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Original articles

Lactose and Lactase—Who Is Lactose Intolerant and Why?

Montgomery, Robert K*; Krasinski, Stephen D; Hirschhorn, Joel N; Grand, Richard J§

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Journal of Pediatric Gastroenterology and Nutrition: December 2007 - Volume 45 - Issue - p S131-S137
doi: 10.1097/MPG.0b013e31812e68f6
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Abstract

INTRODUCTION

Lactase-phlorizin hydrolase (LPH, EC 3.2.1.108-EC3.2.1.62) is the enzyme responsible for the digestion of milk lactose into the absorbable monosaccharides, glucose, and galactose. It is an excellent model for the study of intestine-specific gene expression and intestinal differentiation because in adult mammals, LPH is expressed only in the small intestine and is confined to absorptive enterocytes on the villi and not the proliferating cells of the crypts. LPH demonstrates positional regulation as exhibited by a tightly controlled pattern of expression along the proximal to distal axis of the intestine in both animals and humans, with high levels in the mid-intestine and reduced levels in the duodenum and distal ileum (1–4). This review focuses on 6 major topics: lactose as a substrate, the characteristics of LPH protein, the synthesis and processing of LPH, LPH development and lactase phenotypes, the molecular expression of the lactase gene, and the molecular regulation of lactase gene expression.

LACTOSE

Lactose (comprising glucose and galactose in a β1–4 configuration) is a major source of carbohydrate for all mammalian neonates. Lactose is synthesized only in the mammary gland and is present both as free and lactosyl adducts in milk glycolipids. There has been evolutionary conservation of lactose in virtually all mammalian milks, but with varying concentrations according to species. Of the milks studied, human milk contains the highest content of lactose (7%). Milk in the Pinnipediae does not contain lactose, but it is high in lipid content (5).

CHARACTERISTICS OF LPH

In humans LPH is a 160-kDa transmembrane glycoprotein with a C-terminus (26 amino acids) that is intracellular and an N-terminus found on the luminal surface of the lipid bilayer of the microvillus membrane (MVM). The transmembrane-spanning region is a short sequence of hydrophobic amino acids (19 residues). LPH is a multifunctional enzyme with several substrate specificities, and has the capacity to hydrolyze, in addition to lactose, lactosylceramide, cellobiose, cellotriose, and phlorizin. LPH is not inducible, and its enzymatic activity is not reduced by a lactose-free diet. LPH expression is developmentally regulated (2).

The primary sequence of LPH protein in the human, rabbit, and rat reveals a 4-fold internal homology (designated domains I–IV), most likely due to 2 independent duplication events during evolution (Fig. 1) (6). Domains II–IV of all of the LPH cDNAs show homologies of 38% to 55%. Of the 4 domains, domain I has diverged the most; only a stretch of about 90 residues shows high homology with domains II–IV; however, high homology between the domains I of the species already mentioned suggests an analogous function. Indeed, it has been shown that domains I and II together serve to regulate protein folding in the endoplasmic reticulum; they are not glycosylated and they lack enzymatic activity. Domains III and IV are heavily glycosylated, are inserted into the microvillus membrane, and contain the 2 active sites in which each nucleophile is a glutamate residue. The amino acid sequence around the active site glutamic acid (E) in domain III is PIYITENG, whereas that of domain IV is PIYVTENG. Interestingly, the closest match for the active site ENG sequence is a family of bacterial glycosidases, not a mammalian or eukaryote enzyme (7,8).

FIG. 1
FIG. 1:
Primary structure of lactase-phlorizin hydrolase is evolutionarily conserved. High sequence homology between domains suggests that the gene arose through 2 duplications of an ancestral gene.

SYNTHESIS AND PROCESSING OF LPH

Nascent LPH (∼195 kDa) is synthesized in the endoplasmic reticulum and undergoes cotranslational, dolichol-dependent, high-mannose glycosylation yielding a molecular mass of ∼215 kDa. Complex glycosylation of domains III and IV occurs in the Golgi yielding a structure of ∼220 kDa. Subsequently, there is cleavage of a small N-terminal propiece, and of domains I and II that serve as a chaperone for the remaining molecule that is inserted into the MVM. A final extracellular cleavage event (perhaps by pancreatic proteases) removes a small residue from the N-terminus producing the final, mature LPH enzyme (9) (Fig. 2). Additional characteristics of this process are dimerization of the monomeric pro-LPH in the endoplasmic reticulum, and intracellular transport of LPH to the MVM in specific post-Golgi vesicles not associated with lipid rafts. The role of the extracellular cleavage product is unknown (10,11).

FIG. 2
FIG. 2:
Synthesis and processing of lactase-phlorizin hydrolase.

Of relevance to the apical localization of LPH in the MVM is the finding that LPH mRNA also is localized to the apical pole of enterocytes in rats, mice, and humans. The exact role of targeted mRNAs in intestinal epithelial cells remains to be elucidated; a cell culture model of this process has been described (12–15).

LPH DEVELOPMENT AND LACTASE PHENOTYPES

Lactase enzyme activity exhibits distinct patterns of expression in both nonhuman mammals and humans that are regulated mainly at the level of LPH transcription (Fig. 3). In animals LPH mRNA, protein, and activity are low until just before birth, when they rise dramatically. Levels then remain elevated until weaning, when they decline to <10% of the neonatal values; reduced activity is maintained throughout adult life. The decline is regulated by several mechanisms: a reduction in the rate of LPH transcription, a consequent decrease in LPH-protein synthesis and enzyme activity along the proximal to distal gradient, and virtual extinction of expression in the duodenum and distal ileum. The mid-jejunum retains measurable LPH mRNA, LPH protein synthesis, and lactase activity (16–20).

FIG. 3
FIG. 3:
Developmental patterns (transcriptionally regulated) of lactase-phlorizin hydrolase expression in humans and other mammals. Schematic diagram of developmental changes in lactase-specific activity changes from fetal life through birth and weaning.

The developmental pattern of lactase expression in the human fetus is different from that in the well-studied rodent models. Before 24 weeks of gestation, intestinal lactase activity is low. It then begins to increase, and, during the third trimester, lactase activity increases markedly until levels in term neonates are at or above those of infants of ages 2 to 11 months. As in other mammals, lactase in humans exhibits a characteristic proximal-to-distal pattern of expression, with activity greater in the mid-jejunum and decreasing activity both proximally and distally, resulting in minimal activity in the duodenum and terminal ileum (21,22).

In most human populations lactase activity decreases during mid-childhood (at an average of about 5 years of age, but with a broad range), resulting in low levels from that age onward (lactase nonpersistence) (23–25). This pattern is similar to that seen in all other mammals examined, but the time course is markedly extended in humans (26). In contrast, a minority of the human population, especially people of northern European extraction and a few other population groups around the world, retain high levels of activity throughout adult life (lactase persistence) (23–25). Persistence of elevated lactase activity has been clearly shown to be a relatively recent human evolutionary event, arising within the last 8000 to 10,000 years, coincident with the development of dairying (27,28). Of those humans with lactase nonpersistence, a small number has been shown to have an abnormality in the intracellular processing of newly synthesized LPH protein, indicating posttranscriptional control of this pathway (29–31). However, in most other humans, as in all of the mammals studied, the primary mechanism of both the nonpersistence and persistence phenotypes is regulation of gene transcription (29,32,33). Examples of lactase phenotypes are shown in Table 1.

TABLE 1
TABLE 1:
Lactase phenotypes in selected human populations

MOLECULAR GENETICS AND REGULATION

Considerable effort has been devoted to the elucidation of the molecular mechanisms involved in the transcriptional regulation responsible for lactase nonpersistence in animals and the 2 human phenotypes, although to date only hypotheses are possible. Nevertheless, any hypothesis for the mechanism of lactase persistence must account for both the presence of high levels of lactase-enzyme activity in all genetic groups during infancy and early childhood, and the low levels or near absence in the majority of the world's adult population. In 1995 Wang et al (34) suggested that the data available pointed to cis-regulation of both the human persistence and nonpersistence phenotypes. That hypothesis also predicted that trans-acting factors would be involved because cis-acting elements could not by themselves account for both high and low levels of expression in the same individual at different ages.

We subsequently derived a model that takes into account both of these needs (Fig. 4). Accordingly, lactase gene expression depends on a positive regulator that directs developmental increases in transcription and maintains expression in those populations that are lactase persistent. In contrast, in populations that are lactase nonpersistent, a powerful repressor appears at around age 5 years that downregulates lactase expression as a consequence of binding to an appropriate cis-element in the 5′-flanking region of the lactase gene. This repressor remains active for life. Populations that are lactase persistent fail to bind this repressor because of a single nucleotide polymorphism (SNP) at the critical site. Although the actual number of cis-acting elements, repressors, or polymorphisms may differ from this simple model, it serves as the basis for further study of the genetic regulation of lactase-gene expression. Indeed, presently research is taking 2 pathways, examining molecular genetics at the population level and molecular regulation in intestinal cell lines and transgenic animals.

FIG. 4
FIG. 4:
Hypothesis for molecular regulation of lactase-phlorizin hydrolase expression in humans displaying lactase persistence and nonpersistence.

The human lactase gene, located on chromosome 2q21, comprises 17 exons and covers approximately 49 kb, giving rise to a messenger RNA of slightly more than 6 kb. From initiation codon to stop codon, human lactase mRNA encodes 1927 amino acids forming the complete translation product. Details of the protein structure and function are described above. Initial analyses of the gene identified several SNPs within both the coding region and the 5′-flanking region (6). None was considered to have functional significance. Subsequent analysis has led to the identification of additional SNPs and several other features unique to the human gene that may be of relevance to the mechanisms of LPH persistence or nonpersistence.

The first 100 bp of the proximal promoters of the mammals analyzed to date (rat, mouse, pig, and human) are virtually identical and appear to be similarly regulated (Fig. 5). Binding sites for Cdx-2, HNF-1, and GATA factors are in similar positions relative to the transcriptional start site (35–39). In addition, a number of other transcription factors have been found to interact with the lactase 5′-flanking sequence, albeit some in more distal loci—including HOXC11, HNF-3, C/EBP, and FREAC-2 and -3 (40–43)—and to activate promoter-reporter constructs in transfection assays. In contrast, Pdx-1 appears to reduce expression in vitro (44). Because HNF-1α and GATA factors are found together only in the intestine and colon, we have extensively studied the interactions between these 2 transcription factors in the activation of lactase-gene function. In mouse intestine the expression patterns of HNF-1α and GATA-4 closely correlate with lactase expression (37). In transfection assays GATA-4 activates lactase-gene transcription both independently and cooperatively with HNF-1α. HNF-1α binding to DNA, but not GATA-4 binding, is necessary for functional synergy, as is GATA-4 and HNF-1α physical interaction. Physical association is mediated by the C-terminal zinc finger and basic region of GATA-4, and the homeodomain of HNF-1α; and HNF-1α activation domains, but not those of GATA-4, are required for functional synergy (37). GATA-5 and HNF-1α physically associate both in vitro and in vivo, and this interaction is necessary for cooperative activation of the lactase promoter (38).

FIG. 5
FIG. 5:
Comparison of the genomic sequence of proximal promoters of human, pig, rat, and mouse lactase. Transcriptional and translational start sites are indicated. Binding sites for the transcription factors Cdx2, GATA, and HNF1 are indicated.

Studies in transgenic mice have indicated that approximately 1 kb of the 5′-flanking sequence in the pig, and 2 kb of the 5′-flanking sequence in the rat, are sufficient to direct appropriate tissue, cell, and villus expression, as well as the developmental decline at weaning (45–47). Such studies are being conducted in our laboratory with 3.3 kb of the human promoter. If human-lactase nonpersistence is regulated as in other mammals, then regulatory elements are likely to be in the equivalent 5′-flanking region.

In contrast to the other mammals analyzed, the first 1000 bp of the 5′-flanking region of the human lactase gene contains 2 Alu sequences of approximately 300 bp each, whereas the more distal region contains additional repetitive DNA sequences, making it difficult to directly compare this more distal regulatory region to those of other mammals. Whether these inserted repetitive DNA segments affect lactase expression is unknown. Furthermore, exon 17 of MCM6, a cell cycle regulatory gene, ends 3.5 kb from the start site of the human lactase gene (48). The transcriptional start site of the MCM6 gene lies approximately 39 kb 5′ of the lactase transcriptional start site. The 2 genes are close together, but the available evidence indicates that their regulation is independent.

In 2002 Enattah and colleagues (49) identified a C>T SNP at −13.9 kb upstream of the lactase transcriptional start site that correlated strongly with lactase persistence, and its converse with lactase nonpersistence. All 99 individuals with low lactase activity were homozygous for a C at this SNP, whereas all 137 individuals with lactase persistence carried either a C/T or T/T. A similar but not perfect association was found with a G>A SNP at −22 kb. No other variants were as tightly associated with lactase persistence as were these 2 SNPs. Interestingly, other haplotypes previously had been associated with lactase persistence and nonpersistence (50). In a second publication by the same group (51), these data were confirmed and extended by demonstrating that all of the chromosomes carrying a T at −13.9 kb also had an A at −22 kb. Subsequently, these authors used genetic testing to screen 329 children and adolescents from varying genetic populations (Finns, other whites, Africans) for expression of the C and T alleles and intestinal lactase activity. Low lactase activity was tightly correlated with a C/C −13.9 genotype (52). The findings were confirmed in an independent study among other population groups whose lactase status was known (53). Although all of the authors are careful not to draw a causal relationship between the C/C genotype and lactase-gene expression, data from African groups who are pastoralists and milk consumers indicate that the C/C allele can be present in people who are known to be lactase persistent (Table 2). Indeed, these groups also lacked significant expression of the T allele (54). This suggests that either lactase persistence has arisen multiple times or that the T allele is not the causal variant. To test the functional implications of these polymorphisms, 2 studies used the T and C alleles in transient transfection assays, attempting to find function for the T allele and no function for the C allele. Although 1 study did demonstrate enhancer-like stimulation of a promoter-reporter construct containing the T allele, the C allele also produced activation; inactivity of the construct containing the C allele was not found (55). Similar findings were obtained in the second study, although the activation responses were far less robust (56). Thus, it remains unclear whether the SNPs directly affect expression of lactase or are simply markers for lactase persistence or nonpersistence.

TABLE 2
TABLE 2:
Comparison of selected lactase phenotype and genotype data in populations of traditional milk drinkers

No matter what the causal significance of these SNPs turns out to be, the 2 polymorphic sites reside on a large background haplotype of approximately 1.2 Mb (27,53) (Fig. 6). This long, common haplotype appears to have arisen rapidly due to recent selection occurring within the past 5000 to 10,000 years, and would be consistent with an advantage to lactase persistence in the setting of dairy farming. Indeed, the signals of selection observed are among the strongest yet seen for any gene in the genome (27). In support of this concept, it has recently been shown that cow's milk–protein polymorphisms track closely with the patterns of lactose tolerance known to exist throughout northern Europe and peoples of northern European ancestry (57).

FIG. 6
FIG. 6:
Map of the 1.2-Mb region surrounding the lactase gene (LCT) locus on human chromosome 2.

CONCLUSIONS

Several important concepts emerge from the studies reviewed. First, the lactase proximal promoter (ie, the first 100 bp upstream of the lactase transcriptional start site) is necessary but not sufficient for normal lactase expression, and more distal regions of the gene are required, but these need further characterization. Indeed, the role of the distal promoter in humans is under study in several laboratories. Complex transcription factor interactions are key to the activation of lactase transcription, but the causal factors and the cis-elements to which they bind remain to be elucidated. A clear population genetic association of lactase persistence with the −13.9T and −22A SNPs in the distal 5′-flanking region of the lactase gene has been confirmed in subjects of northern European heritage. However, data on lactase persistence in certain African groups who are homozygotes for the −13.9C allele are not consistent with this variant being the only cause of lactase persistence (54). Accordingly, continued detailed genotype/phenotype analysis will be necessary to reveal the correct molecular regulation of both lactase persistence and lactase nonpersistence.

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Keywords:

Lactase-phlorizin hydrolase; Single nucleotide polymorphism; Phenotype

© 2007 Lippincott Williams & Wilkins, Inc.