Cloning of a pig homologue of the human lactoferrin receptor: expression and localization during intestinal maturation in piglets

Abstract

The presence of a small intestinal lactoferrin receptor (SI-LfR) has been suggested in the pig, but remains to be identified. LfR has been suggested to play a key role in the internalization of lactoferrin (Lf) and to facilitate absorption of iron bound to Lf. The aim of this study was to identify the pig SI-LfR cDNA, determine its mRNA and protein expression during different stages of intestinal development. The coding region of the pig LfR cDNA was cloned by PCR using conserved sequences among species. LfR mRNA expression and protein abundance were measured in proximal small intestine from piglets at 1 week (pre-weaning), 3 weeks (weaning) and 6 months (post-weaning) of age by quantitative real-time PCR (Q-PCR) and Western blot, respectively. Intestinal brush border membrane vesicles (BBMV) were also isolated to examine LfR abundance on the apical membrane. We determined the pig SI-LfR open reading frame (ORF) consists of 972 bp, resulting in a protein with a molecular mass approximately 135 kD and approximately 35 kD under non-reducing and reducing conditions, respectively. Using Q-PCR, we determined LfR expression significantly increased with age in the duodenum and reciprocally decreased in the jejunum. Intestinal LfR protein expression was maintained at all timepoints in the jejunum; however, in the duodenum LfR abundance reached maximum levels at 6 months. In BBMV fractions, LfR abundance significantly increased with age. Taken together our findings demonstrate the presence of a human SI-LfR homologue in pig, with mRNA and protein expression concomitantly regulated in the duodenum and inversely regulated in the jejunum. These findings suggest a mechanism by which pig Lf can be internalized in the intestine.

Figure 1

Conserved region alignment of putative lactoferrin receptors from Rattus norvegicus (GenBank accession no. NM_001034946), Mus musculus (GenBank accession no. NM_010584), Homo sapiens (GenBank accession no. NM_017625), Pan troglodytes (GenBank accession no. XM_513928), and Xenopus tropicalis (GenBank accession no. NM_203598). Asterisks indicate identical nucleotide sequences identified and underline sections indicates sequences used to generate primers.

Figure 2

LfR mRNA expression in pig duodenum and jejunum. The extracted mRNA was analyzed by Q-PCR. PCR products were quantified for relative levels of mRNA using image analysis by comparing LfR with β-actin. At week 1, LfR expression was significantly lower in duodenum compared to jejunum (Student’s t-test, P<0.05); however, this difference disappeared with age. LfR expression significantly increased with age in duodenum and decreased in jejunum. Values are means ± SD, different letters indicate significant difference over time.

Figure 3

SDS-PAGE analysis of 3 weeks old pig duodenum and characterization of antibody specificity. Lane 1: protein (18 μg) resolved without beta-mercaptoethanol in the sample buffer; Lane 2: protein (18 μg) resolved with beta-mercaptoethanol in the sample buffer; Lane 3: protein (18 μg) resolved with beta-mercaptoethanol in the sample buffer, and anti-serum (1:1000) incubated with excess antigen (0.8 mg/ml peptide) for 2 h prior to blotting of nitrocellulose membranes with electrophoresed proteins.

Figure 4

LfR protein expression in total homogenates of pig duodenum and jejunum. Proteins (18 μg) were resolved by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with LfR anti-serum. LfR expression in duodenum increased after 1 week of age, but did not change in jejunum. Values are means ± SD, different letters indicate significant difference over time. All data were normalized to β-actin (n=3).

Figure 5

LfR protein expression in BBMV from pig duodenum and jejunum. Proteins (10 μg) were resolved by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with LfR anti-serum. LfR expression in BBMV demonstrated an increase with age at both locations. Values are means ± SD, different letters indicate significant difference over time. All data were normalized to β-actin (n=3).