Nescient helix-loop-helix 1 (Nhlh1) is a novel activating transcription factor 5 (ATF5) target gene in olfactory and vomeronasal sensory neurons in mice.

Activating transcription factor 5 (ATF5) is a transcription factor that belongs to the cAMP-response element-binding protein/ATF family and is essential for the differentiation and survival of sensory neurons in mouse olfactory organs. However, transcriptional target genes for ATF5 have yet to be identified. In the present study, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed to verify ATF5 target genes in the main olfactory epithelium and vomeronasal organ in the postnatal pups. ChIP-qPCR was conducted using hemagglutinin (HA)-tagged ATF5 knock-in olfactory organs. The results obtained demonstrated that ATF5-HA fusion proteins bound to the CCAAT/enhancer-binding protein-ATF response element (CARE) site in the enhancer region of nescient helix-loop-helix 1 (Nhlh1), a transcription factor expressed in differentiating olfactory and vomeronasal sensory neurons. Nhlh1 mRNA expression was downregulated in ATF5-deficient (ATF5-/-) olfactory organs. The LIM/homeobox protein transcription factor Lhx2 co-localized with ATF5 in the nuclei of olfactory and vomeronasal sensory neurons and bound to the homeodomain site proximal to the CARE site in the Nhlh1 gene. The CARE region of the Nhlh1 gene was enriched by the active enhancer marker, acetyl-histone H3 (Lys27). The present study identified Nhlh1 as a novel target gene for ATF5 in murine olfactory organs. ATF5 may upregulate Nhlh1 expression in concert with Lhx2, thereby promoting the differentiation of olfactory and vomeronasal sensory neurons.

Activating transcription factor 5 (ATF5) controls intestinal tuft and goblet cell expansion upon succinate-induced type 2 immune responses in mice.

Intestinal tuft cells, a chemosensory cell type in mucosal epithelia that secrete interleukin (IL)-25, play a pivotal role in type 2 immune responses triggered by parasitic infections. Tuft cell-derived IL-25 activates type 2 innate lymphoid cells (ILC2) to secrete IL-13, which, in turn, acts on intestinal stem or transient amplifying cells to expand tuft cells themselves and mucus-secreting goblet cells. However, the molecular mechanisms of tuft cell differentiation under type 2 immune responses remain unclear. The present study investigated the effects of the deletion of activating transcription factor 5 (ATF5) on the type 2 immune response triggered by succinate (a metabolite of parasites) in mice. ATF5 mRNAs were expressed in the small intestine, and the loss of the ATF5 gene did not affect the gross morphology of the tissue or the basal differentiation of epithelial cell subtypes. Succinate induced marked increases in tuft and goblet cell numbers in the ATF5-deficient ileum. Tuft cells in the ATF5-deficient ileum are assumed to be a subtype of intestinal tuft cells (Tuft-2 cells) marked by the transcription factor Spib. Exogenous IL-25 induced similar increases in tuft and goblet cell numbers in wild-type and ATF5-deficient ilea. IL-13 at a submaximal dose enhanced tuft cell differentiation more in ATF5-deficient than in wild-type intestinal organoids. These results indicate that the loss of ATF5 enhanced the tuft cell-ILC2 type 2 immune response circuit by promoting tuft cell differentiation in the small intestine, suggesting its novel regulatory role in immune responses against parasitic infections.

Spatiotemporal changes of tissue glycans depending on localization in cardiac aging.

Heart failure is caused by various factors, making the underlying pathogenic mechanisms difficult to identify. Since cardiovascular disease tends to worsen over time, early diagnosis is key for treatment. In addition, understanding the qualitative changes in the heart associated with aging, where information on the direct influences of aging on cardiovascular disease is limited, would also be useful for treatment and diagnosis. To fill these research gaps, the focus of our study was to detect the structural and functional molecular changes associated with the heart over time, with a focus on glycans, which reflect the type and state of cells. METHODS: We investigated glycan localization in the cardiac tissue of normal mice and their alterations during aging, using evanescent-field fluorescence-assisted lectin microarray, a technique based on lectin-glycan interaction, and lectin staining. RESULTS: The glycan profiles in the left ventricle showed differences between the luminal side (medial) and wall side (lateral) regions. The medial region was characterized by the presence of sialic acid residues. Moreover, age-related changes in glycan profiles were observed at a younger age in the medial region. The difference in the age-related decrease in the level of α-galactose stained with Griffonia simplicifolia lectin-IB4 in different regions of the left ventricle suggests spatiotemporal changes in the number of microvessels. CONCLUSIONS: The glycan profile, which retains diverse glycan structures, is supported by many cell populations, and maintains cardiac function. With further research, glycan localization and changes have the potential to be developed as a marker of the signs of heart failure.

Regulation of alcohol oxidase gene expression in methylotrophic yeast Ogataea minuta.

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of heterologous glycoproteins. However, it remained unknown how the AOX1 promoter is regulated in O. minuta. To elucidate regulation mechanisms of the AOX1 promoter, we adopted an assay system to quantitate AOX1 promoter activity using the PHO5 gene, which encodes an acid phosphatase, of Saccharomyces cerevisiae. The promoter activity assay revealed that glycerol, as well as glucose, cause strong catabolite repression of AOX1 expression in O. minuta. To investigate what factors are involved in transcription of the AOX1 promoter in O. minuta, we cloned three putative transcription factor genes, TRM1, TRM2, and MPP1, as homologues of other methylotrophic yeast species. Deletion mutants of these genes all showed decreased induction of the AOX1 promoter when methanol was added as the sole carbon source, indicating that these genes are indeed involved in AOX1 promoter regulation in O. minuta. Double deletion and constitutive expression of these transcription factor genes indicated that TRM1 and MPP1 regulate the transcription of AOX1 in the same pathway, while TRM2 regulates it in another pathway. By reverse transcription-qPCR, we also found that these two pathways compensate for each other and have crosstalk mechanisms with each other. A possible model for regulation of the AOX1 promoter in O. minuta was shown.

ATF5 deficiency causes abnormal cortical development.

Activating transcription factor 5 (ATF5) is a member of the cAMP response element binding protein (CREB)/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5-/-) mice exhibited behavioural abnormalities, including abnormal social interactions, reduced behavioural flexibility, increased anxiety-like behaviours, and hyperactivity in novel environments. ATF5-/- mice may therefore be a useful animal model for psychiatric disorders. ATF5 is highly expressed in the ventricular zone and subventricular zone during cortical development, but its physiological role in higher-order brain structures remains unknown. To investigate the cause of abnormal behaviours exhibited by ATF5-/- mice, we analysed the embryonic cerebral cortex of ATF5-/- mice. The ATF5-/- embryonic cerebral cortex was slightly thinner and had reduced numbers of radial glial cells and neural progenitor cells, compared to a wild-type cerebral cortex. ATF5 deficiency also affected the basal processes of radial glial cells, which serve as a scaffold for radial migration during cortical development. Further, the radial migration of cortical upper layer neurons was impaired in ATF5-/- mice. These results suggest that ATF5 deficiency affects cortical development and radial migration, which may partly contribute to the observed abnormal behaviours.

Functional validation of epitope-tagged ATF5 knock-in mice generated by improved genome editing of oviductal nucleic acid delivery (i-GONAD).

Activating transcription factor 5 (ATF5) is a stress-responsive transcription factor that belongs to the cAMP response element-binding protein (CREB)/ATF family, and is essential for the differentiation and survival of sensory neurons in murine olfactory organs. However, the study of associated proteins and target genes for ATF5 has been hampered due to the limited availability of immunoprecipitation-grade ATF5 antibodies. To overcome this issue, we generated hemagglutinin (HA)-tag knock-in mice for ATF5 using CRISPR/Cas9-mediated genome editing with one-step electroporation in oviducts (i-GONAD). ATF5-HA fusion proteins were detected in the nuclei of immature and some mature olfactory and vomeronasal sensory neurons in the main olfactory epithelium and vomeronasal organ, respectively, as endogenous ATF5 proteins were expressed, and some ATF5-HA proteins were found to be phosphorylated. Chromatin immunoprecipitation (ChIP) experiments revealed that ATF5-HA bound to the CCAAT/enhancer-binding protein (C/EBP)-ATF response element site in the promotor region of receptor transporting protein 1 (Rtp1), a chaperone gene responsible for proper olfactory receptor expression. These knock-in mice may be used to examine the expression, localization, and protein-protein/-DNA interactions of endogenous ATF5 and, ultimately, the function of ATF5 in vivo.

Co-expression of C/EBPγ and ATF5 in mouse vomeronasal sensory neurons during early postnatal development.

The differentiation of sensory neurons involves gene expression changes induced by specific transcription factors. Vomeronasal sensory neurons (VSNs) in the mouse vomeronasal organ (VNO) consist of two major subpopulations of neurons expressing vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo, which differentiate from a common neural progenitor. We previously demonstrated that the differentiation and survival of VSNs were inhibited in ATF5 transcription factor-deficient mice (Nakano et al. Cell Tissue Res 363:621-633, 2016). These defects were more prominent in V2r/Gαo-type than in V1r/Gαi2-type VSNs; however, the molecular mechanisms responsible for the differentiation of V2r/Gαo-type VSNs by ATF5 remain unclear. To identify a cofactor involved in ATF5-regulated VSN differentiation, we investigated the expression and function of CCAAT/enhancer-binding protein gamma (C/EBPγ, Cebpg), which is a major C/EBP family member expressed in the mouse VNO and dimerizes with ATF5. The results obtained showed that C/EBPγ mRNAs and proteins were broadly expressed in the postmitotic VSNs of the neonatal VNO, and their expression decreased by the second postnatal week. The C/EBPγ protein was expressed in the nuclei of approximately 70% of VSNs in the neonatal VNO, and 20% of the total VSNs co-expressed C/EBPγ and ATF5 proteins. We examined the trans-acting effects of C/EBPγ and ATF5 on V2r transcription and found that the co-expression of C/EBPγ and ATF5, but not C/EBPγ or ATF5 alone, increased Vmn2r66 promoter reporter activity via the C/EBP:ATF response element (CARE) in Neuro2a cells. These results suggest the role of C/EBPγ on ATF5-regulated VSN differentiation in early postnatal development.

Mating type switching, formation of diploids, and sporulation in the methylotrophic yeast Ogataea minuta.

Ogataea minuta is a methylotrophic yeast that is closely related to Ogataea (Hansenula) polymorpha. Like other methylotrophic yeasts, O. minuta also possesses strongly methanol-inducible genes, such as AOX1. We have focused on O. minuta as a host for the production of therapeutic glycoproteins. However, genetic methods, which are required for the construction of strains by breeding, have not yet been established in this organism. In this study, we investigated the O. minuta mechanisms of mating and sporulation, which would facilitate genetic analysis in this species. Specifically, we determined DNA sequences around the MAT locus in O. minuta strain NBRC 10746, and found that two MAT loci were flanked by a pair of inverted repeat sequences, as reported in O.polymorpha (Maekawa and Kaneko, PLOS Genet., 10, e1004796, 2014). As in O. polymorpha, mating type in O. minuta appears to be switched by inversion of the chromosomal region between the two MAT loci. We successfully obtained O. minuta diploid cells, which showed vegetative growth on rich medium. The size of the diploid cells was 1.3-fold larger than haploid cells of this species. Diploid cells formed ascospores, which contained 2-4 spores, under nutrient starvation conditions. Phenotypes of the resultant haploid cells exhibited Mendelian segregation, indicating that genetic approaches are applicable to O. minuta.

Lipid moiety of glycosylphosphatidylinositol-anchored proteins contributes to the determination of their final destination in yeast.

Yeasts have two classes of glycosylphosphatidylinositol (GPI)-anchored proteins; one is transferred to the cell wall, whereas the other is retained on the plasma membrane. The lipid moieties of the GPI in Saccharomyces cerevisiae consist of either phosphatidylinositol (PI) or inositolphosphorylceramide (IPC). Cwh43p is involved in the remodeling of lipid from PI to IPC. We found that the GPI lipid moiety of Cwp2p in wild-type cells is PI. To elucidate the physiological role of the lipid remodeling by Cwh43p, we investigated the distribution of Gas1p and Cwp2p by immunoblotting and found that Gas1p with the PI-form GPI lipid moiety in cwh43∆ mutant cells tends to be localized to the cell wall, suggesting that the IPC species in the GPI lipid moiety contributes to the retention of GPI-anchored proteins on the plasma membrane. We also found that CWH43 is genetically related to TED1, which encodes a protein involved in the removal of the ethanolamine phosphate from the second mannose residue in GPI glycan moieties. We propose possible models for the physiological function of Cwh43p and Ted1p in the transfer of GPI-anchored proteins from the plasma membrane to the cell wall.

Comprehensive Behavioral Analysis of Activating Transcription Factor 5-Deficient Mice.

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of basic leucine zipper transcription factors. We previously reported that ATF5-deficient (ATF5-/-) mice demonstrated abnormal olfactory bulb development due to impaired interneuron supply. Furthermore, ATF5-/- mice were less aggressive than ATF5+/+ mice. Although ATF5 is widely expressed in the brain, and involved in the regulation of proliferation and development of neurons, the physiological role of ATF5 in the higher brain remains unknown. Our objective was to investigate the physiological role of ATF5 in the higher brain. We performed a comprehensive behavioral analysis using ATF5-/- mice and wild type littermates. ATF5-/- mice exhibited abnormal locomotor activity in the open field test. They also exhibited abnormal anxiety-like behavior in the light/dark transition test and open field test. Furthermore, ATF5-/- mice displayed reduced social interaction in the Crawley's social interaction test and increased pain sensitivity in the hot plate test compared with wild type. Finally, behavioral flexibility was reduced in the T-maze test in ATF5-/- mice compared with wild type. In addition, we demonstrated that ATF5-/- mice display disturbances of monoamine neurotransmitter levels in several brain regions. These results indicate that ATF5 deficiency elicits abnormal behaviors and the disturbance of monoamine neurotransmitter levels in the brain. The behavioral abnormalities of ATF5-/- mice may be due to the disturbance of monoamine levels. Taken together, these findings suggest that ATF5-/- mice may be a unique animal model of some psychiatric disorders.

Two Alkaloids from Bulbs of Lycoris sanguinea MAXIM. Suppress PEPCK Expression by Inhibiting the Phosphorylation of CREB.

In the fasting state, gluconeogenesis is upregulated by glucagon. Glucagon stimulates cyclic adenosine monophosphate production, which induces the expression of key enzymes for gluconeogenesis, such as cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), which are involved in gluconeogenesis through the protein kinase A/cAMP response element-binding protein (CREB) pathway. Using a luciferase reporter gene assay, a methanol extract of the bulbs of Lycoris sanguinea MAXIM. var. kiushiana Makino was found to suppress cAMP-enhanced PEPCK-C promoter activity. In addition, two alkaloids, lycoricidine and lycoricidinol, in the extract were identified as active constituents. In forskolin-stimulated human hepatoma cells, these alkaloids suppressed the expression of a reporter gene under the control of cAMP response element and also prevented increases in the endogenous levels of phosphorylated CREB and PEPCK mRNA expression. These results suggest that lycoricidine and lycoricidinol suppress PEPCK-C expression by inhibiting the phosphorylation of CREB and may thus have the potential to prevent excessive gluconeogenesis in type 2 diabetes. Copyright © 2016 John Wiley & Sons, Ltd.

Activating transcription factor 5 (ATF5) is essential for the maturation and survival of mouse basal vomeronasal sensory neurons.

Activating transcription factor 5 (ATF5) is a member of the CREB/ATF family of transcription factors, which is highly expressed in olfactory chemosensory tissues, the main olfactory epithelium and vomeronasal epithelium (VNE) in mice. The vomeronasal sensory neurons in the VNE detect pheromones in order to regulate social behaviors such as mating and aggression; however, the physiological role of ATF5 in the vomeronasal sensory system remains unknown. In this study, we found that the differentiation of mature vomeronasal sensory neurons, assessed by olfactory marker protein expression, was inhibited in ATF5-deficient VNE. In addition, many apoptotic vomeronasal sensory neurons were evident in ATF5-deficient VNE. The vomeronasal sensory neurons consist of two major types of neuron expressing either vomeronasal 1 receptor (V1r)/Gαi2 or vomeronasal 2 receptor (V2r)/Gαo. We demonstrated that the differentiation, survival and axonal projection of V2r/Gαo-type rather than V1r/Gαi2-type vomeronasal sensory neurons were severely inhibited in ATF5-deficient VNE. These results suggest that ATF5 is one of the transcription factors crucial for the vomeronasal sensory formation.

Activating transcription factor 5 is required for mouse olfactory bulb development via interneuron.

Activating transcription factor 5 (ATF5) is a stress response transcription factor of the cAMP-responsive element-binding/ATF family. Earlier, we reported that ATF5 expression is up-regulated in response to stress, such as amino acid limitation or arsenite exposure. Although ATF5 is widely expressed in the brain and the olfactory epithelium, the role of ATF5 is not fully understood. Here, the olfactory bulbs (OBs) of ATF5-deficient mice are smaller than those of wild-type mice. Histological analysis reveals the disturbed laminar structure of the OB, showing the thinner olfactory nerve layer, and a reduced number of interneurons. This is mainly due to the reduced number of bromodeoxyuridine-positive proliferating cells in the subventricular zone, where the interneuron progenitors are formed and migrate to the OBs. Moreover, the olfaction-related aggressive behavior of ATF5-deficient mice is reduced compared to wild-type mice. Our data suggest that ATF5 plays a crucial role in mouse OB development via interneuron.

N-terminal hydrophobic amino acids of activating transcription factor 5 (ATF5) protein confer interleukin 1ß (IL-1ß)-induced stabilization.

Activating transcription factor 5 (ATF5) is a stress-response transcription factor that responds to amino acid limitation and exposure to cadmium chloride (CdCl2) and sodium arsenite (NaAsO2). The N-terminal amino acids contribute to the destabilization of the ATF5 protein in steady-state conditions and serve as a stabilization domain in the stress response after CdCl2 or NaAsO2 exposure. In this study, we show that interleukin 1ß (IL-1ß), a proinflammatory cytokine, increases the expression of ATF5 protein in HepG2 hepatoma cells in part by stabilizing the ATF5 protein. The N-terminal domain rich in hydrophobic amino acids that is predicted to form a hydrophobic network was responsible for destabilization in steady-state conditions and served as an IL-1ß response domain. Furthermore, IL-1ß increased the translational efficiency of ATF5 mRNA via the 5' UTRα and phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). ATF5 knockdown in HepG2 cells up-regulated the IL-1ß-induced expression of the serum amyloid A 1 (SAA1) and SAA2 genes. Our results show that the N-terminal hydrophobic amino acids play an important role in the regulation of ATF5 protein expression in IL-1ß-mediated immune response and that ATF5 is a negative regulator for IL-1ß-induced expression of SAA1 and SAA2 in HepG2 cells.

The 5'-untranslated region regulates ATF5 mRNA stability via nonsense-mediated mRNA decay in response to environmental stress.

We previously reported that activating transcription factor 5 (ATF5) mRNA increases in response to amino acid limitation, and that this increase is dependent on mRNA stabilization. The ATF5 gene allows transcription of mRNAs with two alternative 5'-UTRs, 5'-UTRα and 5'-UTRß, derived from exon 1α and exon 1ß. 5'-UTRα contains the upstream open reading frames uORF1 and uORF2. Phosphorylation of eukaryotic initiation factor 2α during the integrated stress response had been previously shown to lead to bypassing of uORF2 translation and production of ATF5 protein. Translation of uORF2 is expected to result in translational termination at a position 125 nucleotides upstream of the exon junction, and this fits the criterion of a nonsense-mediated decay target mRNA. We investigated the potential role of 5'-UTRα in the control of mRNA stabilization, and found that 5'-UTRα reduced the stability of ATF5 mRNA. 5'-UTRα-regulated destabilization of mRNA was suppressed by knockdown of the nonsense-mediated decay factors Upf1 and Upf2. Mutation of the downstream AUG (uAUG2) rendered mRNA refractory to Upf1 and Upf2 knockdown. Moreover, 5'-UTRα-regulated down-regulation was hindered by amino acid limitation and tunicamycin treatment, and stress-induced phosphorylation of eukaryotic initiation factor 2α was involved in stabilization of ATF5 mRNA. These studies show that ATF5 mRNA is a naturally occurring normal mRNA target of nonsense-mediated decay, and provide evidence for linkage between stress-regulated translational regulation and the mRNA decay pathway. This linkage constitutes a mechanism that regulates expression of stress response genes.

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast.

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)-anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI-anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A2 Per1p and O-acyltransferase Gup1p play essential roles in diacylglycerol-type lipid remodelling of GPI-anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphoryl-ceramide by Cwh43p. In this report, we constructed double-mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI-anchored proteins. Based on our analyses of single- and double-mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso-phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag-tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.

Phenolic diterpenes from rosemary suppress cAMP responsiveness of gluconeogenic gene promoters.

The cAMP/protein kinase A/cAMP response element (CRE)-binding protein pathway is important for various physiological aspects including regulation of gluconeogenic gene expression. Rosemary, a well-known herb, has been reported to decrease blood glucose levels. We found that methanol extracts of rosemary suppressed forskolin (FSK)-stimulated luciferase expression under the control of CRE, as well as the promoters for cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and glucose-6-phosphatase (G6Pase) catalytic subunit genes in human hepatoma HepG2 cells. Three abietane-type diterpenes and two flavonoids were isolated from the rosemary extracts. Among these, 7-O-methylrosmanol (1) and royleanonic acid (3) effectively suppressed FSK-induced luciferase expression under the control of the CRE, PEPCK-C and G6Pase gene promoters. PEPCK-C and G6Pase, which play a key role in the homeostatic regulation of blood glucose levels, are important for managing type II diabetes mellitus. Therefore, the ability of rosemary and its components to suppress cAMP responsiveness of the PEPCK-C or G6Pase gene may contribute to its antihyperglycemic activity.

Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane.

Porcine pancreatic α-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680-4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na(+)/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycan-specific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic α-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic α-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other.

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p.

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.