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Characterization of expression in mice of a transgene containing 3.3 kb of the human lactase-phlorizin hydrolase (LPH) 5' flanking sequence.

ABSTRACT: The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator.Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22.In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28 days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7 days and declines significantly by 28 days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells.Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.

SUBMITTER: Baffour-Awuah NY

PROVIDER: S-EPMC3408868 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Characterization of expression in mice of a transgene containing 3.3 kb of the human lactase-phlorizin hydrolase (LPH) 5' flanking sequence.

Baffour-Awuah Nana Yaa NY Delemarre Eveline E Fujiwara Yuko Y Heijmans Jarom J Boerwinkel David D Algra Selma S Fialkovich John J JJ Islam Shabana S Bosse Tjalling T Grand Richard J RJ Krasinski Stephen D SD Montgomery Robert K RK

Digestive diseases and sciences 20101118 1

<h4>Background and aim</h4>The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator.<h4>Methods</h4>Four ...[more]

PMID: 21086165

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Project description:Background and aimsThe mechanism of the developmental downregulation of the lactase-phlorizin hydrolase (LPH) gene underlying adult-type hypolactasia is unknown. We have determined the functional significance of the recently identified two single nucleotide polymorphisms (SNPs), C/T(-13910) and G/A(-22018), associated with adult-type hypolactasia by studying LPH mRNA levels in intestinal biopsy samples with different genotypes.MethodsIntestinal biopsy samples were taken from 52 patients with abdominal complaints. Hypolactasia was diagnosed by determining lactase and sucrase activities and calculating their ratio (L/S ratio). The functional effect of the C/T(-13910) and G/A(-22018) genotype on expression of LPH mRNA was demonstrated in patients heterozygous for the C/T(-13910) and G/A(-22018) polymorphism and an informative expressed SNP located in the coding region of the LPH mRNA. Reverse transcription-polymerase chain reaction followed by solid phase minisequencing was used for accessing the relative expression levels of the LPH alleles using informative SNPs located in exons 1, 2, 6, 10, 13, or 17 as markers.ResultsStatistically significant differences between the three different genotypes CC(-13910) GG(-22018), CT(-13910) GA(-22018), and TT(-13910) AA(-22018) and their respective L/S ratios were observed. Relative quantitation of the expressed LPH alleles showed that the persistent allele represented 92 (6)% (mean (SEM), range 78-99%; n=14) of the expressed LPH mRNA. The patient with the homozygous persistent TT(-13910) AA(-22018), as well as hypolactasic patients with CC(-13910) GG(-22018), showed equal expression of both alleles (47 (1)%; n=7).ConclusionsExpression of LPH mRNA in the intestinal mucosa in individuals with T(-13910) A(-22018) alleles is several times higher than that found in individuals with C(-13910), G(-22018) alleles. These findings suggest that the two SNPs, C/T(-13910) and G/A(-22018), associated with adult-type hypolactasia, are associated with the transcriptional regulation of the LPH gene. The presence of the T(-13910) A(-22018) allele also shows significant elevation of the L/S ratio.

Project description:Previous studies [Büller, Montgomery, Sasak & Grand (1987) J. Biol. Chem. 262, 17206-17211] have demonstrated that lactase-phlorizin hydrolase is inserted into the microvillus membrane (MVM) as a large precursor of approx. 220 kDa, which then undergoes two proteolytic cleavage steps to become the 130 kDa mature MVM protein. In order to assess the role of glycosylation in intracellular transport, the processing of this enzyme has been studied in the presence of castanospermine, an inhibitor of N-linked oligosaccharide modification and subsequent treatment with two endoglycosidases, endo-beta-N-acetyl-glucosaminidase (endo-H) and peptide:N-glycosidase-F (N-glycanase). We now show that the intracellular precursor (205 kDa) undergoes carbohydrate processing (220 kDa) and transport to the MVM where its further proteolytic cleavage is as described. Treatment of the intracellular 205 kDa precursor with either endo-H which cleaves only high-mannose N-linked oligosaccharides, or with N-glycanase, which cleaves both high-mannose and complex N-linked oligosaccharides, results in the conversion of the 205 kDa protein band to one of 195 kDa. These data suggest that the 205 kDa precursor contains only high-mannose N-linked carbohydrates, and that the unglycosylated nascent protein is 195 kDa. In the presence of castanospermine, an intracellular precursor of approx. 210 kDa is observed. When treated with endo-H or N-glycanase, this form also produces a protein of 195 kDa. The transport of the intracellular precursor to the MVM and further proteolytic processing is not blocked by the inhibitor. However, all MVM forms of lactase-phlorizin hydrolase show an increase of approx. 5 kDa. Treatment of these three MVM forms with endo-H indicates the increased presence of high mannose oligosaccharides in comparison with non-castanospermine-treated forms. The susceptibility to endo-H of the 130 kDa MVM band synthesized in the absence of castanospermine implies the presence of high-mannose N-linked oligosaccharides in the mature form of lactase-phlorizin hydrolase. Incubation of these MVM forms with N-glycanase further reduces their electrophoretic mobility, indicating the presence of complex N-linked oligosaccharides in the MVM forms, in contrast with the intracellular precursor. Altered glycosylation reduces but does not abolish intracellular transport of lactase-phlorizin hydrolase to the MVM.

Project description:We have previously identified and purified a novel beta-glucosidase, designated PNGH (pyridoxine-5'-beta-D-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5'-beta-D-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase-phlorizin hydrolase), the beta-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568-784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

Dataset Information

Characterization of expression in mice of a transgene containing 3.3 kb of the human lactase-phlorizin hydrolase (LPH) 5' flanking sequence.

Publications

Characterization of expression in mice of a transgene containing 3.3 kb of the human lactase-phlorizin hydrolase (LPH) 5' flanking sequence.

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