Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.108 (lactase)
 2,133 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human lactase-phlorizin hydrolase (LPH, EC 3.2.1.23/62) is synthesized as a single-chain precursor glycoprotein (pro-LPH) with a relative molecular mass of just over 200 kDa. Maturation to the mature enzyme (m-LPH, 160 kDa) occurs after passage of pro-LPH through the Golgi complex and involves the proteolytic removal of a 849 amino acid propeptide. The role of this propeptide as well as its removal is not fully understood and the proteolytic enzyme or enzymes involved are unknown. We studied the potential role of five different members of the family of subtilisin-like proprotein processing proteases in the maturation process of human LPH using a vaccinia virus based coexpression system in pig kidney PK(15) cells. Infected/transfected PK(15) cells expressed full-length pro-LPH but no maturation to m-LPH was observed. Coexpression of human pro-LPH with human furin, human PC1/PC3, human PC2, human PACE4 and mouse PC6A in PK(15) cells did not result in maturation of the enzyme. Cleavage and secretion of von Willebrand factor precursor (pro-vWF) was used as a positive control. None of the five proprotein processing proteases tested were capable of cleaving human pro-LPH, strongly suggesting that they are not involved in the maturation of this enzyme.
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PMID:Human lactase-phlorizin hydrolase is not processed by furin, PC1/PC3, PC2, PACE4 and PC5/PC6A of the family of subtilisin-like proprotein processing proteases. 866 47

Human lactase-phlorizin hydrolase (LPH), a brush border membrane hydrolase of the small intestine, is synthesized as a precursor molecule that undergoes proteolytic cleavage to yield mature LPH (LPHbeta) by a trypsin-like protease (Naim et al., 1987, 1991). Arg868-Ala869 has been previously proposed to be the putative cleavage site for this processing step. Site-directed mutagenesis of this monobasic site does not lead to the generation of an uncleaved proLPH species, which strongly suggests the existence of an additional cleavage site. Further analyses of LPH synthesized in different cell lines lend support to this hypothesis. Biosynthetic labeling of human intestinal biopsy samples in the presence of trypsin reveals an LPHbeta species that is slightly smaller than the intracellularly cleaved molecule. When the proLPH molecule is screened for potential cleavage sites, two dibasic pairs are revealed upstream of the N-terminal end of brush border LPH at Lys851-Arg852 and Arg830-Lys831. Treatment of proLPH with trypsin for different periods of time supports the idea of at least two cleavage steps, whereby Arg868-Ala869 represents the final cleavage site that generates LPHbeta. We propose that the initial cleavage of proLPH takes place intracellularly at a site further away from Arg868-Ala869, to generate LPHbeta initial; LPHbeta is subsequently cleaved extracellularly in the gut lumen, presumably by trypsin, at Arg868-Ala869 to mature brush border LPH (LPHbeta initial).
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PMID:Maturation of human intestinal lactase-phlorizin hydrolase: generation of the brush border form of the enzyme involves at least two proteolytic cleavage steps. 866 96

Despite extensive study in both humans and nonhuman mammals the mechanisms which regulate intestinal lactase activity, particularly during development, are incompletely understood. Our previous studies of human adults are consistent with an important role of lactase-phlorizin hydrolase (LPH) mRNA abundance in determining the lactase persistence/nonpersistence phenotypes. Our intent in the present study was to determine the role of LPH mRNA in the regulation of lactase in children. We therefore studied duodenal mucosal biopsies from 39 children undergoing diagnostic upper endoscopy in whom significant small intestinal and nutritional disease was excluded. We found no relationship between the level of LPH mRNA and lactase enzymatic activity. Our observations suggest the importance of posttranscriptional mechanisms in lactase regulation in human children.
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PMID:Heterogeneity of intestinal lactase activity in children: relationship to lactase-phlorizin hydrolase messenger RNA abundance. 872 45

Analysis of the quaternary structure of human intestinal lactase-phlorizin hydrolase (LPH) by chemical cross-linking and sucrose-gradient centrifugation reveals that the brush border form of LPH (LPH beta; 160-kDa) is a homodimeric molecule. Dimerization ensures in the ER when LPH is still exclusively found as an uncleaved mannoserich precursor (pro-LPHb; 215-kDa). This is supported by the following observations. (i) Biosynthetically labeled intestinal biopsy specimens as well as transfected COS-1 cells expressing pro-LPH contain monomeric and dimeric forms of pro-LPHb; the complex glycosylated pro-LPH (pro-LPHc; 230-kDa) as well as the cleaved mature LPH beta species in intestinal biopsy samples are discerned exclusively as dimers. (ii) Dimeric forms of pro-LPHh could be also detected when cells were biosynthetically labeled at 15 degrees C, at which temperature the egress of pro-LPH from the ER is blocked. Dimerization is essential for the transport competence of pro-LPH and is strongly associated with the presence of an intact transmembrane domain. Mutant pro-LPH-mact lacking the complete transmembrane domain persists as a monomeric, mannose-rich and transport-incompetent molecule that is not secreted into the exterior milieu, accumulates most likely in the ER and is ultimately degraded. Further, deletion of the cytoplasmic tail in the pro-LPH-ct mutant leads to marked reduction in the proportion of dimeric as well as complex glycosylated pro-LPH-ct. Finally, dimerization is linked to the acquisition of LPH to its biological function, since only dimers of wild type pro-LPH or pro-LPH-ct are enzymatically active, while their monomeric counterparts as well as pro-LPH-mact are not.
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PMID:Dimerization of lactase-phlorizin hydrolase occurs in the endoplasmic reticulum, involves the putative membrane spanning domain and is required for an efficient transport of the enzyme to the cell surface. 883 4

Recent studies suggest the importance of two transcription factors, Cdx-2 and NF-LPH-1, in the regulation of sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH) gene expression, respectively. Cdx-2 accounts for the tissue specificity of sucrase expression (16), and NF-LPH-1 varies with postnatal changes in lactase activity, suggesting a role in its developmental regulation (22). We used electrophoretic mobility shift assays to study the relationship of Cdx-2 and NF-LPH-1 to SI and LPH gene expression in Caco-2 cells to provide evidence regarding the role of these factors in the development of sucrase and lactase with cellular differentiation. We found that Cdx-2 levels correlated with SI expression and that NF-LPH-1 did not correlate with LPH expression. These studies suggest a role for Cdx-2 but not for NF-LPH-1 in the development of carbohydrase expression in these cells.
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PMID:Regulation of sucrase and lactase in Caco-2 cells: relationship to nuclear factors SIF-1 and NF-LPH-1. 889 92

The cytosolic beta-glucosidase (EC 3.2.1.21) present in the livers of mammalian species is distinguished by its broad specificity for sugars and its preference for hydrophobic aglycones. We purified the cytosolic beta-glucosidase from guinea pig liver and sequenced 142 amino acid residues contained within 12 trypsin digest fragments. Using degenerate oligonucleotide primers deduced from the peptide sequences, a 622 bp cytosolic beta-glucosidase cDNA was amplified by reverse-transcriptase PCR, using total guinea pig liver RNA as template. The 'rapid amplification of cDNA ends (RACE)' method [Frohman (1993) Methods Enzymol. 218, 340-356] was used to synthesize the remaining segments of the full-length cDNA. The complete cDNA contained 1671 nucleotides with an open reading frame coding for 469 amino acid residues. The amino acid sequence deduced from the cDNA sequence included the amino acid sequences of all 12 trypsin digest fragments derived from the purified enzyme. Amino acid sequence analysis indicates that the guinea pig liver cytosolic beta-glucosidase is a Family 1 beta-glycosidase and that it is most closely related to mammalian lactase-phlorizin hydrolase. These results suggest that the cytosolic beta-glucosidase and lactase-phlorizin hydrolase diverged from a common evolutionary precursor.
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PMID:Primary structure of the cytosolic beta-glucosidase of guinea pig liver. 892 Sep 87

Human lactase-phlorizin hydrolase (EC 3.2.1.23/62) is a major disaccharidase in the microvillus membrane of small intestinal epithelial cells. The enzyme is synthesized as a single-chain precursor protein and undergoes proteolytic processing during maturation. We studied proteolytic processing of human lactase-phlorizin hydrolase in transfected COS-1, Caco-2, and MDCK cells using metabolic labeling, surface immunoprecipitation, protease sensitivity assays, and microsequencing. Furthermore, we generated mutated forms of the enzyme to alter potential proteolytic cleavage sites and expressed these in Caco-2 and COS-1 cells. Since the N-terminal amino acid of microvillus lactase-phlorizin hydrolase corresponds to Ala869 in the precursor protein, it has been speculated that processing occurs at position Arg868-Ala869. Substitution of Arg868 with isoleucine, lysine, or glutamic acid had no effect on the proteolytic processing of pro-LPH in Caco-2 cells. As in wild-type enzyme a processed 160-kDa form was generated. These data are not consistent with a primary proteolytic processing at position Arg868-Ala869. Using amino-terminal amino acid sequencing of this processed form isolated from stable transfected MDCK cells we identified the cleavage site at Arg734-Leu735. Treatment of pro-lactase-phlorizin hydrolase expressed in COS-1 and MDCK cells by trypsin yielded a 145-kDa form with an identical amino terminal as the mature microvillus enzyme isolated from intestinal mucosa (Ala869). These data provide unambiguous evidence of a two-step processing of human lactase-phlorizin hydrolase. The first cleavage occurs intracellularly after a dibasic site (Arg734-Leu735) and yields the 160-kDa intermediate form. In a second step the intermediate form inserted into the microvillus membrane is trimmed to the mature enzyme by luminal trypsin.
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PMID:Proteolytic processing of human lactase-phlorizin hydrolase is a two-step event: identification of the cleavage sites. 895 Oct 31

To gain insight into the postnatal growth delay induced by ethanol in utero, we characterized functional impairments of the small intestine of neonatal rats prenatally exposed to ethanol using a well-described model of gestational alcoholism (25% ethanol w/v in the drinking water). Expression of the intestinal enzymes-lactase-phlorizin hydrolase (LPH) and intestinal alkaline phosphatase (IAP)-that are critical for enteral nutrition of neonates was studied. Characteristic patterns of LPH and IAP expression along the proximal-distal (horizontal) and crypt-villus (vertical) axes of the small intestine, as well as the intracellular localization of LPH and IAP mRNAs and immunoreactive proteins within absorptive enterocytes, were not altered by prenatal exposure to ethanol. However, a 10- to 15-fold increase in the number of LPH and IAP mRNA molecules per absorptive enterocyte was found throughout the intestine of ethanol-exposed neonates, compared with controls, whereas lactase and alkaline phosphatase activities per enterocyte remained unchanged. These findings suggest that ethanol in utero alters the mRNA abundance of epithelial enzymes in newborn rat small intestine. Changes in mRNA abundance could be an important aspect of enterocyte adaptation to high ethanol concentrations in gastrointestinal amniotic fluid of ethanol-exposed fetuses.
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PMID:Prenatal ethanol exposure alters the expression of intestinal hydrolase mRNAs in newborn rats. 898 19

Polarized transport of proteins is contingent on the presence of specific protein structures or motifs that function as sorting signals. Our model protein to analyze and to identify such signals is that of lactase-phlorizin hydrolase (LPH), a strictly polarized brush border membrane protein of small intestinal epithelial cells. It is synthesized as a large pro-LPH precursor molecule, which is proteolytically processed to yield the mature brush border enzyme (LPHbeta). Pro-LPH as well as LPHbeta are correctly sorted to the brush border membrane. In this paper we examine the location of putative sorting signals in the pro-LPH molecule. Expression of a cDNA encoding the LPHbeta mature form in the absence of the LPHalpha species in Madin-Darby canine kidney (MDCK) cells reveal an LPHbeta molecule that is not as transport-competent as wild type pro-LPH. The proportion of complex glycosylated LPHbeta constitutes not more than 10% of the total synthesized protein. This form displays a similar trypsin sensitive pattern as wild type intestinal LPHbeta suggesting comparable folding patterns of the two species. Complex glycosylated LPHbeta is sorted to the apical membrane more efficiently than wild type pro-LPH. We conclude that the apical sorting signals for pro-LPH are exclusively found in the LPHbeta mature domain.
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PMID:The apical sorting of lactase-phlorizin hydrolase implicates sorting sequences found in the mature domain. 901 26

Morphogenesis, initiation of differentiation marker gene expression, and their correlation with CCAT/enhancer binding protein (C/EBP) expression were analyzed in the developing fetal rat small intestine. Expressions of mRNAs for lactase-phlorizin hydrolase (LPH), intestinal alkaline phosphatase (IALP), carbamoyl-phosphate synthetase (CPS), and three isoforms of C/EBP were simultaneously determined by Northern blot analysis from 15 to 19 days of gestation. At 17 days of gestation, prior to villus formation as demonstrated by light and electron microscopy, only CPS and C/EBPalpha, -beta, and -delta expression could clearly be detected. Both LPH and IALP mRNA were definitely detectable in proximal and middle intestine on day 18, as soon as the stratified epithelium of the early intestine had been transformed into a single layer of columnar epithelium lining villi. This distribution was confirmed by in situ hybridization for LPH mRNA. During the period of transformation when the columnar epithelium and villi were forming, no LPH or IALP mRNA was detectable in the immature distal one-third of the fetal intestine. Preceding villus morphogenesis, immunostaining demonstrated nuclear localization of C/EBPalpha protein in intestinal epithelial cells, with continued expression in all enterocytes through 19 days of gestation. Enhanced expression of C/EBPalpha mRNA and protein began 24 h prior to the initiation of the differentiation markers, suggesting that it may play a role in regulation of fetal intestinal differentiation.
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PMID:Increased C/EBP in fetal rat small intestine precedes initiation of differentiation marker mRNA synthesis. 912 74


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