Dataset Information

Structure of the chromosomal gene and cDNAs coding for lactase-phlorizin hydrolase in humans with adult-type hypolactasia or persistence of lactase.

ABSTRACT: Lactase-phlorizin hydrolase (LPH) splits lactose in the small intestine. LPH activity is high in the suckling; in many human populations the activity declines in adults, leading to adult-type hypolactasia, whereas in other populations the high LPH activity persists in adults. In the present work, we compared LPH sequences at the gene and cDNA level among adult subjects with high and low LPH activity. The complete intron-exon organization, including the sequences of all 17 exons and of the borders of all introns (as well as about 1,000 bp of 5' flanking region), was established for the cloned chromosomal LPH gene of a subject with persistence of lactase. Using PCR, we directly sequenced the exons of a hypolactasic subject. Except for silent mutations and the unknown linkage phase at two heterozygous positions, both coding sequences were identical. We further examined the LPH mRNA of a hypolactasic subject by S1 mapping and by sequencing a set of overlapping PCR products produced from cDNA templates. Except for allelic differences, the LPH sequence of the hypolactasic subject was identical to that of the LPH cDNAs of three subjects with persistence of lactase (one cDNA isolated previously by cloning and two characterized in the present work by PCR). No allele was peculiar to the hypolactasic subject. We conclude that humans with high or low levels of lactase can code for identical LPH enzymes.

SUBMITTER: Boll W

PROVIDER: S-EPMC1683064 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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Structure of the chromosomal gene and cDNAs coding for lactase-phlorizin hydrolase in humans with adult-type hypolactasia or persistence of lactase.

Boll W W Wagner P P Mantei N N

American journal of human genetics 19910501 5

Lactase-phlorizin hydrolase (LPH) splits lactose in the small intestine. LPH activity is high in the suckling; in many human populations the activity declines in adults, leading to adult-type hypolactasia, whereas in other populations the high LPH activity persists in adults. In the present work, we compared LPH sequences at the gene and cDNA level among adult subjects with high and low LPH activity. The complete intron-exon organization, including the sequences of all 17 exons and of the border ...[more]

PMID: 1902057

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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.

Dataset Information

Structure of the chromosomal gene and cDNAs coding for lactase-phlorizin hydrolase in humans with adult-type hypolactasia or persistence of lactase.

Publications

Structure of the chromosomal gene and cDNAs coding for lactase-phlorizin hydrolase in humans with adult-type hypolactasia or persistence of lactase.

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