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 (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

The roles of various domains of intestinal lactase-phlorizin hydrolase (pro-LPH) on its folding, dimerization, and polarized sorting are investigated in deletion mutants of the ectodomain fused or not fused with the membrane-anchoring and cytoplasmic domains (MACT). Deletion of 236 amino acids immediately upstream of MACT has no effect on the folding, dimerization, transport competence, or polarized sorting of the mutant LPH1646MACT. By contrast, LPH1646, an anchorless counterpart of LPH1646MACT, is not transported beyond the ER and persists as a mannose-rich monomer during its entire life cycle. The further deletion of 87 amino acids generates a correctly folded but transport-incompetent monomeric LPH1559MACT mutant. The results strongly suggest that dimerization and transport of pro-LPH implicate a stretch of 87 amino acids in the ectodomain between LPH1646MACT and LPH1559MACT. In addition, dimerization of pro-LPH requires at least two further criteria: (i) a correctly folded ectodomain of pro-LPH and (ii) the presence of the transmembrane region. Neither of these requirements alone is sufficient for dimerization. Finally, the sorting of pro-LPH appears to be mediated by signals located between the cleavage site of pro-LPH and the LPH1646MACT mutant.
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PMID:Protein domains implicated in intracellular transport and sorting of lactase-phlorizin hydrolase. 959 32

Human lactase-phlorizin hydrolase (human-LPH) is synthesized as a large precursor (prepro-LPH), then cleaved to a pro-LPH of 220 kDa which is further cut to a "mature-like LPH" of a size close to that of mature LPH, i.e. about 150 kDa (in the processing of rabbit pro-LPH the intermediate has a mass of approximately 180 kDa). By coexpression of human prepro-LPH with furin in COS-7 cells we show that furin generates a mature-like LPH. Radioactive amino acid sequence analysis reveals that furin recognizes the motif R-T-P-R832, a protein convertase consensus, to generate a NH2 terminus located 36 amino acids upstream of the NH2 terminal found in vivo at Ala869. This intermediate is ultimately cleaved to the mature LPH form by other proteases including the pancreatic ones. These data demonstrate that human pro-LPH, like the rabbit enzyme, is processed to the mature enzyme by furin or furin-like enzymes through at least an intermediate form that has, however, an apparent mass close to that of the mature enzyme.
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PMID:Processing of human intestinal prolactase to an intermediate form by furin or by a furin-like proprotein convertase. 979 47

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.
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PMID:Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase. 1069 80

Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism-based glycosidase inhibitors. When LPH is treated with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside (1) and 2', 4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside (2), known mechanism-based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (D-glucal and D-galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active-site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett. 435, 225-228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem. 267, 18744-18752].
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PMID:Differential mechanism-based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase. 1110 9

We have identified a novel mouse gene klph (Klotho-LPH related protein; where LPH stands for lactase-phlorizin hydrolase) that encodes a novel mammalian family 1 glycosidase-like protein. KLPH was a putative type I membrane protein that consists of N-terminal signal sequence, glycosidase domain, transmembrane region and short cytoplasmic tail. Despite its overall structural similarity to other family 1 glycosidases, the glutamic acid for the acid-base catalyst was not conserved in this protein. klph mRNA was predominantly expressed in the kidney and skin. Epitope-tagged KLPH was localized to the perinuclear tubular network structure of the endoplasmic reticulum in cultured cells.
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PMID:Identification of a novel mouse membrane-bound family 1 glycosidase-like protein, which carries an atypical active site structure. 1208 82

Lactase phlorizin hydrolase is a small intestinal-specific brush border protein commonly used as a specific marker of differentiated enterocytes. A number of transcription factors involved in the enterocyte-specific expression of lactase phlorizin hydrolase have been identified. An upstream regulatory region, which we have named the "LPH enhancer", located at position -894 to -798 in the porcine lactase phlorizin hydrolase gene, is necessary for high differentiation-dependent LPH expression in intestinal cells. The LPH enhancer was studied by mutation analysis, transfection experiments and electrophoretical mobility shift assays. The LPH enhancer is active in intestinal cells (Caco-2) and not in non-intestinal cells (HeLa). The LPH enhancer is only able to enhance expression when it is located in front of an intestinal-specific promoter such as the lactase phlorizin hydrolase promoter or the sucrase-isomaltase promoter. In front of an SV40-derived promoter the LPH enhancer has no stimulatory effect. In addition to the lack of promoter-promiscuity, the LPH enhancer is not a classical enhancer in the sense that it is not orientation-independent and it cannot function when located 3' of a reporter gene. The LPH enhancer contains at least three cis-elements (at -894 to -880, -880 to -875 and -833 to -814) with functional importance for the LPH enhancer activity.
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PMID:An enhancer activates the pig lactase phlorizin hydrolase promoter in intestinal cells. 1259 46

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.
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PMID:Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-D-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene. 1497 28

Glycosidase activity influences the intestinal absorption of glycosides. Our previous study in rats suggested that disaccharide conjugates might be prototypes for pre-prodrugs aiming at the Na(+)/glucose co-transporter-mediated transport of prodrugs (drug glucoside) as a novel absorption pathway. One of the crucial factors is the formation of a glucoside drug from the disaccharide conjugate. Since there is a large species difference in metabolism, it is necessary to examine the cells and/or enzymes derived from human tissue to confirm this concept. In this paper, we kinetically characterized the glycosidase activity of disaccharide conjugates in Caco-2 cells. Disaccharide conjugates of p-nitrophenol (p-NP) (p-NP beta-cellobioside, p-NP beta-lactoside and p-NP beta-maltoside) were hydrolysed to p-NP beta-glucoside. beta-glucosidase or beta-galactosidase (lactase/phloridzin hydrolase, LPH) and alpha-glucosidase (sucrase-isomaltase) had different pH-dependent activities for disaccharide conjugates. At neutral pH, LPH has low affinity and low capacity, and sucrase-isomaltase has high affinity and high capacity, whereas at acid pH, LPH has high affinity and low capacity, and sucrase-isomaltase has low affinity and high capacity. The hydrolysis clearance calculated with Vmax/Km indicated that sucrase-isomaltase activity is much higher than LPH activity at either neutral or acid pH in Caco-2 cells. Since the hydrolysis rate of the disaccharide conjugate was highly dependent on the pH value and type of glycoside linkage, the appropriate selection of a glycoside form after consideration of these differences is the key to designing a sugar-conjugate prodrug.
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PMID:Kinetic characterization of glycosidase activity from disaccharide conjugate to monosaccharide conjugate in Caco-2 cells. 1590 56

Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23-62) is a brush border membrane (BBM)-associated enzyme in intestinal cells that hydrolyse lactose, the most important sugar in milk. Impairing in lactase activity during rotavirus infection has been described in diseased infants but the mechanism by which the functional lesion occurs remains unknown. We undertook a study to elucidate whether rotavirus impairs the lactase enzymatic activity in BBM of human enterocyte cells. In this study we use cultured human intestinal fully differentiated enterocyte-like Caco-2 cells to demonstrate how the lactase enzymatic activity at BBM is significantly decreased in rhesus monkey rotavirus (RRV)-infected cells. We found that the decrease in enzyme activity is not dependent of the Ca(2+)- and cAMP-dependent signalling events triggered by the virus. The LPH biosynthesis, stability, and expression of the protein at the BBM of infected cells were not modified. We provide evidence that in RRV-infected cells the kinetic of lactase enzymatic activity present at the BBM was modified. Both BBM(control) and BBM(RRV) have identical K(m) values, but hydrolyse the substrate at different rates. Thus, the BBM(RRV) exhibits almost a 1.5-fold decreased V(max) than that of BBM(control) and is therefore enzymatically less active than the latter. Our study demonstrate conclusively that the impairment of lactase enzymatic activity at the BBM of the enterocyte-like Caco-2 cells observed during rotavirus infection results from an inhibitory action of the secreted non-structural rotavirus protein NSP4.
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PMID:An NSP4-dependant mechanism by which rotavirus impairs lactase enzymatic activity in brush border of human enterocyte-like Caco-2 cells. 1750 19


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