Hypomorphic variants of lactase-phlorizin hydrolase in congenital lactase deficiency are trafficking incompetent and functionally inactive.

Patients with the rare autosomal recessive disorder congenital lactase deficiency (CLD) present with severe, potentially life-threatening symptoms shortly after birth. Several variants have been characterized within the gene for lactase-phlorizin hydrolase (LCT) that are associated with CLD. Here, we analyze at the biochemical and cellular levels LCT mutants harboring the genetic variants p.Y1390*, p.E1612*, p.S1150Pfs*19, p.S1121L, p.R1587H, and p.S688P. Our data unequivocally demonstrate that these mutants are absolutely transport incompetent, some of which are readily degraded, and are enzymatically inactive. The current study contributes to and expands our understanding on the pathogenesis of CLD at the molecular level.

Digestive enzyme expression in the large intestine of children with short bowel syndrome in a late stage of adaptation.

BACKGROUND AND AIMS: Intestinal adaptation in short bowel syndrome (SBS) includes morphologic processes and functional mechanisms. This study investigated whether digestive enzyme expression in the duodenum and colon is upregulated in SBS patients. METHOD: Sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH), and neutral Aminopeptidase N (ApN) were analyzed in duodenal and colonic biopsies from nine SBS patients in a late stage of adaptation as well as healthy and disease controls by immunoelectron microscopy (IEM), Western blots, and enzyme activities. Furthermore, proliferation rates and intestinal microbiota were analyzed in the mucosal specimen. RESULTS: We found significantly increased amounts of SI, LPH, and ApN in colonocytes in most SBS patients with large variation and strongest effect for SI and ApN. Digestive enzyme expression was only partially elevated in duodenal enterocytes due to a low proliferation level measured by Ki-67 staining. Microbiome analysis revealed high amounts of Lactobacillus resp. low amounts of Proteobacteria in SBS patients with preservation of colon and ileocecal valve. Colonic expression was associated with a better clinical course in single cases. CONCLUSION: In SBS patients disaccharidases and peptidases can be upregulated in the colon. Stimulation of this colonic intestinalization process by drugs, nutrients, and pre- or probiotics might offer better therapeutic approaches.

Congenital Lactase Deficiency: Mutations, Functional and Biochemical Implications, and Future Perspectives.

Congenital lactase deficiency (CLD) is a severe autosomal recessive genetic disorder that affects the functional capacity of the intestinal protein lactase-phlorizin hydrolase (LPH). This disorder is diagnosed already during the first few days of the newborn's life due to the inability to digest lactose, the main carbohydrate in mammalian milk. The symptoms are similar to those in other carbohydrate malabsorption disorders, such as congenital sucrase-isomaltase deficiency, and include severe osmotic watery diarrhea. CLD is associated with mutations in the translated region of the LPH gene that elicit loss-of-function of LPH. The mutations occur in a homozygote or compound heterozygote pattern of inheritance and comprise missense mutations as well as mutations that lead to complete or partial truncations of crucial domains in LPH, such as those linked to the folding and transport-competence of LPH and to the catalytic domains. Nevertheless, the identification of the mutations in CLD is not paralleled by detailed genotype/protein phenotype analyses that would help unravel potential pathomechanisms underlying this severe disease. Here, we review the current knowledge of CLD mutations and discuss their potential impact on the structural and biosynthetic features of LPH. We also address the question of whether heterozygote carriers can be symptomatic for CLD and whether genetic testing is needed in view of the severity of the disease.

Distinguishing the differences in ß-glycosylceramidase folds, dynamics, and actions informs therapeutic uses.

Glycosyl hydrolases (GHs) are carbohydrate-active enzymes that hydrolyze a specific ß-glycosidic bond in glycoconjugate substrates; ß-glucosidases degrade glucosylceramide, a ubiquitous glycosphingolipid. GHs are grouped into structurally similar families that themselves can be grouped into clans. GH1, GH5, and GH30 glycosidases belong to clan A hydrolases with a catalytic (ß/α)8 TIM barrel domain, whereas GH116 belongs to clan O with a catalytic (α/α)6 domain. In humans, GH abnormalities underlie metabolic diseases. The lysosomal enzyme glucocerebrosidase (family GH30), deficient in Gaucher disease and implicated in Parkinson disease etiology, and the cytosol-facing membrane-bound glucosylceramidase (family GH116) remove the terminal glucose from the ceramide lipid moiety. Here, we compare enzyme differences in fold, action, dynamics, and catalytic domain stabilization by binding site occupancy. We also explore other glycosidases with reported glycosylceramidase activity, including human cytosolic ß-glucosidase, intestinal lactase-phlorizin hydrolase, and lysosomal galactosylceramidase. Last, we describe the successful translation of research to practice: recombinant glycosidases and glucosylceramide metabolism modulators are approved drug products (enzyme replacement therapies). Activity-based probes now facilitate the diagnosis of enzyme deficiency and screening for compounds that interact with the catalytic pocket of glycosidases. Future research may deepen the understanding of the functional variety of these enzymes and their therapeutic potential.

Characterization of Mucosal Disaccharidases from Human Intestine.

In this study, we used a brush border membrane (BBM) preparation from human small intestine to analyze the proportion and the activity of major intestinal disaccharidases, including sucrase-isomaltase (SI), maltase-glucoamylase (MGAM) and lactase-phlorizin hydrolase (LPH). SI, MGAM and LPH respectively constituted 8.2%, 2.7% and 1.4% of total BBM protein. The activity of SI and LPH decreased threefold after purification from the brush border membrane, which highlights the effect of membrane microdomains on the functional capacity of these enzymes. All of the disaccharidases showed optimal activity at pH 6, over 50% residual activity between pH 5 to pH 7, and increasing activity with rising temperatures up to 45 °C, along with a stable functional structure. Therefore the enzymes can withstand mild intraluminal pH alterations with adequate function, and are able to increase their activity with elevated core body temperature. Our data provide a functional measure for characterization of intestinal disaccharidases under different physiological and pathological conditions.

Structural determinants for transport of lactase phlorizin-hydrolase in the early secretory pathway as a multi-domain membrane glycoprotein.

BACKGROUND: Lactase phlorizin-hydrolase (LPH) is a membrane anchored type I glycoprotein of the intestinal epithelium that is composed of four homologous structural domains. The role of each distinct domain in the intramolecular organization and function of LPH is not completely understood. METHODS: Here, we analyzed the early events of LPH biosynthesis and trafficking by directed restructuring of the domain compositions. RESULTS: Removal of domain I (LPH∆1) results in a malfolded ER-localized protein. By contrast, LPH without domain II (LPH∆2) is normally transported along the secretory pathway, but does not dimerize nor is enzymatically active. Interestingly a polypeptide stretch in domain II between L735-R868 exerts an intriguing role in modulating the trafficking behavior of LPH and its biological function. In fact, association of this stretch with transport-competent LPH chimeras results in their ER-arrest or aberrant trafficking. This stretch harbors a unique N-glycosylation site that is responsible for LPH retention in the ER via association with calnexin and facilitates proper folding of domains I and III before ER exit of LPH. Notably, a similar N-glycosylation site is also found in domain IV with comparable effects on the trafficking of LPH-derived molecules. CONCLUSIONS: Our study provides novel insights into the intramolecular interactions and the sequence of events involved in the folding, dimerization and transport of LPH. GENERAL SIGNIFICANCE: Elucidation of the structural-functional relevance of the domains in pro-LPH is crucial in unravelling and understanding the molecular basis of carbohydrate malabsorption disorders that are associated with lactase deficiency or lactase malfunction.

Glucosylceramidases and malignancies in mammals.

Sphingolipids represent a major class of lipids that are essential constituents of eukaryotic cells. They are predominantly located in plasma membrane microdomains, and play an important structural role in regulating membrane fluidity. They are also bioactive effectors involved in diverse key cellular functions such as apoptosis and proliferation. The implication of some sphingolipids in cancer is well established whereas that of some others is still a matter of intense investigation. Glucosylceramide is the backbone of more than 300 structurally different glycosphingolipids including gangliosides and sulfatides, and is essential for mammalian development. Therefore, glucosylceramidases (also named GBA1, GBA2 and GBA3 ß-glucosidases), the enzymes that hydrolyse ß-glucosylceramide, play important functions. GBA1 is a lysosomal hydrolase whose deficiency causes Gaucher disease, the most prevalent inherited lysosomal storage disorder. GBA2 is a ubiquitous non-lysosomal glucosylceramidase whose mutations have been associated with some forms of hereditary spastic paraplegia. GBA3 is a cytosolic ß-glucosidase, mostly present in the kidney, liver, spleen, intestine and lymphocytes of mammals, the function of which is still unclear. Whereas glucosylceramide synthase is implicated in multidrug resistance, the role of glucosylceramide breakdown in cancer is not yet fully appreciated. Defective GBA1 enzyme activity in humans, i.e., Gaucher disease, is associated with an increased risk of multiple myeloma and other malignancies. Putative molecular links between Gaucher disease and cancer, which might implicate the malignant cell and/or its microenvironment, are reviewed. The functions of GBA2 and GBA3 in cancer progression are also discussed.

The Diverse Forms of Lactose Intolerance and the Putative Linkage to Several Cancers.

Lactase-phlorizin hydrolase (LPH) is a membrane glycoprotein and the only ß-galactosidase of the brush border membrane of the intestinal epithelium. Besides active transcription, expression of the active LPH requires different maturation steps of the polypeptide through the secretory pathway, including N- and O-glycosylation, dimerization and proteolytic cleavage steps. The inability to digest lactose due to insufficient lactase activity results in gastrointestinal symptoms known as lactose intolerance. In this review, we will concentrate on the structural and functional features of LPH protein and summarize the cellular and molecular mechanism required for its maturation and trafficking. Then, different types of lactose intolerance are discussed, and the molecular aspects of lactase persistence/non-persistence phenotypes are investigated. Finally, we will review the literature focusing on the lactase persistence/non-persistence populations as a comparative model in order to determine the protective or adverse effects of milk and dairy foods on the incidence of colorectal, ovarian and prostate cancers.

Small intestinal hydrolysis of plant glucosides: higher glucohydrolase activities in rodents than passerine birds.

Glycosides are a major group of plant secondary compounds characterized by one or more sugars conjugated to a lipophilic, possibly toxic aglycone, which is released upon hydrolysis. We compared small intestinal homogenate hydrolysis activity of three rodent and two avian species against four substrates: amygdalin and sinigrin, two plant-derived glucosides, the sugar lactose, whose hydrolysis models some activity against flavonoid and isoflavonoid glucosides, and the disaccharide sugar maltose (from starch), used as a comparator. Three new findings extend our understanding of physiological processing of plant glucosides: (1) the capacity of passerine birds to hydrolyze plant glucosides seems relatively low, compared with rodents; (2) in this first test of vertebrates' enzymic capacity to hydrolyze glucosinolates, sinigrin hydrolytic capacity seems low; (3) in laboratory mice, hydrolytic activity against lactose resides on the enterocytes' apical membrane facing the intestinal lumen, but activity against amygdalin seems to reside inside enterocytes.

Triple Recycling Processes Impact Systemic and Local Bioavailability of Orally Administered Flavonoids.

Triple recycling (i.e., enterohepatic, enteric and local recycling) plays a central role in governing the disposition of phenolics such as flavonoids, resulting in low systemic bioavailability but higher gut bioavailability and longer than expected apparent half-life. The present study aims to investigate the coexistence of these recycling schemes using model bioactive flavonoid tilianin and a four-site perfused rat intestinal model in the presence or absence of a lactase phlorizin hydrolase (LPH) inhibitor gluconolactone and/or a glucuronidase inhibitor saccharolactone. The result showed that tilianin could be metabolized into tilianin glucuronide, acacetin, and acacetin glucuronide, which are excreted into the bile and luminal perfusate (highest in the duodenum and lowest in the colon). Gluconolactone (20 mM) significantly reduced the absorption of tilianin and the enteric and biliary excretion of acacetin glucuronide. Saccharolactone (0.1 mM) alone or in combination of gluconolactone also remarkably reduced the biliary and intestinal excretion of acacetin glucuronide. Acacetin glucuronides from bile or perfusate were rapidly hydrolyzed by bacterial ß-glucuronidases to acacetin, enabling enterohepatic and enteric recycling. Moreover, saccharolactone-sensitive tilianin disposition and glucuronide deconjugation, which was more active in the small intestine than the colon, points to the small intestinal origin of the deconjugation enzyme and supports the presence of local recycling scheme. In conclusion, our studies have demonstrated triple recycling of a bioactive phenolic (i.e., a model flavonoid), and this recycling may have an impact on the site and duration of polyphenols pharmacokinetics in vivo.

Genetic origins of lactase persistence and the spread of pastoralism in Africa.

In humans, the ability to digest lactose, the sugar in milk, declines after weaning because of decreasing levels of the enzyme lactase-phlorizin hydrolase, encoded by LCT. However, some individuals maintain high enzyme amounts and are able to digest lactose into adulthood (i.e., they have the lactase-persistence [LP] trait). It is thought that selection has played a major role in maintaining this genetically determined phenotypic trait in different human populations that practice pastoralism. To identify variants associated with the LP trait and to study its evolutionary history in Africa, we sequenced MCM6 introns 9 and 13 and ~2 kb of the LCT promoter region in 819 individuals from 63 African populations and in 154 non-Africans from nine populations. We also genotyped four microsatellites in an ~198 kb region in a subset of 252 individuals to reconstruct the origin and spread of LP-associated variants in Africa. Additionally, we examined the association between LP and genetic variability at candidate regulatory regions in 513 individuals from eastern Africa. Our analyses confirmed the association between the LP trait and three common variants in intron 13 (C-14010, G-13907, and G-13915). Furthermore, we identified two additional LP-associated SNPs in intron 13 and the promoter region (G-12962 and T-956, respectively). Using neutrality tests based on the allele frequency spectrum and long-range linkage disequilibrium, we detected strong signatures of recent positive selection in eastern African populations and the Fulani from central Africa. In addition, haplotype analysis supported an eastern African origin of the C-14010 LP-associated mutation in southern Africa.

A comparative study on the metabolism of Epimedium koreanum Nakai-prenylated flavonoids in rats by an intestinal enzyme (lactase phlorizin hydrolase) and intestinal flora.

The aim of this study was to compare the significance of the intestinal hydrolysis of prenylated flavonoids in Herba Epimedii by an intestinal enzyme and flora. Flavonoids were incubated at 37 °C with rat intestinal enzyme and intestinal flora. HPLC-UV was used to calculate the metabolic rates of the parent drug in the incubation and LC/MS/MS was used to determine the chemical structures of metabolites generated by different flavonoid glycosides. Rates of flavonoid metabolism by rat intestinal enzyme were quicker than those of intestinal flora. The sequence of intestinal flora metabolic rates was icariin>epimedin B>epimedin A>epimedin C>baohuoside I, whereas the order of intestinal enzyme metabolic rates was icariin>epimedin A>epimedin C>epimedin B>baohuoside I. Meanwhile, the LC/MS/MS graphs showed that icariin produced three products, epimedin A/B/C had four and baohuoside I yielded one product in incubations of both intestinal enzyme and flora, which were more than the results of HPLC-UV due to the fact LC/MS/MS has lower detectability and higher sensitivity. Moreover, the outcomes indicated that the rate of metabolization of flavonoids by intestinal enzyme were faster than those of intestinal flora, which was consistent with the HPLC-UV results. In conclusion, the metabolic pathways of the same components by intestinal flora and enzyme were the same. What's more, an intestinal enzyme such as lactase phlorizin hydrolase exhibited a more significant metabolic role in prenylated flavonoids of Herba Epimedi compared with intestinal flora.

A novel local recycling mechanism that enhances enteric bioavailability of flavonoids and prolongs their residence time in the gut.

Recycling in the gastrointestinal tract is important for endogenous substances such as bile acids and for xenobiotics such as flavonoids. Although both enterohepatic and enteric recycling mechanisms are well recognized, no one has discussed the third recycling mechanism for glucuronides: local recycling. The intestinal absorption and metabolism of wogonin and wogonoside (wogonin-7-glucuronide) was characterized by using a four-site perfused rat intestinal model, and hydrolysis of wogonoside was measured in various enzyme preparations. In the perfusion model, the wogonoside and wogonin were interconverted in all four perfused segments. Absorption of wogonoside and conversion to its aglycon at the upper small intestine was inhibited in the presence of a glucuronidase inhibitor (saccharolactone) but was not inhibited by lactase phlorizin hydrolase (LPH) inhibitor gluconolactone or antibiotics. Further investigation indicated that hydrolysis of wogonoside in the blank intestinal perfusate was not correlated with bacterial counts. Kinetic studies indicated that K(m) values from blank duodenal and jejunal perfusate were essentially identical to the K(m) values from intestinal S9 fraction but were much higher (>2-fold) than those from the microbial enzyme extract. Lastly, jejunal perfusate and S9 fraction share the same optimal pH, which was different from those of fecal extract. In conclusion, local recycling of wogonin and wogonoside is the first demonstrated example that this novel mechanism is functional in the upper small intestine without significant contribution from bacteria ß-glucuronidase.

Theodore E. Woodward Award: lactase persistence SNPs in African populations regulate promoter activity in intestinal cell culture.

Lactase-phlorizin hydrolase, lactase, is the intestinal enzyme responsible for the digestion of the milk sugar lactose. The majority of the world's human population experiences a decline in expression of the lactase gene by late childhood (lactase non-persistence). Individuals with lactase persistence, however, continue to express high levels of the lactase gene throughout adulthood. Lactase persistence is a heritable autosomal dominant condition and has been strongly correlated with several single nucleotide polymorphisms (SNPs) located ∼14 kb upstream of the lactase gene in different ethnic populations: -13910*T in Europeans and -13907*G, -13915*G, and -14010*C in several African populations. The coincidence of the four SNPs clustering within 100 bp strongly suggests that this region mediates the lactase non-persistence/persistence phenotype. Having previously characterized the European SNP, we aimed to determine whether the African SNPs similarly mediate a functional role in regulating the lactase promoter. Human intestinal Caco-2 cells were transfected with lactase SNP/promoter-reporter constructs and assayed for promoter activity. The -13907*G and -13915*G SNPs result in a significant enhancement of lactase promoter activity relative to the ancestral lactase non-persistence genotype. Such differential regulation by the SNPs is consistent with a causative role in the mechanism specifying the lactase persistence phenotype.

Role of intestinal hydrolase in the absorption of prenylated flavonoids present in Yinyanghuo.

PURPOSE: Yinyanghuo (Herba Epimdii) is a traditional Chinese herb containing prenylated flavonoids as its active constituents. The aim of this study was to examine the significance of the intestinal hydrolysis of prenylated flavonoids by lactase phlorizin hydrolase (LPH), an enzyme at the brush border membrane of intestinal cells. METHODS: A four-site perfused rat intestinal model was used. The concentration of the flavonoids of interest and their metabolites in different intestinal segements were analyzed by HPLC, and the apparent permeabilities were calculated. A lactase phlorizin hydrolase inhibitor (gluconolactone) was employed to investigate the mechanism of the intestinal absorption, and the metabolites of the four flavonoids were identified using LC/MS/MS. RESULTS: Diglycosides (icariin) or triglycosides (epimedin A, epimedin B, and epimedin C) were hydrolyzed rapidly in duodenum and jejunum producing one or two metabolites, while a monoglycoside (baohuoside I) was absorbed directly. When co-perfused with glucono-lactone, both the hydrolysis of diglycosides and triglycosides were significantly inhibited, with inhibition rates for icariin (62%, 50%, 40%, 46%), epimedin A, (55%, 26%, 21%, 14%); epimedin B (42%, 40%, 74%, 22%), and epimedin C (42%, 40%, 52%, 35%) in duodenum, jejunum, ileum, and colon, respectively. Also the metabolites of icariin, epimedin A, epimedin B, and epimedin C were identified as baohuoside I (one of two), sagittatoside A, sagittatoside B, and 2"-O-rhamnosylicariside II, respectively. CONCLUSIONS: The results showed that lactase phlorizin hydrolase was a major determinant of the intestinal absorption of prenylated flavonoids present in Yinyanghuo.

Characterization of expression in mice of a transgene containing 3.3 kb of the human lactase-phlorizin hydrolase (LPH) 5' flanking sequence.

BACKGROUND AND AIM: The regulation of human intestinal lactase-phlorizin hydrolase remains incompletely understood. One kb of pig and 2 kb of rat 5'-flanking sequence controls correct tissue, cell, topographic, and villus LCT expression. To gain insight into human LCT expression, transgenic mouse lines were generated from 3.3 kb of human LPH 5' flanking sequence from a lactase persistent individual fused to a human growth hormone (hGH) reporter bounded by an insulator. METHODS: Four lines were identified in which reporter expression was specifically detectable in the intestine and no other organ, two of which demonstrated hGH expression specific to small and large intestine. Quantitative RT-PCR was carried out on proximal to distal segments of small intestine at fetal days 16.5 and 18.5 and at birth, postnatal days 7 and 28 in line 22. RESULTS: In fetal intestine, hGH expression demonstrated a proximal to distal gradient similar to that in native intestine. There was no significant difference between hGH expression levels at 7 and 28 days in segment 3, the midpoint of the small intestine, where expression of endogenous lactase is maximal at 7 days and declines significantly by 28 days. Distal small intestine displayed high levels of hGH expression in enteroendocrine cells, which were shown to be a subset of the PYY cells. CONCLUSIONS: Thus, a 3.3-kb LPH 5' flanking sequence construct from a lactase persistent individual is able to maintain postnatal expression in transgenic mice post weaning.

Effect of milk formula protein content on intestinal barrier function in a porcine model of LBW neonates.

Our study aimed at investigating the impact of the level of protein in milk formula on intestinal structure, barrier function, and its nervous regulation in normal and LBW neonates using a porcine model. Normal birth weight (NBW) or LBW piglets were fed from d7 to d28 of age either with a high protein (HP) or with an adequate protein (AP) formula or stayed with their mother [mother fed (MF)]. The proximal jejunum and distal ileum were sampled at d28 for morphometry analysis and ex vivo permeability measurement in Ussing chambers. Formula feeding induced a trophic effect on the jejunum and ileum of both NBW and LBW piglets, which exhibited longer villi than MF animals, irrespective of the type of formula. In NBW piglets, intestinal permeability was not altered by formula feeding. On the contrary, LBW piglets fed with HP formula, but not AP, exhibited a greater ileal permeability than MF piglets. Feeding the HP formula also disturbed jejunal and ileal regulation of permeability by acetylcholine and vasoactive intestinal peptide (VIP) in LBW compared with MF LBW piglets. In conclusion, the level of protein in formulas did not modify intestinal structure and function in NBW individuals but dramatically modified intestinal barrier function physiology in LBW individuals.

Resveratrol from transgenic alfalfa for prevention of aberrant crypt foci in mice.

Transgenic alfalfa (Medicago sativa L.), which accumulated resveratrol-glucoside (RG), was incorporated into diets and fed to female, 6-wk-old CF-1 mice for 5 wk. Mice fed diets containing transgenic alfalfa with supplemented alpha -galactosidase had significantly fewer azoxymethane (AOM)-induced aberrant crypt foci (ACF) in their colon relative to mice fed the transgenic alfalfa diets without added alpha -galactosidase (P = 0.02). Resveratrol-aglycone (Rag) was detected in the colon of 100% of mice fed transgenic alfalfa diets with supplemented alpha -galactosidase and in 60% of mice fed transgenic alfalfa without alpha -galactosidase (P < 0.05). Colonic concentrations of Rag (< 0.5 nmol/g tissue) in mice fed transgenic alfalfa with alpha -galactosidase (0.22 +/- 0.18 nmol/g tissue) tended to be higher than in animals fed diets without alpha -galactosidase (0.1 +/- 0.08 nmol/g tissue; P = 0.09). The use of N-(Bn-butyl)-deoxygalactonojirimycin, an inhibitor of lactase-phlorizin hydrolase (LPH), in transport studies with everted jejunal sacs from CF-1 mice (N = 8) suggested that LPH is involved in the intestinal deglycosylation of RG. Our collective findings suggest that RG from transgenic alfalfa is metabolized and absorbed in the upper intestine and does not reach the colon in sufficient amounts to inhibit ACF.

Structural hierarchy of regulatory elements in the folding and transport of an intestinal multidomain protein.

Human intestinal lactase-phlorizin hydrolase, LPH, encompasses four homologous domains, which presumably have evolved from two subsequent duplications of one ancestral gene. The profragment, LPHalpha, comprises homologous domains I and II and functions as an intramolecular chaperone in the context of the brush-border LPHbeta region of LPH. Here, we analyze the inter-relationship between homologous domains III and IV of LPHbeta and their implication in the overall structure, function, and trafficking of LPH. In silico analyses revealed potential domain boundaries for these domains as a basis for loop-out mutagenesis and construction of deletion or individual domain forms of LPH. Removal of domain IV, which contains lactase, results in a diminished phlorizin hydrolase activity, lack of dimerization in the endoplasmic reticulum (ER), but accelerated transport kinetics from the ER to the Golgi apparatus. By contrast, deletion of domain III, which harbors phlorizin hydrolase, generates a malfolded protein that is blocked in the ER. Interestingly, homologous domain III is transport-competent per se and sorted to the apical membrane in polarized Madin-Darby canine kidney cells. Nevertheless, it neither dimerizes nor acquires complete phlorizin hydrolase activity. Our data present a hierarchical model of LPH in which the homologous domain III constitutes (i) a fully autonomous core domain within LPH and (ii) another intramolecular chaperone besides the profragment LPHalpha. Nevertheless, the regulation of the trafficking kinetics and activity of domain III and entire LPH including elevation of the enzymatic activities require the correct dimerization of LPH in the ER, an event that is accomplished by the non-autonomous domain IV.

Analysis of behavior of sodiated sugar hemiacetals under low-energy collision-induced dissociation conditions and application to investigating mutarotation and mechanism of a glycosidase.

Analysis of anomericity is one of the most important issues in the structure elucidation of carbohydrates. Mass spectrometry (MS)-based methods are of particular interest and important to address the issue related to resolving anomericity of monosaccharide units in a glycan. However, direct analysis of hemiacetals has not been possible by MS because of the nonavailability of information regarding the gas-phase behavior of such ion species. We addressed this issue by using stage-discriminated energy-resolved mass spectrometry (ERMS) at the stages of MS(n) and MS(n+1) and showed that such analysis can be made. This was achieved by proving that individual anomers can be identified and that the equilibrium of sodium adducted ion species of alpha- and beta-anomers can be negated in the gas phase under collision-induced dissociation (CID) conditions. On the basis of these results, we could 1) observe the mutarotation of lactose and 2) speculate the hydrolysis mechanism of endo-glycosylceramidase by using mass spectrometry.