Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4

Abstract

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody.

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“…As shown in Figure 1, the 31 kDa antigen in the soluble fraction, 53 and 40 kDa antigens in insoluble fraction were detected by AE6F4 antibody. These antigens were previously reported 14-3-3 proteins (Shoji et al, 1994a;Kawamoto et al, 1995), CK8 and CK19 (Ichikawa et al, 1997), respectively. To confirm whether carbohydrate chains in these antigens participate in antigen recognition by AE6F4 antibody, the protein-transferred nitrocellulose membrane was treated with sodium periodate to destruct the carbohydrate chain structure.…”

“…Shoji et al revealed that the AE6F4 antibody recognizes a 31 kDa antigen in the cytosolic fraction of A549 cells and partial amino acid sequence of this 31 kDa antigen shows a 90% homology to the 14-3-3 protein family (Shoji et al, 1994b). Kawamoto et al reported that the 31 kDa antigen cDNA cloned from A549 cells is identical to cytosolic phospholipase A2 (cPLA2) which is a member of the 14-3-3 protein family (Kawamoto et al, 1995).…”

“…These data suggested that AE6F4 antibody cross-reactive to 14-3-3 proteins, CK8 and CK19. Our previous results of immunoblotting analysis with AE6F4 antibody against sodium periodate treated antigens suggested that AE6F4 antibody reacts with both carbohydrate part of 14-3-3 proteins and peptide epitopes on CK8 and CK19 (Kawamoto et al, 1995;Ichikawa et al, 1997). An accumulation of knowledge about the cross-reactivity of AE6F4 antibody is necessary in order to apply to diagnoses and medical treatments.…”

Prognostic significance of cytosolic phospholipase A2 expression in patients with colorectal cancer

“…cPLA2 expression is increased in several human cancers including small bowel, lung, bile duct and liver [12,23-25]. Proliferation of prostate cancer is also dependant on the action of cPLA2 [26].…”

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“…BD9D12 and AE6F4 hybridoma cell lines were established by an in vitro immunization method (Kawahara et al, 1992). AE6F4 mAb recognizes 14-3-3 protein in lung cancer cells (Shoji et al, 1994a;1994b;Kawamoto et al, 1995) and cytokeratin 8 (Ichikawa et al, 1997). HB4C5 mAb has been applied to RI imaging of lung cancer tissues (Kusakabe et al, 1994) and AE6F4 to diagnosis of lung cancer cells in sputa (Shoji et al, 1994a).…”