Biomedical subjects

J N Freund

Publications and source records attributed to J N Freund.

At least 19 recordsLinked to original sources

PubMed2001

Functional interference between thyroid hormone receptor alpha (TRalpha) and natural truncated TRDeltaalpha isoforms in the control of intestine development.

Thyroid hormone is known to participate in the control of intestine maturation at weaning. Its action is mediated by the thyroid hormone nuclear receptors, encoded by the TRalpha and TRbeta genes. Since previous studies have shown that TRbeta plays a minor role in the gut, we focused here our analysis on the TRalpha gene. The TRalpha locus generates the TRalpha1 receptor together with the splicing variant TRalpha2 and the truncated products TRDeltaalpha1 and TRDeltaalpha2, which all lack an intact ligand binding domain. The TRDeltaalpha isoforms are transcribed from an internal promoter located in intron 7, and their distribution is restricted to a few tissues including those of the intestine. In order to define the functions of the different isoforms encoded by the TRalpha locus in the intestinal mucosa, we produced mice either lacking all known TRalpha products or harboring a mutation which inactivates the intronic promoter. We performed a detailed analysis of the intestinal phenotypes in these mice and compared it to that of the previously described TRalpha(-/-) mice, in which TRalpha isoforms are abolished but the TRDeltaalpha isoforms remain. This comparative analysis leads us to the following conclusions: (i) the TRalpha1 receptor mediates the T3-dependent functions in the intestine at weaning time and (ii) the TRDeltaalpha products negatively control the responsiveness of the epithelial cells to T3. Moreover, we show that TRDeltaalpha proteins can interfere with the transcription of the intestine-specific homeobox genes cdx1 and cdx2 and that their activity is regulated by TRalpha1. Altogether these data demonstrate that cooperation of TRalpha and TRDeltaalpha products is essential to ensure the normal postnatal development of the intestine and that mutations in the TRalpha locus can generate different phenotypes caused by the disruption of the equilibrium between these products.

Animals↗

PubMed2001

Differentially expressed endoderm and mesenchyme genes along the fetal rat intestine.

Developmental studies have shown that morphological and functional regionalization occurring along the mammalian intestine is defined at early fetal stages and that some aspects of this patterning are dependent on epithelial-mesenchymal cell interactions. The molecular basis of these processes are largely unknown. In this study, a differential display approach was used to identify genes differentially expressed along the longitudinal axis in the intestinal endoderm and mesenchyme moieties of 14-day-old rat fetuses at a stage prior to morpho-functional differentiation of the gut. Fifty-eight genes were identified, 36 being identical or similar to known genes and 13 corresponding to ESTs or genome sequences with unknown function. Nine cDNAs could not be assigned to any previously described nucleotide sequence. The selected genes are involved in several aspects of cell physiology, including metabolic pathways, cytoskeleton organization, signal transduction, protein biosynthesis, and regulation of gene transcription.

Animals↗

PubMed2000

Wnt/(beta)-catenin signaling regulates the expression of the homeobox gene Cdx1 in embryonic intestine.

During mammalian development, the Cdx1 homeobox gene exhibits an early period of expression when the embryonic body axis is established, and a later period where expression is restricted to the embryonic intestinal endoderm. Cdx1 expression is maintained throughout adulthood in the proliferative cell compartment of the continuously renewed intestinal epithelium, the crypts. In this study, we provide evidence in vitro and in vivo that Cdx1 is a direct transcriptional target of the Wnt/(beta)-catenin signaling pathway. Upon Wnt stimulation, expression of Cdx1 can be induced in mouse embryonic stem (ES) cells as well as in undifferentiated rat embryonic endoderm. Tcf4-deficient mouse embryos show abrogation of Cdx1 protein in the small intestinal epithelium, making Tcf4 the likely candidate to transduce Wnt signal in this part of gut. The promoter region of the Cdx1 gene contains several Tcf-binding motifs, and these bind Tcf/Lef1/(beta)-catenin complexes and mediate (beta)-catenin-dependent transactivation. The transcriptional regulation of the homeobox gene Cdx1 in the intestinal epithelium by Wnt/(beta)-catenin signaling underlines the importance of this signaling pathway in mammalian endoderm development.

3T3 Cells↗

PubMed1999

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras.

Downregulation of the colon tumour-suppressor homeobox gene Cdx-2 by oncogenic ras Constitutive activation of the ras proto-oncogene is a frequent and early event in colon cancers, but the downstream nuclear targets are not fully understood. The Cdx-1 and Cdx-2 homeobox genes play crucial roles in intestinal cell proliferation and differentiation. In addition, Cdx-2 is a colonic tumour-suppressor gene, whereas Cdx-1 has oncogenic potential. Here, we show that constitutive activation of ras alters Cdx-1 and Cdx-2 expression in human colonic Caco-2 and HT-29 cells that harbour a normal ras proto-oncogene. Oncogenic ras downregulates Cdx-2 through activation of the PKC pathway and a decline in activity of the Cdx-2 promoter AP-1 site. This decline results from a PKC-dependent decrease in the relative expression of c-Jun, an activator of Cdx-2 transcription, compared to c-Fos, an inhibitor of Cdx-2. Unlike Cdx-2, Cdx-1 is upregulated by oncogenic ras and this effect is mediated by activation of the MEK1 pathway. These results indicate that oncogenic ras activation has opposite effects on Cdx-1 and Cdx-2 expression through distinct signalling pathways and they provide the first evidence for a functional link between ras activation and the downregulation of the Cdx-2 tumour-suppressor gene in colon cancer cells.

Avian Proteins↗

PubMed1999

Involvement of T3Ralpha- and beta-receptor subtypes in mediation of T3 functions during postnatal murine intestinal development.

BACKGROUND & AIMS: Thyroid hormones are implicated in intestinal development. Their effects are mediated by nuclear receptors, which are transcriptional regulators activated upon binding of triiodothyronine. The aim of this study was to define the involvement of the receptor subtypes during intestinal development. METHODS: We used strains of knockout mice lacking T3Ralpha, T3Rbeta, or both receptors, encoded by T3Ralpha and T3Rbeta genes. RESULTS: Morphological features and expression of digestive enzymes and of two intestinal regulators, Cdx-1 and Cdx-2, were compared in wild-type and T3Ralpha, T3Rbeta, and T3Ralphabeta knockout animals. T3Ralpha-/- mice had abnormal intestinal morphology, assessed by a decrease in the number of epithelial cells along the crypt-villus axis and a decrease in proliferating crypt cells. Expression of Cdx-1 and Cdx-2, and of the digestive enzymes, was down-regulated. These parameters can be partially reversed by T3 injection. A similar (jejunum) or more severe (ileum) phenotype was found in T3Ralphabeta double mutants. In contrast, no changes occurred in T3Rbeta mice. CONCLUSIONS: These data describe for the first time a direct effect of TH through the T3Ralpha-receptor subtypes on postnatal intestinal mucosa maturation. They also suggest that T3Rbeta receptors are dispensable but can partially substitute for T3Ralpha.

Animals↗

PubMed1999

Production of low-lactose milk by ectopic expression of intestinal lactase in the mouse mammary gland.

We have investigated, in mice, an in vivo method for producing low-lactose milk, based on the creation of transgenic animals carrying a hybrid gene in which the intestinal lactase-phlorizin hydrolase cDNA was placed under the control of the mammary-specific alpha-lactalbumin promoter. Transgenic females expressed lactase protein and activity during lactation at the apical side of mammary alveolar cells. Active lactase was also secreted into milk, anchored in the outer membrane of fat globules. Lactase synthesis in the mammary gland caused a significant decrease in milk lactose (50-85%) without obvious changes in fat and protein concentrations. Sucklings nourished with low-lactose milk developed normally. Hence, these data validate the use of transgenic animals expressing lactase in the mammary gland to produce low-lactose milk in vivo, and they demonstrate that the secretion of an intestinal digestive enzyme into milk can selectively modify its composition.

Animals↗

PubMed1999

Promotion of intestinal carcinogenesis by dietary methionine.

The metabolism of the polyamines spermidine and spermine is known to be enhanced in rapidly proliferating cells. Methionine is a precursor of the aminopropyl moieties of these amines. Therefore, it was of interest to study the effects of a methionine supplemented diet on polyamine metabolism and preneoplastic changes occurring in the intestinal tract of rats treated with the chemical carcinogen azoxymethane (AOM). Adult Wistar rats received 15 mg AOM/kg body wt (i.p.) once each week for 2 weeks. Thereafter, the rats were randomly divided into two groups and received controlled isoenergetic diets containing the same amount of folate, choline and vitamin B12 during 12 weeks: one group was kept on a standard diet; the other was fed the same diet, except that 1% L-methionine was added at the expense of carbohydrates. After 12 weeks, the administration of the methionine-supplemented diet stimulated the turnover rate of ileal epithelial cells, indicating enhanced crypt cell proliferation. Furthermore, in this group, a 2-fold increase in the number of aberrant hyperproliferative crypts and the appearance of tumors was observed in the colon. These effects were accompanied by the increased formation of spermidine and spermine due to the enhancement of S-adenosylmethionine decarboxylase activity and by the upregulation of Cdx-1, a homeobox gene with oncogenic potentials. The experimental data do not support the view of a chemopreventive effect of dietary methionine supplementation on intestinal carcinogenesis in rats, even at an early phase of preneoplastic development, but rather suggest that methionine promotes intestinal carcinogenesis.

Animals↗

PubMed1998

Intestinal epithelial-mesenchymal cell interactions.

Intestinal morphogenesis, as well as maintenance of the stem cell population and of the steady state between cell proliferation and differentiation, results from controlled cell interactions. There is growing evidence that the mesenchymal cells control epithelial cell behavior via their own expression and induction in the epithelial cells of key regulatory genes. This heterologous cross talk involves basement membrane molecules and paracrine factors. New in vitro/in vivo cellular models allowed us to analyze various mesenchymal cell phenotypes and to show that they exhibit different inductive properties on epithelial cells and that their proliferation and metabolic properties are differentially modulated by cytokines. Finally the epithelial-mesenchymal unit is controlled by hormonal and exogenous factors.

Animals↗

PubMed1998

Cellular and molecular partners involved in gut morphogenesis and differentiation.

The intestinal mucosa represents an interesting model to study the cellular and molecular basis of epithelial-mesenchymal cross-talk participating in the development and maintenance of the digestive function. This cross-talk involves extracellular matrix molecules, cell-cell and cell-matrix adhesion molecules as well as paracrine factors and their receptors. The cellular and molecular unit is additionally regulated by hormonal, immune and neural inputs. Such integrated cell interactions are involved in pattern formation, in proximodistal regionalization, in maintenance of a gradient of epithelial proliferation and differentiation, and in epithelial cell migration. We focus predominantly on two aspects of these integrated interactions in this paper: (i) the role of basement membrane molecules, namely laminins, in the developmental and spatial epithelial behaviour; and (ii) the importance of the mesenchymal cell compartment in these processes.

Animals↗

PubMed1998

The Cdx-1 and Cdx-2 homeobox genes in the intestine.

The past years have witnessed an increasing number of reports relative to homeobox genes in endoderm-derived tissues. In this review, we focus on the caudal-related Cdx-1 and Cdx-2 homeobox genes to give an overview of the in vivo, in vitro, and ex vivo approaches that emphasize their primary role in intestinal development and in the control of intestinal cell proliferation, differentiation, and identity. The participation of these genes in colon tumorigenesis and their identification as important actors of the oncogenic process are also discussed.

Animals↗

PubMed1998

Subepithelial fibroblast cell lines from different levels of gut axis display regional characteristics.

The intestine is characterized by morphofunctional differences along the proximodistal axis. The aim of this study was to derive mesenchymal cell lines representative of the gut axis. We isolated and cloned rat intestinal subepithelial myofibroblasts raised from 8-day proximal jejunum, distal ileum, and proximal colon lamina propria. Two clonal cell lines from each level of the gut were characterized. They 1) express the specific markers vimentin, smooth muscle alpha-actin, and smooth muscle myosin heavy chain, revealed by immunofluorescence microscopy and 2) distinctly support endodermal cell growth in a coculture model, depending on their regional origin, and 3) the clones raised from the various proximodistal regions maintain the same pattern of morphogenetic and growth and/or differentiation factor gene expression as in vivo: hepatocyte growth and/or scatter factor and transforming growth factor-beta 1 mRNAs analyzed by RT-PCR were more abundant, in the colon and ileal clones and mucosal connective tissue, respectively. In addition, epimorphin mRNA studied by Northern blot was also the highest in one ileal clone, in which it was selectively upregulated by all-trans retinoic acid (RA) treatment. Epimorphin expression in isolated 8-day intestinal lamina propria was higher in the distal small intestine and proximal colon than in the proximal small intestine. In conclusion, we isolated and characterized homogeneous cell subtypes that can now be used to approach the molecular regulation of the epithelium-mesenchyme-dependent regional specificity along the gut.

Animals↗

PubMed1998

Lactase is unchanged in suckling mice fed with lactose-free milk.

At weaning, mammals switch from milk to complex adult food, and change from a lactose-rich to a lactose-free diet. At the same time, the small intestine matures resulting in changes in lactase expression and the onset of sucrase. The aim of this study was to analyze the effect of premature and specific depletion of lactose on maturation of the small intestine and on lactase expression in suckling mice. For this purpose, from postnatal days 10 to 16, suckling mice were fed by transgenic alpha-lactalbumin-deficient females that produce lactose-free milk. Pups fed with lactose-free milk had a lower body weight than controls fed by wildtype females. They also displayed hypotrophy of intestinal muscle layers, but no obvious alterations in the morphology of the intestinal epithelium. The level of lactase activity as well as the longitudinal distribution of corresponding mRNA were unchanged compared to suckling animals nourished with normal lactose-containing milk. Finally, there was no premature onset of sucrase expression. We conclude that feeding suckling mice for six days with lactose-free milk does not provoke any premature maturation of the small intestine. Thus, decreasing lactose intake is not a major cause for the modifications of lactase expression which occur at weaning.

Animals↗

PubMed1997

Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation.

To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell-cell and cell-substratum interactions and in transduction process: indeed, E-cadherin, integrin-beta4 subunit, laminin-gamma2 chain, hemidesmosomal protein, APC, and alpha-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures. Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-alpha1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-alpha1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix-mediated intestinal cell differentiation.

Adenocarcinoma↗

PubMed1997

Functional diversity and interactions between the repeat domains of rat intestinal lactase.

Lactase-phlorizin hydrolase (LPH), a major digestive enzyme in the small intestine of newborns, is synthesized as a high-molecular-mass precursor comprising four tandemly repeated domains. Proteolytic cleavage of the precursor liberates the pro segment (LPHalpha) corresponding to domains I and II and devoid of known enzymic function. The mature enzyme (LPHbeta) comprises domains III and IV and is anchored in the brush border membrane via a C-terminal hydrophobic segment. To analyse the roles of the different domains of LPHalpha and LPHbeta, and the interactions between them, we have engineered a series of modified derivatives of the rat LPH precursor. These were expressed in cultured cells under the control of a cytomegalovirus promoter. The results show that recombinant LPHbeta harbouring both domains III and IV produces lactase activity. Neither domain III nor IV is alone sufficient to generate active enzyme, although the corresponding proteins are transport-competent. Tandem duplication of domains III or IV did not restore lactase activity, demonstrating the separate roles of both domains within LPHbeta. Further, the development of lactase activity did not require LPHalpha; however, LPHalpha potentiated the production of active LPHbeta but the individual LPHalpha subdomains I and II were unable to do so. Lactase activity and targeting required the C-terminal transmembrane anchor of LPH; this requirement was terminal transmembrane anchor or LPH; this requirement was not satisfied by the signal/anchor region of another digestive enzyme: sucrase-isomaltase. On the basis of this study we suggest that multiple levels of intramolecular interactions occur within the LPH precursor to produce the mature enzyme, and that the repeat domains of the precursor have distinct and specific functions in protein processing, substrate recognition and catalysis. We propose a functional model of LPHbeta in which substrate is channelled from an entry point located within domain II to the active site located in domain IV.

Animals↗

PubMed1997

Identification of homologues of the mammalian intestinal lactase gene in non-mammals (birds and molluscs).

Mammalian intestinal lactase hydrolyses a variety of beta-glycosides and is processed from a precursor comprising four tandem domains exhibiting sequence similarity, suggestive of multiple duplication events in the evolutionary past. The aim of the present study was to investigate whether genes homologous to the lactase gene exist in animals other than mammals. A reverse transcriptase-PCR strategy using a degenerate mixture of oligonucleotides was developed to search for the presence of transcripts similar in sequence to the mammalian lactase mRNA in the digestive tracts of a bird (the chicken) and an invertebrate (the mussel). Partial cDNAs corresponding to the 3' end of intestinal mRNAs were identified in both animals. In chicken, two cDNAs were isolated, corresponding to 6.5 kb transcripts that used two distinct polyadenylation sites. In mussels, three cDNAs were obtained and classified into two categories. One class of cDNA hybridized to a major mRNA of 3.5 kb and to minor species of 4.5 kb and 6 kb. The second class of cDNA hybridized to a 13 kb transcript, which was approximately twice as large as the mammalian lactase mRNA. Peptide sequences predicted from the chicken and mussel cDNAs confirmed that the proteins are related to mammalian lactase. They also suggested that the chicken protein and one mussel protein are integral molecules anchored in the cell membrane by a C-terminal transmembrane anchor, like lactase. These data provide evidence that proteins phylogenetically related to the mammalian-specific lactase are widespread in the animal kingdom, and that these proteins are expressed in the intestinal tract.

Amino Acid Sequence↗

PubMed1997

Mesenchyme-mediated effects of retinoic acid during rat intestinal development.

In previous experiments we showed that intestinal development was dependent upon epithelial-mesenchymal cell interactions. The aim of this study was to investigate the possible role of retinoic acid (RA), a morphogenetic and differentiating agent, on the gut epithelial-mesenchymal unit. For this purpose we first analyzed the effects of a physiological dose of RA on 14-day fetal rat intestine using short-term organ culture experiments, or long-term grafts under the skin of nude mice. In these conditions, RA accelerated villus outgrowth and epithelial cell differentiation as assessed by the onset of lactase expression, and it also stimulated muscle and crypt formation. In order to analyze potential effects of RA mediated by mesenchymal cells, we isolated and characterized gut mucosa mesenchyme-derived cell cultures (mesenchyme-derived intestinal cell lines, MIC). These cells were shown to express mRNAs for retinoid binding proteins similar to those expressed in situ in the intestinal mesenchyme. MIC cells co-cultured with 14-day intestinal endoderms promoted endodermal cell adhesion and growth, and the addition of exogeneous RA enhanced epithelial cell polarization and differentiation assessed by cytokeratin and lactase immunostaining. Such a differentiating effect of RA was not observed on endodermal cells when cultured without a mesenchymal feeder layer or maintained in conditioned medium from RA-treated MIC cells. In the co-cultures, immunostaining of laminin and collagen IV with polyclonal antibodies, as well as alpha1 and beta1 laminin chains mRNAs (analyzed by RT-PCR) increased concurrently with the RA-enhanced differentiation of epithelial cells. It is worth noting that this stimulation by RA was also obvious on the mesenchymal cells cultured alone. These results show that RA plays a role in intestinal morphogenesis and differentiation. In addition, they indicate that RA acts on the mesenchymal cell phenotype and suggest that RA may modify the mesenchymal-epithelial cell interactions during intestinal development.

Animals↗

PubMed1997

Changing intestinal connective tissue interactions alters homeobox gene expression in epithelial cells.

In segmented organs, homeobox genes are involved in axial patterning and cell identity. Much less is known about their role in non-segmented endoderm derivatives such as the digestive epithelium. Using a xenograft model of fetal intestinal anlagen implanted under the skin of nude mice, we have investigated whether the expression of five homeobox genes (HoxA-4, HoxA-9, HoxC-8, Cdx-1 and Cdx-2) is modified when intestinal epithelium undergoes normal development or displays heterodifferentiation in association with heterotopic mesenchyme. In homotypic associations of fetal endoderm and mesenchyme that recapitulate normal development, the overall pattern of homeobox gene expression was maintained: HoxA-9 and HoxC-8 were the highest in the colon and ileum, respectively, and HoxA-4 was expressed all along the intestine; Cdx-1 and Cdx-2 exhibited an increasing gradient of expression from small intestine to colon. Yet, grafting per se caused a faint upregulation of HoxA-9 and HoxC-8 in small intestinal regions in which these genes are not normally expressed, while the endoderm-mesenchyme dissociation-association step provoked a decay of Cdx-1 in the colon. In heterotopic associations of colonic endoderm with small intestinal mesenchyme, the colonic epithelium exhibited heterodifferentiation to a small intestinal-like phenotype. In this case, we observed a decay of HoxA-9 expression and an upregulation of HoxC-8. Additionally, heterodifferentiation of the colonic epithelium was accompanied by a downregulation of Cdx-1 and Cdx-2 to a level similar to that found in the normal small intestine. To demonstrate that mesenchyme-derived cells can influence Cdx-1 and Cdx-2 expression in the bowel epithelium, fetal jejunal endoderm was associated with intestinal fibroblastic cell lines that either support small intestinal-like or colonic-like morphogenesis. A lower expression of both homeobox genes was shown in grafts presenting the small intestinal phenotype than in those showing glandular colonic-like differentiation. Taken together, these results suggest that homeobox genes participate in the control of the positional information and/or cell differentiation in the intestinal epithelium. They also indicate that the level of Cdx-1 and Cdx-2 homeobox gene expression is influenced by epithelial-mesenchymal cell interactions in the intestinal mucosa.

Animals↗

PubMed1995

Verification of the lactase site of rat lactase-phlorizin hydrolase by site-directed mutagenesis.

BACKGROUND & AIMS: Lactase-phlorizin hydrolase (LPH) is an intestinal microvillus membrane glycoprotein that hydrolyzes lactose and phlorizin. These enzymatic activities have been assigned to glutamic acid (E) residues 1271 and 1747 in rabbit LPH. The aim of this study was to determine directly if this assignment was correct and if these two amino acids are the only nucleophiles required for LPH enzyme activity. METHODS: Site-directed mutagenesis of a full-length rat LPH complementary DNA was used to convert the rat homologues E1274 and E1750 to aspartic acid or glycine. Mutants were analyzed by enzyme activity assays. RESULTS: All tested activities of E1274D and E1274G were virtually unaffected. In contrast, mutations E1750D and E1750G resulted in total loss of lactase and cellobiose activities, leaving only low ONP-glc and ONP-gal hydrolase activities detectable. A double mutant containing both E1274G and E1750G had no activity. CONCLUSIONS: These studies directly confirm that the two previously identified glutamic acids are essential to the enzymatic activity of rat LPH. Rat lactase activity is not associated with the E1274 site. This study provides the first evidence that rat LPH has its major catalytic site at E1750, representing all of the lactase and the majority of the phlorizin hydrolase activity.

Amino Acid Sequence↗