Results

Molecular cloning of the Acdp gene family
To clone the mouse Acdp genes, the human ACDP cDNA and predicted protein sequences were used to search the mouse EST database with the blastn and tblastn programs. Mouse EST markers corresponding to each Acdp member were then identified. For example, EST H3086H12-5 corresponds to Acdp1, W98010 for Acdp2, 603299135F1 for Acdp3 and BG083791 for Acdp4. A modified oligo-dT with a M13 tail was used for the RT reaction. A forward primer from each EST marker and the M13 primer (olig-dT tail) were used to amplify the 3' UTR sequence for each corresponding Acdp gene from the RT products. To obtain 5'-end coding sequences for the Acdp genes, we conducted a series nested PCR with combinations of mouse and human primers. The 5' UTR sequences were identified by directly sequencing BAC DNA containing the corresponding Acdp genes. The BAC clones were identified by screening a CITB mouse BAC DNA library (Research Genetics). The 5' UTR sequences obtained from above were further confirmed by RT-PCR. The Acdp1 gene contains 3,631 bp of nucleotide sequence and encodes a predicted protein with 951 amino acids (AA). The other three Acdp genes (Acdp2, 3 and 4) contain 3,244 bp, 2,684 bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 874 amino acids, 713 amino acids and 771 amino acids, respectively.

Tissue distribution
Northern blot analyses of the Acdp gene family were carried out using membranes purchased from Origene. A total of 12 mouse tissues were included in the study (Fig. 1). Due to sequence homologies between each Acdp member within the conserved domain, probes for Northern bolts were PCR fragments from the last exon and the 3' untranslated region sequences. The mouse Acdp messages showed almost the same tissue distributions as the human ACDP genes. Acdp1 message is highly expressed in the brain, while kidney and testis also showed low levels of expression. Acdp2 showed higher expressions in the brain, kidney and liver. However, the Acdp2 transcript was not present in the skeleton muscle and skin, and it showed very low levels of expression in the rest of tissues. Acdp3 and Acdp4 showed different levels of expression in all tissues tested; the highest expressions for Acdp3 were observed in the brain, kidney, liver and heart, and the highest expressions for Acdp4 were observed in the kidney, small intestine and testis. The expression levels for Acdp3 and 4 in skeleton muscle were barely detectable; however, β-actin showed normal expression suggesting that the results were not a consequence of bad RNA quality (data not shown). The ubiquitous expression pattern may be taken as another indication of the functional importance of Acdp proteins in fundamental biological processes in addition to the sequence conservation in evolutionarily divergent species.
Figure 1  Northern blot analyses of the Acdp gene family. S. muscle represents skeletal muscle, Sm. Int. represents small intestine. Multiple Choice Northern Blot filters were purchased from Origene.

Chromosomal location
Radiation hybrid mapping indicated that the Acdp1 gene maps to chromosome 19 between markers D19Mit119 (34.3 cR proximal)and D19Mit112 (13.6 cR distal). The Acdp2 gene maps slightly more distal to the Acdp1 on chromosome 19 between D19Mit9 (2.4 cR proximal) and D19Mit38 (15.1 cR distal). The Acdp3 and Acdp4 genes map to chromosome 1 within one BAC clone (RP23-294I17), proximal to marker D1Mit171 (17.4 cR). These regions are syntenic to the human counterparts.

Sequence homology and molecular characteristics
The mouse Acdp genes showed very strong homologies of both nucleotide and AA sequences to the human ACDP genes (Table 1). The highest homologies were observed between the human ACDP2 and the mouse Acdp2 gene (91% of nucleotide identity, 97% of AA identity and 99.4% of AA homology). In addition, the 5' UTR nucleotide sequences (20 bp of nucleotides before start codon) also showed high homologies to the human homologs, for example, the Acdp2 5' UTR sequence showed 95% identities to its human homolog. However, the homologies in the 3' UTR sequences (20 bp of nucleotides after stop codon) were much lower (40–55%) for all Acdp genes except Acdp4 (90% identity to its human homolog). The ancient conserved domain (ACD) has 55.3% of AA identity and 83.3% of homology between all mouse and human ACDP proteins (Fig. 2). The ACD domain is evolutionarily conserved in divergent species ranging from bacteria, yeast, C. elegans, D. melanogaster, mouse to human (Fig. 3). Particularly, as shown in Fig. 3, Acdp proteins showed very strong AA homology to bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and cobalt efflux [7]. High AA homology was also observed between the Acdp proteins and the yeast Amip3 protein (35% AA identity with 56% homology). The Amip3 is likely to be a homologous to the bacteria CorC protein. The Amip3 mutants confer resistance to copper toxicity (Personal communication with Dr. V.C. Culotte, John Hopkins Bloomberg, School of Public Health). The evolutionary relationships among those proteins are illustrated by a phylogenetic tree constructed based on the AA homology of proteins (Fig. 4).
Table 1  Nucleotide and amino acid homologies (%) between human ACDP and mouse Acdp members.
Acdp  Acdp1  Acdp2  Acdp3  Acdp4
DNA in coding region  89  91  86  87
AA identity  94  97  86  89
AA homology  98.8  99.4  94  96
5' UTR  85  95  80  80
3' UTR  40  55  50  90
Figure 2  Amino acid sequence homology alignment for all of the ACDP and Acdp genes within the ACD domain. The sequence data for the Acdp genes have been deposited in GenBank under accession number AF202994 (Acdp1), AF216961 (Acdp2), AF216964 (Acdp3) and AF216963 (Acdp4). Identical amino acids or amino acids with very strong homologies among all proteins were shaded black. Identical amino acids or amino acids with very strong homologies in most of the proteins were shaded grey. Dot lines represent gaps for the alignment.
Figure 3  Amino acid sequence alignment showing the conservation of ACD domain in various species. Amip3 is a protein from Saccharomyces cerevisiae (NP_014581). CanG is a protein from Candida glabrata (AAF33142). NeuC (EAA31204) is a hypothetical protein from Neurospora crassa. DroM is a gene product from D. melanogaster. The accession number for this gene is CG40084 in BDGP (Berkeley Drosophila Genome Project). AnoG represents a protein from the anopheles gambiae str. (EAA01004). CaeE (AAK77203) is a hypothetical protein from the Caenohabditis elegans. CorC represents bacteria magnesium and cobalt efflux protein from the Shewanella oneidensis. XyFD is a hypothetical protein from the Xylella fastidiosa Dixon (ZP_00038107).
Figure 4  Phylogenetic tree showing relationships among proteins containing the ACD domain from figure 2 and 3. The phylogenetic tree was constructed according to the calculation of the best match for the selected sequences. Abbreviations for each protein are the same as presented in figure 3. We found that all mouse Acdp members contain four distinct transmembrane domains (Fig. 5), two CBS domains and a DUF21 domain that are found in bacteria CorC and yeast Amip3 proteins. CBS domains are small intracellular modules that are mostly found in 2 or four copies within a protein. Pairs of CBS domains dimerise to form a stable globular domain [8]. DUF21 (CD: pfam01959.9) is a newly defined domain with unknown function. This domain is a transmembrane region and found to be located in the N-terminus of the proteins adjacent to two intracellular CBS domains . A cNMP-binding domain (cyclic nucleotide-monophosphate-binding domain) was found in all Acdp members.
Figure 5  Four transmembrane domains within Acdp4 protein. Transmembrane domains were predicted by the TMHMM program . The plot shows the posterior probabilities of inside /outside/TM helix. At the top of the plot (between 1 and 1.2) the N-best prediction is shown. The plot is obtained by calculating the total probability that a residue sits in helix, inside, or outside summed over all possible paths through the model. In addition, Acdp1 contains an Alanine-rich region (2–10: AAAAAAAAA), a Leucine-rich region (204–257: LLRVRPRLYGPGGDLLPPAWLRALGALLLLALSALF SGLRLSLLSLDPVELRVL), a Proline-rich region (78–130: PGPPVPAAPVPAPSLA PGENGTGDWAPRLVFIEEPPGAGGAAPSAVPTRPPGP), and two amidation sites (917–920: MGKK; 926–929: SGRK). Acdp2 has a glycine-rich region (201–222: GAGGSGSASGTVGGKGGAGVAG). Acdp3 possesses a large alanine-rich region (2–261) and a large leucine-rich region (201–299). Acdp4 contains a leucine zipper pattern (185–206: LVMVLLVLSGIFSGLNLGLMAL) and an amidation site (7–10: GGRR).

Antibody production, Western results and subcellular localization
Peptides from Acdp1 N- (TSFLLRVYFQPGPPATAAPVPSPT) and C- (TQQLTLSPAAVPTR) terminuses, conserved peptide from ACD domain of Acdp1 (HNIVDILFVKDLAFVDPDDCTPLLTVTRF) were commercially synthesized (Sigma Genosys). These antigenic sites were predicted by software from Sigma Genosys and polyclonal antibodies for each peptide were produced by immunizing rabbits. To test the specificity of the antibodies, we conducted Western-blot analysis of mouse brain tissue extracts. As shown in Fig. 6A, the antibody produced by C-terminal peptide specifically detected Acdp1 (lane 3). The antibody generated by N-terminal peptide recognized Acdp4 in addition to Acdp1, although the reactivity to latter was significantly higher (Fig. 6A, lane 2). As expected, the antibody produced by the conserved sequence peptide detected all Acdp proteins (Fig. 6A, lane 1). To further determine the specificity of the antibody against the Acdp1 C-terminus, we analyzed extracts of HEK293, 3T3 and PC12 cells. The results are shown in Fig. 6B. Apparently, this antibody detected a specific band of Acdp1 in all cell lysates. Of note, shown in Fig. 6B are signals of 10 μg extracts of HEK293 cell lysates, 100 μg extracts of 3T3 and PC12 cell lysates. Thus, the expression levels of Acdp1 in these cell types vary a lot, with the highest expression in HEK293 cells. Nevertheless, these immunoblot results support our analysis of brain tissue extracts that the antibody against Acdp1 C-terminus specifically recognizes Acdp-1.
The specificity of the Acdp1 C-terminus antibody suggests the possibility of using it to localize Acdp-1 within cells. Since Northern blot revealed almost exclusive expression of Acdp1 in the brain, we examined its subcellular localization in hippocampus neurons isolated from mouse embryos. The neurons were cultured on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes in dishes. Immunostaining was using the Acdp1 C-terminus specific antibody. Confocal imaging revealed that Acdp1 is predominantly localized on the plasma membrane. A series of sections of a cell at the thickness of 0.5 micrometer clearly showed membrane location of Acdp1-immunoreactivity (Fig. 7), which is consistent with the observation of transmembrane domains within the Acdp proteins.
Figure 6  Fig. 6A: Immunoblot analysis of Acdp proteins in brain tissue extracts. Immunoblotting were carried out using a Western blotting detection system (ECL) (PIERCE). Lane 1, probed with antibody generated by conserved peptide. From top to bottom, each band corresponding to Acdp1 (115 kD), Acdp2 (100 kD), Acdp4 (90 kD) and Acdp3 (80 kD). Lane 2 and 3, probed with the Acdp1 antibodies generated by N-terminal and C-terminal peptides, respectively. Fig. 6B: Immunoblot analysis of Acdp1 in HEK293, 3T3 and PC12 cells. The blots were probed with the antibody against the C-terminus of Acdp-1. Lane 1, 10 μg of HEK293 cell lysates. Lane 2 and 3, 100 μg of 3T3 and PC12 cell lysates.
Figure 7  Subcellular localization of Acdp1 in hippocampus neurons. A series of confocal images from a cultured neuron stained with an anti-Acdp1 antibody. The step of each imaging section is 0.5 μm, from the surface of the neuron (0 μm) to the middle plan (4.5 μm).