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Poult Sci 2008. 87:768-776. doi:10.3382/ps.2007-00369
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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY

Cloning, Sequencing, Characterization, and Expressions of Divalent Metal Transporter One in the Small Intestine of Broilers1

S. P. Bai*,{dagger}, L. Lu*,{dagger}, X. G. Luo*,{dagger},2 and B. Liu*,{dagger}

* Mineral Nutrition Research Division, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, P. R. China; and {dagger} State Key Laboratory of Animal Nutrition, Beijing 100094, P. R. China

2 Corresponding author: wlysz{at}263.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Divalent metal transporter 1 (DMT1) is an electrogenic transporter of divalent Mn that is expressed at a high level on the brush-border membrane of enterocytes. This study is the first reported isolation of the 2 full-length cDNA sequences of the small intestinal DMT1 gene of broilers by rapid amplification of cDNA ends. The chicken DMT1 isoform I cDNA was 1,972 bp and contained a 1,695-bp open reading frame encoding a 564-amino acid protein, and the chicken DMT1 isoform II cDNA was 1,775 bp and contained a 1,593-bp open reading frame encoding a 530-amino acid protein. The 2 chicken DMT1 isoform transcripts differed in their 3'-translated regions and untranslated regions. The identities of the amino acid sequence deduced from the full-length cDNA sequence of the chicken DMT1 isoform I with DMT1 not containing the iron-responsive element of the mouse, rat, and human were 82, 82, and 80%, respectively. The identities of the amino acid sequence deduced from the full-length cDNA sequence of the chicken DMT1 isoform II with those of the mouse, rat, and human were 84, 84, and 83%, respectively. Analyses of hydrophobicity, transmembrane region, and signal peptides of DMT1 proteins deduced by nucleotide sequences suggested that chicken DMT1 isoforms are transmembrane proteins with several conserved peptide sequences, such as N-linked glycosylation signals (N-X-S/ T, where X designates any amino acid) and the consensus transport motif. The total mRNA levels of the 2 chicken DMT1 isoforms, the mRNA levels of chicken DMT1 isoform I, and the mRNA levels of chicken DMT1 isoform II in the duodenum and jejunum were higher (P < 0.002) than that in the ileum by real-time reverse transcription PCR assay. There was no significant difference (P > 0.26) between the duodenum and jejunum for the above 3 indices. The mRNA level of the chicken DMT1 isoform I was higher (P < 0.001) than that of the chicken DMT1 isoform II in each small intestinal segment of Mn-deficient broilers.

Key Words: divalent metal transporter 1 • nucleotide sequence • mRNA level • small intestine • broiler


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Manganese is essential for normal bone formation and amino acid metabolism in poultry (Scott et al., 1976). The utilization of Mn has become an increasing concern because of the extremely rapid growth rate of commercial broiler strains, which puts additional stress on bone structure. However, excess Mn could catalyze the formation of oxygen free radicals that can damage cellular components (Roth, 2006). For these reasons, the broilers must tightly regulate the amount of Mn absorption to provide the sufficient amount for their biological needs without allowing excess Mn to accumulate. The main excretory routes for Mn are bile (Birtenchamps et al., 1966; Abrams et al., 1977), pancreatic secretions (Burnett et al., 1952), and sloughed intestinal cells (Thomson et al., 1971). Endogenous losses of Mn were ~8% of the amount of Mn actually absorbed regardless of intake (Davis et al., 1992). The intestine seems to be the exclusive locus of homeostatic regulation of body Mn stores.

Recently, some of the molecular details of small intestinal Mn transport have been revealed in mammals. Divalent metal transporter 1 (DMT1), also named divalent cation transporter-1 or natural resistance-associated macrophage protein-2 (Nramp2), was found to be an electrogenic transporter of divalent cation, including Mn2+ (Gunshin et al., 1997). Overexpression of DMT1 in cultured cells resulted in increased Mn uptake (Forbes and Gros, 2003), and incubation with anti-DMT1 antibody blocked uptake of bivalent Mn (Conrad et al., 2000). The Belgrade (b) rat, which possesses a point mutation in DMT1 (G185R), exhibited impaired Mn metabolism (Chua and Morgan, 1997). Solubilized Mn that was released from the stomach into the duodenum was transported across the microvilli via the transport protein DMT1 (Canonne-Hergaux et al., 1999; Trinder et al., 2000; Knopfel et al., 2005). Yeast can be used as a model to study metal transmembrane (TM) import and export functions, which are universal steps in metal homeostasis that are highly conserved in evolution (Irazusta et al., 2006). Of 2 members of the Nramp family of metal transporter proteins in Saccharomyces cerevisiae (Smf1p and Smf2p), Smf1p mediated cell-surface uptake of Mn2+ and was required to resist chelator stress; Smf2p enabled efflux from endocytic vesicles, which was required in all growth conditions for the distribution of Mn2+ to the mitochondria and Golgi to sustain Mn2+-dependent activities (superoxide dismutase and sugar transferase, respectively; Cohen et al., 2004), including some that were required for yeast growth (Paidhungat and Garrett, 1998; Eguez et al., 2004).

To our knowledge, DMT1 cDNA sequences have been cloned and sequenced from the human, mouse, and rat (Vidal et al., 1993; Gruenheid et al., 1995; Gunshin et al., 1997). However, in poultry, DMT1 cDNA sequences in the small intestine have not yet been reported. Therefore, in this study, we report the cloning and sequencing of the chicken DMT1, characterization of the proteins deduced from the cDNA sequences, and their expression in the small intestinal segments of broilers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Birds, Diets, and Treatments
The Chinese Academy of Agricultural Sciences Animal Care Committee approved the protocol used in the experiment. A total of 20 one-day-old male Arbor Acres commercial broilers (Arbor Acres Poultry Breeding Co., Beijing, China) were housed in electrically heated, thermostatically controlled cages with fiberglass feeders, with a 24-h constant light schedule. The birds were allowed ad libitum access to feed and tap water that contained 95.90 µg of Ca/mL, 40.73 µg of Mg/mL, 0 µg of Mn/ mL, and 1.3 µg of Zn/mL. The birds received a Mn-supplemented corn-soybean meal diet (Table 1Go) formulated to meet or exceed all nutrient requirements of broilers before the age of 22 d, as recommended by the NRC (1994). The level of Mn in the diet was 116.80 mg/kg by analysis. In previous studies (Ji et al., 2006a,b), we demonstrated, by using everted intestinal sacs and ligated loops, that the main absorption site of Mn was the ileum in the small intestine of Mn-deficient broilers. Therefore, to investigate its molecular basis, all birds were fed the basal diet containing 18.40 mg of Mn/kg (Table 1Go) for 1 wk from d 22, which could deplete the body Mn stores (Ji et al., 2006a,b). Ten birds were used for collecting the duodenal segments as the materials for studying DMT1 cloning. Another 10 birds were used for the collection of duodenal, jejunal, and ileal segments for assays of DMT1 mRNA levels. There were 10 replicate segments for each small intestinal segment.


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  		Table 1. Composition of the diets for broilers in the experiment
 
Sample Collections and Total RNA Extractions
Small intestinal tissues were taken from 10 birds that were euthanized at 28 d of age with an intravenous overdose injection of sodium pentobarbital (40 mg/kg). The small intestine was removed, and 5-cm segments were dissected from the duodenum, jejunum, and ileum, respectively. Initial preparation involved flushing all tissue samples with ice-cold 0.9% NaCl to remove the intestinal contents. The mucosal samples were then collected by scraping. All mucosal samples were immediately frozen in liquid nitrogen and stored at –70°C until analysis.

The total RNA was extracted from the mucosa of the different intestinal segments in the male broilers by using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Before the first-strand cDNA synthesis, DNAase I treatment of the total RNA was necessary if the total RNA samples were seriously contaminated with genomic DNA.

Cloning and Sequencing of the Small Intestinal DMT1 cDNA Sequences of Chickens
Rapid amplification of cDNA ends (RACE) was carried out according to the method of Bu et al. (2001) with some modifications. The 3' ends of the chicken DMT1 transcripts were amplified by using 3' RACE. Total RNA (1 µg) extracted from the broiler duodenum was reverse transcribed by using Superscript III RT reverse transcription (Invitrogen) and RACE-1 primer (5'-GGCCACGCG TCGACTAGTACTTTTTTTTTTTTTTTTT-3') according to the manufacturer’s instructions. All the subsequent PCR reactions contained the following components unless otherwise stated: 2.5 µL of PCR buffer (Tiangen Bio. Co., Beijing, China), 1.5 mmol/L of MgCl2, 12.5 pmol of each primer, and 5 units of LongTaq polymerase (Tiangen Bio. Co.) in 25-µL reaction volume. The PCR reactions were carried out by using a DNA thermal cycler (PTC-200, Bio-Rad, Hercules, CA) with the following Touchdown PCR cycle conditions: 5-min denaturation at 95°C, 16 cycle of 1-min denaturation at 94°C, 1-min annealing at 70°C (lower 1°C/cycle), and 2-min extension at 72°C; 20 cycles of 1-min denaturation at 94°C, 1-min annealing at 55°C, and 2-min extension at 72°C, followed by a final 10-min extension at 72°C. By referring to the housekeeping amino acid sequences IESDLQS and FLDKYGLR of DMT1 in the mammalian species, gene-specific primers (GSP) were designed for 3' RACE. The first-round PCR was carried out with 12.5 pmol each of GSP1 (5'-ATCGAGTCCGACCTGCAGTC-3') and universal amplification primer (UAP; 5'-GGCCACGCGTCGACTAGTAC-3) by using 1 µL of the reverse transcription reaction products as template. The amplification products were diluted to 0.5 mL with double-distilled water. The second-round PCR was performed by using 12.5 pmol of GSP2 (5'-TTCCTGGACAAATATGGTCTGC-3'), 12.5 pmol of UAP, and 1 µL of diluted first-round PCR products. The resulting products were electrophoresed on a 1% Tris-acetate-EDTA agarose gel. The area corresponding to the estimated size of the largest specific bands on the probed membrane were excised and purified by using a DNA Quick Extraction kit (Tiangen Bio. Co.) according to the manufacturer’s instructions. The resulting ~1.3- and ~1.1-kb products were subcloned with the pMD-18T vector system (TaKaRa, Dalian, China) according to the manufacturer’s instructions, and the products were sequenced by using an ABI Prism 3730 genetic analyzer (Applied Biosystems, Foster City, CA). For 5' RACE, all GSP were designed according to the sequencing results of the 3' RACE products. The first strand of cDNA was synthesized by using the primer GSP3 (5'-GCACGACGCGGCAAAGAAG-3'). A poly(A) tail was appended to the 5' end of the first strand of cDNA by using terminal deoxynucleotidyl transferase (Promega, Madison, WI) after being purified by the Microcon YM-100 filter (Millipore, Billerica, MA) at 37°C for 5 min. The poly(dA)-tailed cDNA was then amplified with RACE-1 primer, UAP, and GSP4 (5'-TGCAGGTACATATTGTGCGGC-3') by using an annealing temperature of 60°C. The PCR products were separated by electrophoresis on a 1.5% agarose gel. The extraction, cloning, and sequencing of PCR products were done as described above. In all cases, at least 4 separate clones were sequenced to correct for any PCR errors.

Sequence Analyses of the Nucleotides and Amino Acids of the DMT1
The nucleotide and amino acid sequences of DMT1 isoforms for chickens were subjected to BLAST searching at the National Center for Biotechnology Information. Multiple comparisons of the nucleotide sequences were then performed by BioEdit (http://www.mbio.ncsu.edu/). Characterization of chicken DMT1 isoforms included determinations of their molecular weight, amino acid composition, hydrophobicity, TM region characteristics, and signal peptide analysis. Hydrophobicity was analyzed by using ProtScale and ExPASy, and evidence for TM regions was analyzed by using ExPASy (prediction parameters: TM-helix length between 17 and 33). Parameters used in the ProtScale analysis included a window size of 9, a window weight on the edges of 100%, the linear weight variation model, and no normalization of the scale. Protein signal peptides were analyzed by SignalP 3.0 software (Bendtsen et al., 2004; http://www.cbs.dtu.dk/services/SignalP-3.0/). Analyses used neural networks and hidden Markov models trained on eukaryotes.

DMT1 mRNA Expression in the Different Small Intestinal Segments of Broilers
The DMT1 mRNA levels in the mucosa of different small intestinal segments were analyzed by real-time reverse transcription PCR (RT-PCR) with an ABI Prism 7000 instrument (Applied Biosystems). Briefly, the RNA integrity was confirmed by gel electrophoresis, and the concentration of the total RNA was determined by ultraviolet spectrophotometry (model Cary-100, Varian, Palo Alto, CA). One microgram of the total RNA was reverse transcribed to cDNA by using random hexamer primers (Invitrogen). Divalent metal transporter 1 and β-actin were amplified in ABI SYBR Green PCR Master Mix (Applied Biosystems) from the cDNA with specific primers. The real-time RT-PCR primers were as follows: the 2 chicken DMT1 isoforms forward 5'-AGCCGTTCACCAC TTATTTCG-3', reverse 5'-GGTCCAAATAGGCGATGC TC-3'; chicken DMT1 isoform I forward 5'-AGCCATCCT CAGCGTCATCT-3', reverse 5'-CCTCCATCTCCCACC GTCA-3'; chicken DMT1 isoform II forward 5'-GGCGG TGCGGTCATCCTCCT-3', reverse 5'-ACCCGGCGTAG CGCAGTCAC-3'; and β-actin forward 5'-GAGAAATTG TGCGTGACATCA-3', reverse 5'-CCTGAACCTCTCAT TGCCA-3'. For quantifying DMT1 and β-actin, we used the standard curve method described by Tchernitchko et al. (2002). Relative standard curves were obtained by blotting the cycle threshold obtained following PCR amplification of serial dilutions of a steady quantity of the plasmid containing the corresponding cDNA. Because the expression levels of β-actin mRNA were identical in the different intestinal segments of the chicken, β-actin was used as the endogenous control (Mete et al., 2005). The PCR products of DMT1 were normalized to the β-actin level.

Statistical Analysis
Data on the total mRNA levels of the 2 chicken DMT1 isoforms, the mRNA levels of chicken DMT1 isoform I, and the mRNA levels of chicken DMT1 isoform II in the different small intestinal segments were analyzed by 1-way ANOVA with the GLM procedures of SAS (SAS Institute, 2003). The difference between the chicken DMT1 isoform I mRNA level and the chicken DMT1 isoform II mRNA level in each small intestinal segment was analyzed by the t-test procedure of SAS (SAS Institute, 2003). Significance was set at P < 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Cloning and Sequencing of Full-Length cDNA for Broiler DMT1 Isoforms
By using a combination of RACE and homology cloning, we cloned and sequenced 2 kinds of full-length cDNA sequences for chicken DMT1. The nucleotide sequence analysis using the BLAST software on the National Center for Biotechnology Information server revealed that these genes were homologous to the Nramp family, and they were then deposited into the GenBank database (accession numbers: EF635922 [GenBank] and EF635923 [GenBank] ). The complete cDNA sequences of chicken DMT1 isoforms and their encoded amino acids are presented in Figure 1Go. The results showed that one 1,972-bp cDNA sequence for the chicken DMT1 isoform I contained a 1,695-bp open reading frame encoding a 564-amino acid protein, and the 1,775-bp cDNA sequence for the chicken DMT1 isoform II contained a 1,593-bp open reading frame encoding a 530-amino acid protein. The ATG codon at nucleotide position 105 of the cDNA sequences of the 2 chicken DMT1 isoforms was used as the initiator, because it was positioned within the optimal sequence context (GCCAUGG) for initiation by eukaryotic ribosomes (Kozak, 1986). The 2 chicken DMT1 isoform transcripts differed in their 3'-translated regions and untranslated regions (UTR; Figure 1Go), which demonstrated that there were 2 variants in the 3' ends of the DMT1 cDNA sequences in the small intestine of broilers. Transcriptions of the mammalian Nramp2 genes coding for DMT1 gave rise to 4 variant mRNA transcripts that differed in their distributions and regulations (Hubert and Hentze, 2002; Hentze, et al., 2004). A 5'-end mRNA processing variant started from exon 1A, which was located upstream of the first exon 1B. The 1A exon (which contained a start codon) was followed by a consensus splice sequence and was spliced directly to exon 2. Meanwhile, the exon 1B was not translated, and translation began with a start codon in exon 2, whereas the 1B isoform was ubiquitous, and the 1A isoform was tissue specific and found predominantly in the duodenum and kidney (Hubert and Hentze, 2002). In addition, variable 3'-end processing yielded 2 transcripts that differed in their 3'-translated regions and UTR (Lee, et al., 1998; Canonne-Hergaux, et al., 1999). One of the transcripts contained the iron-responsive element (IRE) in 3'-UTR. The IRE-containing and non-IRE-containing forms differed mainly in their regulation by iron. The reaction of the iron-responsive protein with IRE increased the stability of mRNA of the DMT1-IRE (Hubert and Hentze, 2002). However, in this study, the 2 chicken DMT1 isoforms had high sequence identity to the mammalian DMT1-non-IRE (Figure 2Go), and did not contain the IRE consensus sequence (CNNNNNCAGUG) predicted to form a stem-loop (Gunshin et al., 1997). The regulation mechanisms of the chicken DMT1 isoforms might be different from those of other species and should be studied further in the future.


Figure 1
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  		Figure 1. Complete cDNA sequences and the putative amino acid sequences of chicken divalent metal transporter 1 (DMT1) isoforms I and II (GenBank accession numbers: EF635923 and EF635924) in the small intestine. An asterisk (*) represents the end code. The difference of the 3'-ends in the 2 chicken DMT1 isoform cDNA sequences is underlined.

 

Figure 2
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  		Figure 2. Alignments of the deduced divalent metal transporter 1 (DMT1) amino acid sequences of the chicken, human, mouse, and rat. Coding nucleotide sequences were available from the GenBank database under accession numbers EF635922 (chicken isoform I) and EF635923 (chicken isoform II), AF064484 (human), AK083478 (mouse), and AF400108 (rat). The 12 putative transmembrane regions are underlined and numbered 1 to 12. The consensus transport motif (CTM) is underlined and named the CTM in the fourth intracellular loop.

 
Amino Acid Sequences of Broiler DMT1 Isoforms
Analyses of the molecular weights and amino acid composition of the DMT1 isoform proteins with BioEdit showed that the molecular weights of chicken DMT1 isoforms I and II were 61.56 and 57.90 kDa, respectively. The DMT1 isoform proteins were predicted to be highly hydrophobic, with characteristics of the integral membrane proteins. Indeed, hydrophobic amino acids accounted for 45% of the chicken DMT1 isoform I (Leu, 13%; Ile, 6%; Val, 9%; Ala, 10%; Phe, 7%), and the apolar residue Gly accounted for 8%, whereas charged residues accounted for only 12% (Arg, 4%; Lys, 2%; Asp, 4%; Glu, 2%) of the total amino acid composition. The hydrophobic amino acids accounted for 44% of the chicken DMT1 isoform II (Leu, 12%; Ile,7%; Val, 9%; Ala, 10%; Phe, 6%) and the apolar residue Gly accounted for 8%, whereas charged residues accounted for only 12% (Arg, 4%; Lys, 2%; Asp, 3%; Glu, 3%) of the total amino acid composition (Figure 1Go). Figure 2Go showed the alignment of the amino acids encoded by the chicken DMT1 cDNA sequences and 3 other orthologous sequences of Nramp2. The identities of the amino acid sequence of DMT1 deduced from the cDNA sequence of the DMT1 isoform I of the chicken with those of the mouse, rat, and human were 82, 82, and 80%, respectively, whereas the identities of the amino acid sequence of DMT1 deduced from the DMT1 isoform II of the chicken with those of the mouse, rat, and human were 84, 84, and 83%, respectively.

Hydrophobicity, TM Regions, and Functional Regions of Broiler DMT1 Proteins
Analyses of hydrophobicity and the TM regions in amino acid sequences showed that DMT1 proteins were predicted to be highly hydrophobic, with characteristics of the integral membrane protein. Hydropathy profile analysis of chicken DMT1 proteins with the Kyte-Doolittle algorithm (Kyte and Doolittle, 1982) identified a minimum of 10 and a maximum of 12 highly hydrophobic putative TM domains in the 2 amino acid sequences (underlined in Figure 2Go). The amino and carboxy terminals of proteins would be intracellular. Results from the protein signal peptide analysis indicated that DMT1 proteins were nonsecretory cytoplasmic proteins (data not shown). Interestingly, some of the TM domains predicted in the amino terminal half of chicken DMT1 proteins (TM 1, 2, 4, 5) and 1 (TM 9) in their carboxy terminal half contained 1 charged residue, whereas the TM 3 included 2 such residues. The amino acid sequences of the 2 chicken DMT1 proteins showed 3 potential N-linked glycosylation signals (N-X-S/T; Marshall, 1972; positions 278, 332, and 345), and 2 of them (positions 332 and 345) clustered in the fourth extracellular loop flanked by the 2 TM domains delineated by residues 292 to 309 and 355 to 374. These motifs were imports for the function of DMT1 and were conserved in the mammals (Vidal et al., 1993; Gruenheid et al., 1995; Gunshin et al., 1997). The consensus transport motif was detected at positions 380 to 399 in the fourth intracellular loop, flanked by the 2 TM domains defined by residues 355 to 374 and 408 to 425 (underlined in Figure 2Go). This inner membrane consensus motif has been detected in the predicted intracellular loop of the membrane subunits of several periplasmic transport systems of gram-negative bacteria (Dassa and Hofnung, 1985; Kerppola and Ames, 1992), and was also described in a few eukaryotic proteins (Vidal et al., 1993). All these results strongly suggest that, like their orthologous sequences, the 2 chicken DMT1 isoforms are the integral membrane proteins with the following membrane topology for an even number of 10 or 12 TM domains. The amino terminus of the orthologous proteins would be intracellular, followed by 10 or 12 TM domains arranged in 5 or 6 TM loops, which would position the carboxy terminus to the intracellular phase of the membrane. The positioning of the cluster of N-linked glycosylation signals to a putative extracellular domain of the proteins and the consensus transport motif to an intracellular domain together suggest that the 2 chicken DMT1 isoforms have a potential transport function.

DMT1 mRNA Expression in the Different Small Intestinal Segments of Broilers
In this study, we used real-time RT-PCR to quantify the total mRNA levels of the 2 chicken DMT1 isoforms, the mRNA level of chicken DMT1 isoform I, and the mRNA level of chicken DMT1 isoform II in the different small intestinal segments of broilers (Figure 3Go). The total mRNA levels of the 2 chicken DMT1 isoforms, the mRNA levels of chicken DMT1 isoform I, and the mRNA levels of chicken DMT1 isoform II in the duodenum and jejunum were higher (P < 0.002) than those in the ileum. There was no significant difference (P > 0.26) for the above 3 indices between the duodenum and jejunum. The mRNA level of the chicken DMT1 isoform I was higher (P < 0.001) than that of the chicken DMT1 isoform II in each small intestinal segment of broilers. The DMT1 gene produces 2 alternatively spliced transcripts generated by the differential use of two 3' exons encoding distinct C-termini of the protein as well as distinct 3' UTR in mammals (Lee et al., 1998). One DMT1 isoform (DMT1-IRE) contains an IRE in its 3' UTR, whereas another DMT1 splice isoform (DMT1-non-IRE) does not (Lee et al., 1998). Expression of DMT1-IRE mRNA was highest in the duodenum, and decreased toward the ileum in the small intestine of rats. Divalent metal transporter 1-non-IRE mRNA appeared to be weakly expressed in all small intestinal segments of rats (Hubert and Hentze, 2002). We did not find such an IRE sequence as CNNNNNCAGUG in the mRNA of the 2 chicken DMT1 isoforms; therefore, the mRNA level of each chicken DMT1 isoform in the small intestine of broilers was not comparable to the mRNA level of the homologous gene isoform in mammals. However, our results demonstrated that DMT1 mRNA was expressed ubiquitously in the mucosa of the duodenum, jejunum, and ileum of broilers, which was consistent with the results of Mete et al. (2005) and with findings in mammals (Gunshin et al., 1997; Johnston et al., 2006). The total mRNA level of the 2 DMT1 isoforms in the small intestine of broilers did not entirely agree with the previous findings. Using 4 mynahs and 4 chickens (strains not shown) fed a normal diet, Mete et al. (2005) studied the DMT1 expression profile in the small intestine and found that DMT1 expression was highest in the jejunum of 2 kinds of birds and was lowest in the duodenum, whereas there was no difference in DMT1 expression between the duodenum and ileum in the mynah. In mammals, DMT1 mRNA expression was highest in the duodenum, and decreased toward the colon (Gunshin et al. 1997; Hubert and Hentze, 2002). The animal species and strains used, the positions of sampling, and animal physiological conditions, especially the Mn2+ store in the body, might explain the differences in the above results. For example, both diet and strain, and the interaction between diet and strain significantly influenced DMT1 mRNA expression in the duodenum of the rat (Dupic et al., 2002), and iron treatment changed the DMT1 expression only at the apical surface of Caco-2 cells, but not in the whole-cell homogenates (Johnson et al., 2005).


Figure 3
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  		Figure 3. The total mRNA level of the 2 chicken divalent metal transporter 1 (DMT1) isoforms, the mRNA level of chicken DMT1 isoform I, and the mRNA level of chicken DMT1 isoform II in the different small intestinal segments of Mn-deficient broilers. Segments of the duodenum, jejunum, and ileum were excised, and the mucosal samples were then collected by scraping. Total RNA was harvested, and the level of DMT1 mRNA was determined by real-time reverse transcription PCR. Expression levels of the DMT1 mRNA were normalized by the expression of β-actin, a housekeeping gene. The graph illustrates the mean ± SEM (n = 10). Bars represent the means of the different small intestinal segments of broilers. The total mRNA levels of the 2 chicken DMT1 isoforms, the mRNA levels of chicken DMT1 isoform I, and the mRNA levels of chicken DMT1 isoform II in the duodenum and jejunum were higher (P < 0.002) than those in the ileum. There was no significant difference (P > 0.26) for the above 3 indices between the duodenum and jejunum. The mRNA level of chicken DMT1 isoform I was higher (P < 0.001) than that of chicken DMT1 isoform II in each small intestinal segment of Mn-deficient broilers.

 
It is known that there is a tight linkage of dietary Mn absorption with the body Mn stores in the proximal intestine, where duodenal crypt cells are thought to sense the Mn needs of the body (Thomson et al., 1971). Although Mn absorption is normally restricted to the proximal small intestine, Beitz and Allen (1984) reported that during Mn deficiency, its distal regions showed an adaptive increase in Mn uptake. In previous studies in our laboratory, we found that the ileum was the main site of Mn absorption in Mn-deficient birds with everted intestinal sacs in vitro (Ji et al., 2006a) and with ligated intestinal loops in situ (Ji et al., 2006b). However, the mechanism of Mn absorption by the gut in broilers is not clear. Recently, DMT1 (also named Nramp2 or divalent cation transporter-1) has been found to be an electrogenic transporter of divalent cation, including Mn2+ (Gunshin et al., 1997). Overexpression of DMT1 in cultured cells resulted in increased uptake of Mn (Forbes and Gros, 2003), and incubation with anti-DMT1 antibody blocked uptake of divalent Mn (Conrad et al., 2000). The Belgrade (b) rat, which possesses a point mutation in DMT1 (G185R), exhibited impaired Mn metabolism (Chua and Morgan, 1997). Solubilized Mn that was released from the stomach into the duodenum was transported across the microvilli via the transport protein DMT1 (Canonne-Hergaux et al., 1999; Trinder et al., 2000; Knopfel et al., 2005). Divalent metal transporter 1 plays a pivotal role in Mn absorption in the intestine. High levels of DMT1 mRNA expression in the duodenum and jejunum of broilers suggest that the DMT1 might be the main carriers of Mn absorption in them. However, low expression of DMT1 mRNA in the ileum seems to be contrary to our finding that the ileum was the main site of Mn absorption in Mn-deficient broilers (Ji et al., 2006a,b). This suggests that there might be other pathways for mediating Mn absorption in the ileum segment of the small intestine of broilers. Ionic Mn could diffuse through the channels for Ca2+ in the plasma membrane of the cells (Milanick and Frame, 1991; Fasolato et al., 1993). Alternatively, tight junctions acting as paracellular shunts have been shown to be the main route of passive ion permeation in the rabbit gall bladder epithelium, which has much in common with the intestinal epithelium (Fromter and Diamond, 1972; Oschman, 1980). The ionic Mn possibly passed either intracellularly through aqueous channels in the plasma membranes or paracellularly through tight junctions in the ileum segment of broilers.

In conclusion, 2 kinds of full-length cDNA sequences coding for chicken DMT1 were successfully cloned and sequenced in the present study. To our knowledge, this is the first report in DMT1 of the complete cDNA of the avian species. Analyses of hydrophobicity, the TM region, and signal peptides of DMT1 proteins deduced by nucleotide sequences suggested that the chicken DMT1 isoforms are TM proteins with several conserved peptide sequences in other species. The real-time RT-PCR assay showed that DMT1 was widely expressed in the different intestinal segments of broilers, but it was expressed more abundantly in the duodenum and jejunum than in the ileum.


   FOOTNOTES
 
1 Supported by the National Basic Research Program of China (project no. 2004, CB117501 [GenBank] ; Beijing, China), the Key Program of the National Natural Science Foundation of China (project no. 30530570; Beijing, China), and Chinese Academy of Agricultural Sciences Foundation for First-Place Outstanding Scientists (Chinese Academy of Agricultural Sciences, Beijing, China). Back

Received for publication September 1, 2007. Accepted for publication January 3, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Abrams, E., J. W. Lassiter, W. J. Miller, M. W. Neathery, R. P. Gentry, and D. M. Blackman. 1977. Effect of normal and high manganese diets on the role of bile in manganese metabolism of calve. J. Anim. Sci. 45:1108–1113.[Abstract/Free Full Text]

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S. P. Bai, L. Lu, X. G. Luo, and B. Liu
Kinetics of Manganese Absorption in Ligated Small Intestinal Segments of Broilers
Poult. Sci., December 1, 2008; 87(12): 2596 - 2604.
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