucleotide exchange in every position. Whereas most mutations are of minor functional importance, changes at or near the transcriptional start site reduce the promoter activity considerably.

Collectively, our data suggest that NB-p260, the apoptosis-mediating receptor for natural human anti-NB IgM, represents a novel surface protein of human NB cells containing polypeptide sequences related to the subunit c of the mitochondrial ATP synthase and the heat shock protein HSP90.

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