Human Protein NEFA, a Novel DNA Binding / EF-Hand / Leucine Zipper Protein. Molecular Cloning and Sequence Analysis of the cDNA, Isolation and Characterization of the Protein.

Abstract

The cDNA libraries constructed from the human acute lymphoblastic leukemia 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 hybridization 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 sequence 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 fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The 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 confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) 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.

Serological identification and expression analysis of gastric cancer-associated genes

“…Moreover, comparison of mRNA levels between cancerous and adjacent non-cancerous tissues by RT -PCR showed that the expression of NUCB2 is downregulated in 50% of gastric tumours. NUCB2 is a Ca 2+ binding protein, which also contains putative DNA binding domain, two helix -loop -helix motifs, EF-hands and leucine zipper (Barnikol-Watanabe et al, 1994;Kroll et al, 1999). It interacts with the postmitotic growth suppressor necdin and is proposed to be involved in the regulation of survival and death of postmitotic cells by controlling Ca 2+ homeostasis in the cytoplasm (Taniguchi et al, 2000).…”

Identification of a cell protein (FIP-3) as a modulator of NF-κB activity and as a target of an adenovirus inhibitor of tumor necrosis factor α-induced apoptosis

“…All of these interactions presumably occur in the cytoplasm. FIP-3 also appears in the nucleus, where its leucine zipper or zinc-finger domains might promote interactions with the NF-B transcriptional apparatus (43)(44)(45). Recently, while this manuscript was in preparation, the mouse homologue of FIP-3 (named NEMO) was isolated by its ability to complement cells deficient in NF-B .…”

Role of nesfatin-1 in the metabolism of skeletal tissues

“…Nucleobindin was identified in the 1990s as a protein capable of binding Ca 2+ ions, located in the nucleus, the Golgi apparatus, and the endoplasmic reticulum (3,35). Further investigations revealed two types of this protein: nucleobindin-1 (NUCB1) and nucleobindin-2 (NUCB2).…”