The basophil activation marker defined by antibody 97A6 is identical to the ectonucleotide pyrophosphatase/phosphodiesterase 3

Abstract

IntroductionIn an attempt to generate antibodies against surface antigens expressed on rare hematopoietic cell populations, monoclonal antibody (MoAb) 97A6 was raised, which selectively recognizes basophils, mast cells, and their CD34 ϩ precursor cells. 1 Stimulation of the basophils from donors allergic for acarids allergen with either anti-IgE antiserum or allergen results in a dose-dependent increase of 97A6 antigen expression, suggesting that the detected antigen plays an important role in cell activation. 1 Immunoprecipitation of the proteins recognized by MoAb 97A6 revealed 2 bands of 150 kd and 270 kd, respectively. 1 However, the identity of the molecule remained unknown. In this report, we show that 97A6 affinity-purified lysates consist of ectonucleotide pyrophosphatase phosphodiesterase-3 (E-NPP3), also termed phosphodiesterase I/nucleotide pyrophosphatase-3 (PDNP3), an ectoenzyme previously found in uterus, prostate, and glioma. [2][3][4] Study design Affinity purification of 97A6 antigenCoupling of purified MoAb 97A6 to an N-hydroxy succinimide (NHS)-activated Sepharose column (Pharmacia, Freiburg, Germany) was performed according to the manufacturer's recommendations. Lysates from 3 ϫ 10 9 KU-812 cells were loaded on a previously prepared 97A6-Sepharose affinity column equilibrated with washing buffer (10 mM Tris/HCl, 150 mM NaCl, 0.025% NaN 3 , 0.5% Triton X-100, pH 8.0). Bound 97A6 antigen was eluted with 50 mM triethanolamine, 0.1% Triton X-100, 150 mM NaCl, pH 11.5 and collected in tubes containing 1M Tris/HCl, pH 6.7. The collected fractions were screened on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for appearance of specific bands. In the next step, positive fractions were pooled and used for preparative separations on 7.5% SDS-PAGE. The proteins were visualized by silver staining and the resulting bands were cut for commercial microsequencing. This included tryptic digestion of the proteins, peptide mass fingerprint analysis (MALDI-MS), reverse phase-high-performance liquid chromatography (RP-HPLC), sequencing of the purified peptides, and search in the NCBI database (Toplab, Martinsried, Germany). Generation of E-NPP3 transfectant cell lineThe E-NPP3 complementary DNA (cDNA) was prepared and inserted into plasmids as described previously. 2 The calcium phosphate method was used for transfection of 293 embryonic kidney cells with 15 g plasmid. 5 Transfected cells were grown in tissue culture flasks in the presence of 0.5 mg/mL Geneticin. Six days after transfection the cells were stained with MoAb 97A6-phycoerythrin (PE) and the 97A6 ϩ fraction (9% of total cells) was sorted on a FACSVantage cell sorter (Becton Dickinson, Heidelberg, Germany). After 3 sorting rounds the cells stably expressed E-NPP3 on the cell surface. The transfectant cells were stained with MoAb 97A6-PE and analyzed on a FACSCalibur (Becton Dickinson). Purification of peripheral blood basophilsFicoll-Hypaque-selected buffy coat peripheral blood (PB) cells from healthy donors were stained with 97...

Cellular function and molecular structure of ecto-nucleotidases

“…NPP3 has been shown to promote differentiation and invasion of glial cells but the underlying mechanisms have not been identified [337]. Moreover, the basophil and mast cell-expressed cell surface antigen CD203c was identified as NPP3 [338]. This protein defines the basophil lineage in the bone marrow and shows an elevated distribution on the cell surface of activated basophils.…”

Flow cytometric investigation of peri‐anaesthetic anaphylaxis using CD63 and CD203c

“…CD203c, on the other hand, is reported to be exclusively expressed on basophils, mast cells and their progenitors and to be up-regulated in response to allergen-specific and anti-IgE-induced IgE cross-linking as for CD63. CD203c (neural cell surface differentiation antigen E-NPP3) belongs to the ectonucleotide pyrophosphate ⁄ phosphodiesterases, which also include E-NPP2 (human tumour motility stimulating protein PD-1a (also called autotoxin) [6]. CD203c has recently been used in the investigation of latex allergy [7].…”

“…In both the latter studies, the specificity has been shown to be high (93% and 100%) and the sensitivity low (63% and 54%). More recently, the expression of CD203c, found primarily on basophils, mast cells and CD34 progenitors [6], has been reported to be a more sensitive marker of basophil activation [7] but there is some disagreement about this [8]. There are no studies known to us evaluating CD203c expression in patients with anaesthetic drug allergy.…”

The underestimated role of basophils in Ph+ chronic myeloid leukaemia

“…This antigen is expressed on mature basophils as well as on immature basophil progenitor cells at various stages, including most immature, agranular CD34 +  basophil progenitor cells 43, 44. CML basophils also display CD203c (Figure 1).…”