HUMAN PROTEIN NEFA, A NOVEL DNA BINDING EF-HAND/LEUCINE ZIPPER PROTEIN - MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF THE CDNA, ISOLATION ANDCHARACTERIZATION OF THE PROTEIN/

The cDNA libraries constructed from the human acute lymphoblastic leuk emia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridizatio n with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA seque nce was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs f undamentally from CALLA (CD10) although clones I and II were J5-positi ve in immune screening. The mature protein corresponding to the cDNA w as isolated and characterized from the KM3 cells using polyclonal anti sera raised against the in vitro expressed polypeptide from clone I. T he protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was con firmed by peptide sequences. The new protein contains a basic amino ac id rich putative DNA binding domain (b) with a potential nuclear targe ting signal, two helix-loop-helix (HLH) motif regions, concurrently EF -hand motifs, an acidic amino acid rich region (a) between the EF-hand s, and a leucine zipper (2) motif. This DNA binding protein therefore is characterized by a linked motif ''b/HLH/a/HLH/Z''. The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.