Ergosteryl-ß-glucosidase (Egh1) involved in sterylglucoside catabolism and vacuole formation in Saccharomyces cerevisiae.

Sterylglucosides (SGs) are composed of a glucose and sterol derivatives, and are distributed in fungi, plants and mammals. We recently identified EGCrP1 and EGCrP2 (endoglycoceramidase-related proteins 1 and 2) as a ß-glucocerebrosidase and steryl-ß-glucosidase, respectively, in Cryptococcus neoformans. We herein describe an EGCrP2 homologue (Egh1; ORF name, Yir007w) involved in SG catabolism in Saccharomyces cerevisiae. The purified recombinant Egh1 hydrolyzed various ß-glucosides including ergosteryl ß-glucoside (EG), cholesteryl ß-glucoside, sitosteryl ß-glucoside, para-nitrophenyl ß-glucoside, 4-methylumberifellyl ß-glucoside and glucosylceramide. The disruption of EGH1 in S. cerevisiae BY4741 (egh1Δ) resulted in the accumulation of EG and fragmentation of vacuoles. The expression of EGH1 in egh1Δ (revertant) reduced the accumulation of EG, and restored the morphology of vacuoles. The accumulation of EG was not detected in EGH1 and UGT51(ATG26) double-disrupted mutants (ugt51Δegh1Δ), indicating that EG was synthesized by Ugt51(Atg26) and degraded by Egh1 in vivo. These results clearly demonstrated that Egh1 is an ergosteryl-ß-glucosidase that is functionally involved in the EG catabolic pathway and vacuole formation in S. cerevisiae.

Physiology of digestion and the molecular characterization of the major digestive enzymes from Periplaneta americana.

Cockroaches are among the first insects to appear in the fossil record. This work is part of ongoing research on insects at critical points in the evolutionary tree to disclose evolutionary trends in the digestive characteristics of insects. A transcriptome (454 Roche platform) of the midgut of Periplanetaamericana was searched for sequences of digestive enzymes. The selected sequences were manually curated. The complete or nearly complete sequences showing all characteristic motifs and highly expressed (reads counting) had their predicted sequences checked by cloning and Sanger sequencing. There are two chitinases (lacking mucin and chitin-binding domains), one amylase, two α- and three ß-glucosidases, one ß-galactosidase, two aminopeptidases (none of the N-group), one chymotrypsin, 5 trypsins, and none ß-glucanase. Electrophoretic and enzymological data agreed with transcriptome data in showing that there is a single ß-galactosidase, two α-glucosidases, one preferring as substrate maltase and the other aryl α-glucoside, and two ß-glucosidases. Chromatographic and enzymological data identified 4 trypsins, one chymotrypsin (also found in the transcriptome), and one non-identified proteinase. The major digestive trypsin is identifiable to a major P. americana allergen (Per a 10). The lack of ß-glucanase expression in midguts was confirmed, thus lending support to claims that those enzymes are salivary. A salivary amylase was molecularly cloned and shown to be different from the one from the midgut. Enzyme distribution showed that most digestion occurs under the action of salivary and midgut enzymes in the foregut and anterior midgut, except the posterior terminal digestion of proteins. A counter-flux of fluid may be functional in the midgut of the cockroach to explain the low excretory rate of digestive enzymes. Ultrastructural and immunocytochemical localization data showed that amylase and trypsin are released by both merocrine and apocrine secretion mainly from gastric caeca. Finally, a discussion on Polyneoptera digestive physiology is provided.

Hepatocystin/80K-H inhibits replication of hepatitis B virus through interaction with HBx protein in hepatoma cell.

Hepatitis B virus (HBV) X protein (HBx) is a key player in HBV replication as well as HBV-induced hepatocellular carcinoma (HCC). However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, a combination of affinity purification and mass spectrometry was applied to identify the host factors interacting with HBx in hepatoma cells. Thirteen proteins were identified as HBx binding partners. Among them, we first focused on determining the functional significance of the interaction between HBx and hepatocystin. A physical interaction between HBx and hepatocystin was confirmed by co-immunoprecipitation and Western blotting. Immunocytochemistry demonstrated that HBx and hepatocystin colocalized in the hepatoma cells. Domain mapping of both proteins revealed that the HBx C-terminus (amino acids 110-154) was responsible for binding to the mannose 6-phosphate receptor homology domain (amino acids, 419-525) of hepatocystin. Using translation and proteasome inhibitors, we found that hepatocystin overexpression accelerated HBx degradation via a ubiquitin-independent proteasome pathway. We demonstrated that this effect was mediated by an interaction between both proteins using a HBx deletion mutant. Hepatocystin overexpression significantly inhibited HBV DNA replication and expression of HBs antigen concomitant with HBx degradation. Using the hepatocystin mutant constructs that bind HBx, we also confirmed that hepatocystin inhibited HBx-dependent HBV replication. In conclusion, we demonstrated for the first time that hepatocystin functions as a chaperon-like molecule by accelerating HBx degradation, and thereby inhibits HBV replication. Our results suggest that inducing hepatocystin may provide a novel therapeutic approach to control HBV infection.

Active Ca(2+) reabsorption in the connecting tubule.

The kidney plays a crucial role in the maintenance of the body calcium (Ca(2+)) balance. Ca(2+) is an essential ion in all organisms and participates in a large variety of structural and functional processes. In mammals, active tubular Ca(2+) reabsorption is restricted to the distal part of the nephron, i.e., the late distal convoluted (DCT2) and the connecting tubules (CNT), where approximately 10-15% of the total Ca(2+) is reabsorbed. This active transcellular transport is hallmarked by the transient receptor potential vanilloid 5 (TRPV5) epithelial Ca(2+) channel, regulated by an array of events, and mediated by hormones, including 1,25-dihydroxyvitamin D(3), parathyroid hormone, and estrogen. Novel molecular mechanisms have been identified, such as the direct regulatory effects of klotho and tissue kallikrein on the abundance of TRPV5 at the apical membrane. The newly discovered mechanisms could provide potential pharmacological targets in the therapy of renal Ca(2+) wasting. This review discusses the three basic molecular steps of active Ca(2+) reabsorption in the DCT/CNT segments of the nephron, including apical entry, cytoplasmic transport, and basolateral extrusion of Ca(2+). In addition, an overview of the recently identified mechanisms governing this active Ca(2+) transport through the DCT2/CNT epithelial cells will be presented.

A transglucosidase necessary for starch degradation and maltose metabolism in leaves at night acts on cytosolic heteroglycans (SHG).

The recently characterized cytosolic transglucosidase DPE2 (EC 2.4.1.25) is essential for the cytosolic metabolism of maltose, an intermediate on the pathway by which starch is converted to sucrose at night. In in vitro assays, the enzyme utilizes glycogen as a glucosyl acceptor but the in vivo acceptor molecules remained unknown. In this communication we present evidence that DPE2 acts on the recently identified cytosolic water-soluble heteroglycans (SHG) as does the cytosolic phosphorylase (EC 2.4.1.1) isoform. By using in vitro two-step (14)C labeling assays we demonstrate that the two transferases can utilize the same acceptor sites of the SHG. Cytosolic heteroglycans from a DPE2-deficient Arabidopsis mutant were characterized. Compared with the wild type the glucose content of the heteroglycans was increased. Most of the additional glucosyl residues were found in the outer chains of SHG that are released by an endo-alpha-arabinanase (EC 3.2.1.99). Additional starch-related mutants were characterized for further analysis of the increased glucosyl content. Based on these data, the cytosolic metabolism of starch-derived carbohydrates is discussed.

[Key enzymes in starch synthesis in plants].

Starch, the most common form of stored carbon in plants, is both the major food source for mankind and important raw material for many industries. It is composed of two types of alpha-1,4-linked glucan polymer: essentially unbranched amylose and regularly branched amylopectin, and synthesized in photosynthetic and non-photosynthetic organs. Starch is synthesized via four committed enzyme steps: ADP-Glc pyrophosphorylase, which synthesizes sugar nucleotide precursors; starch synthase, which extends the alpha-1,4-linked glucan chains using ADP-Glc; starch-branching enzymes, which introduce alpha-1,6 branch points to form amylopectin; and starch debranching enzymes, which hydrolyze alpha-1,6 branches in glucans. In this paper, recent advances in biochemical characterizations and gene engineering concerning these enzymes were reviewed, and the achievements in gene engineering involved in manipulation of starch amount and quality were also cited.

Structural insights into substrate specificity and function of glucodextranase.

A glucodextranase (iGDase) from Arthrobacter globiformis I42 hydrolyzes alpha-1,6-glucosidic linkages of dextran from the non-reducing end to produce beta-D-glucose via an inverting reaction mechanism and classified into the glycoside hydrolase family 15 (GH15). Here we cloned the iGDase gene and determined the crystal structures of iGDase of the unliganded form and the complex with acarbose at 2.42-A resolution. The structure of iGDase is composed of four domains N, A, B, and C. Domain A forms an (alpha/alpha)(6)-barrel structure and domain N consists of 17 antiparallel beta-strands, and both domains are conserved in bacterial glucoamylases (GAs) and appear to be mainly concerned with catalytic activity. The structure of iGDase complexed with acarbose revealed that the positions and orientations of the residues at subsites -1 and +1 are nearly identical between iGDase and GA; however, the residues corresponding to subsite 3, which form the entrance of the substrate binding pocket, and the position of the open space and constriction of iGDase are different from those of GAs. On the other hand, domains B and C are not found in the bacterial GAs. The primary structure of domain C is homologous with a surface layer homology domain of pullulanases, and the three-dimensional structure of domain C resembles the carbohydrate-binding domain of some glycohydrolases.

Recent biochemical approaches to post-testicular, epididymal contraception.

Results from recent animal models with implications for putative human male contraceptives acting on the epididymis are reviewed. Inducing sterility by enhancing sperm transport through the epididymis has not been achieved. The induction of infertility in males of several species is easier to achieve by direct actions of drugs on sperm function (e.g. inhibition of sperm-specific isoenzymes of the glycolytic pathway by chloro-compounds) than by indirectly reducing amounts of epididymal secretions normally present in high concentration (e.g. alpha-glucosidase, L-carnitine). The former show promise for the clinic since human spermatozoa are susceptible to inhibition. On the other hand, the infertile male mice of the c-ros knock-out model demonstrate the influence of even a small region of the epididymis on fertility, so that targeting the as yet unknown epididymal factors presumably secreted in limiting amounts by this epididymal segment, is a new lead for a contraceptive. Targeting a specific sperm protein acquired in the testis, but depleted in the epididymis by toxicants that induce rapid infertility, may also lead to the discovery of new contraceptives, but these will require developing new means of organ-specific delivery of contraceptive drugs.

Investigation of the intracellular transport of tyrosinase and tyrosinase related protein (TRP)-1. The effect of endoplasmic reticulum (ER)-glucosidases inhibition.

Melanin biosynthesis is completely inhibited in the B16 melanoma cells following their incubation with inhibitors of the two ER glucosidases. This is primarily due to the inactivation of tyrosinase. Under the same conditions, the DOPA-oxidase activity of TRP-1 was only partially affected. In this report we investigate the effects of the perturbation of N-glycan processing in ER on the transport and activation of tyrosinase and TRP-1. We have localized the DOPA-oxidase activity in normal and inhibited cells and suggest that the first DOPA-reactive compartment of the secretory pathway (trans Golgi network) is also the site of tyrosinase activation. The inhibition of N-glycan processing does not affect the intracellular trafficking of the two melanogenic enzymes that are correctly transported to melanosomes. Immunoprecipitation experiments followed by analysis in SDS-PAGE under non-reducing conditions suggest that in inhibited cells, both tyrosinase and TRP-1 are synthesized in a modified conformation as compared to the normal proteins. These data suggest that the inhibition of melanin synthesis is not due to a defective transport but rather to conformational changes induced in the structure of tyrosinase and TRP-1 during their transit through the ER.

Inhibitors of N-linked oligosaccharide processing glucosidases interfere with oligodendrocyte differentiation in culture.

Previous studies have demonstrated that inhibitors of glycoprotein processing glucosidases interfere with the development of oligodendrocyte properties in primary cultures of embryonic rat brain cells (Bhat, J Neurosci Res 20:158-164, 1988). The present study examines the effect of castanospermine, an inhibitor of the processing glucosidases, on the development and differentiation of isolated oligodendrocyte progenitor cells. Treatment of oligodendrocyte progenitors with castanospermine did not affect the developmental progression of the precursors to become committed oligodendrocytes as revealed by comparable increases in the percentages of cells positive for galactocerebroside (a surface marker for terminally differentiated oligodendrocytes) in control and drug-treated cultures. On the other hand, there was an impairment of the expression of differentiated properties of oligodendrocytes [i.e., sulfolipid synthesis, myelin basic protein (MBP)] and 2'3'-cyclic nucleotide 3'-phosphohydrolase in the drug-treated cultures. Immunocytochemical analysis with anti-MBP antibodies revealed a reduced number of MBP-positive cells in inhibitor-treated cultures. Furthermore, a majority of MBP-positive cells in such cultures displayed immunoreactive MBP in their cell body and not the processes, unlike in control cultures where both cell body and the processes of oligodendrocytes stained intensely for MBP. The strong inhibitory effect of castanospermine on the expression of oligodendrocyte-specific activities was contrasted with a relatively smaller effect of swainsonine, a mannosidase inhibitor on oligodendrocyte differentiation. Both castanospermine and swainsonine, however, effectively blocked the formation of complex-type oligosaccharides, suggesting thereby a lack of correlation between the inhibition of the formation of complex-type oligosaccharides and oligodendrocyte differentiation. It is suggested, therefore, that early trimming reactions involving the removal of glucose residues from the high mannose oligosaccharides in the endoplasmic reticulum may be essential for the cell surface localization and function of glycoproteins critically involved in surface interactions of oligodendrocytes with each other and/or with the substratum.

Effect of inhibitors of N-linked oligosaccharide processing on the cell surface expression of a melanoma integrin.

The role of trimming and processing of N-linked oligosaccharides on the cell surface expression of the melanoma vitronectin receptor, a member of the integrin family of cell adhesion receptors, was examined by using specific glucosidase and mannosidase inhibitors. Inhibition of glucosidases I and II by castanospermine or N-methyldeoxynojirimycin delayed the vitronectin receptor alpha/beta chain heterodimer assembly and alpha chain cleavage and resulted in a decrease in the level of expression cell surface receptor. Conversely, the vitronectin receptor synthesized in the presence of the mannosidase I and II inhibitors, 1-deoxymannojirimycin and swainsonine, was transported normally to the cell surface with its alpha chain N-linked oligosaccharides in an endoglycosidase H-sensitive form. In the presence of swainsonine, time course studies of the cell surface replacement of control, endoglycosidase H-resistant receptor with an endoglycosidase H-sensitive form demonstrated a vitronectin receptor half-life of approximately 15-16 h. These studies provide evidence that the rates of assembly, proteolytic cleavage, and cell surface expression of the melanoma vitronectin receptor are dependent on the initial trimming of glucosyl residues from the alpha chain N-linked oligosaccharides.