The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor

Abstract

The human protein NEFA (DNA binding, EF-hand, Acidic region) has previously been isolated from a KM3 cell line and immunolocalized on the plasma membrane, in the cytoplasma, and in the culture medium. Sequence analysis of a cDNA clone encoding NEFA identified a hydrophilic domain, two EF-hands, and a leucine zipper at the C-terminus. These characters are shared with nucleobindin (Nuc). In this paper we have further characterized NEFA and probed its evolutionary origins. Circular dichroism (CD) spectra of recombinant NEFA indicated a helical content of 51% and showed that the EF-hands are capable of binding Ca2+. Experiments with recombinant NEFA and synthesized peptides revealed that the leucine zipper cannot form a homodimer. The leucine zipper may allow heterodimer formation of NEFA and an unknown protein. Phylogenetic analyses suggest that this protein is derived from a four-domain EF-hand ancestor with subsequent duplications and fusions. The leucine zipper and putative DNA-binding domains of NEFA have evolved secondarily from existing EF-hand sequences. These analyses provide insights into how complex proteins may originate and trace the precursor of NEFA to the common ancestor of eukaryotes.

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer

“…They share 61.65% amino acid identity and a characteristic composition of functional domains -a signal peptide, a Leu/Ile rich region, basic amino acid rich region including putative bipartite nuclear localisation signal, two Ca 2+ binding EF-hand domains separated by acidic amino acid rich region and a leucine zipper (Figure 1) (Barnikol-Watanabe et al 1994;Miura et al 1992). Genes encoding NUCB1 and NUCB2 are located in 19q13.2-q13.4 and 11p15.1-p14, respectively, and are deemed to be derived from a common four-domain EF-hand ancestor (Karabinos et al 1996). Within the cell both are located in the cytoplasmic and Golgi luminal pools, both are secreted into extracellular space and interact with other proteins in a calciumdependent manner (Lavoie et al 2002;Lin et al 2000;Morel-Huaux et al 2002;Taniguchi et al 2000;Islam et al 2006).…”

Calnuc: Emerging roles in calcium signaling and human diseases

“…In addition to these motifs/ regions, NEFA has a probable nuclear localization signal, a basic amino acid region sandwiched between the two EF-hands, and a leucine zipper. Because of its extensive similarity to calnuc (61.56% sequence similarity), NEFA was alternately named as Nucb2 (25).…”

“…Phylogenetic analysis revealed that calnuc2 and calnuc have a common four-domain EF-hand ancestor, CTER (25). EF-hand motifs in proteins such as calmodulin and troponin C seem to have evolved from the CTER domains after undergoing a long process of genetic transformation, involving splicing, gene duplication, translocations, genetic fusions, and other similar methods of genetic evolution (25).…”

“…EF-hand motifs in proteins such as calmodulin and troponin C seem to have evolved from the CTER domains after undergoing a long process of genetic transformation, involving splicing, gene duplication, translocations, genetic fusions, and other similar methods of genetic evolution (25). Calnuc, with its various domains, seems to have also evolved in a similar fashion.…”

Serine Protease Activity of Calnuc

“…Because of their complex structure, understanding their diverse functions is an arduous task. Calnuc is one such Ca 2ϩ binding, Golgi-resident protein with multiple domains, highly conserved in species as varied as Ciona intestinalis to Homo sapiens (3)(4)(5)(6). Earlier reports on calnuc conferred it with a DNA binding property (4), and subsequently the presence of other domains was established including EF-hand Ca 2ϩ -binding sites (3), a cyclooxygenase (COX)-binding site (7,8), a G protein-binding site (9), and a species-specific variable C-terminal region.…”