Identity of neutral alpha-glucosidase AB and the glycoprotein processing enzyme glucosidase II. Biochemical and genetic studies

We have previously partially purified, characterized, and chromosomally mapped a human isozyme of alpha-glucosidase which is active at neutral pH. This isozyme appears as a doublet of enzyme activity on native gel electrophoresis and was termed neutral alpha-glucosidase AB. We now report genetic and biochemical evidence that neutral alpha-glucosidase AB is synonymous with the glycoprotein processing enzyme glucosidase II. We have found that a mutant mouse lymphoma line which is deficient in glucosidase II is also deficient in neutral alpha-glucosidase AB, as defined electrophoretically and quantitatively (less than 0.5% of parental). In contrast, both mutant and parental cell lines exhibited several lysosomal hydrolases which are processed by glucosidase II. We have also further purified the human neutral alpha-glucosidase A component of neutral alpha-glucosidase AB 740-fold from placenta in order to compare its biochemical properties with those described for rat liver and pig kidney glucosidase II. Both glucosidase II and neutral alpha-glucosidase AB are high-molecular mass (greater than 200,000 dalton) anionic glycoproteins which bind to concanavalin A, have a broad pH optima (5.5-8.5), and have a similar Km for maltose (4.8 versus 2.1 mM) and the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (35 versus 19 microM). Similar to human neutral alpha-glucosidase AB, purified rat glucosidase II migrates as a doublet of enzyme activity on native gel electrophoresis. Although rat glucosidase II has been reported to have a subunit size of 67 kDa, pig glucosidase II has been found to have a subunit size of 100 kDa, like the 98-kDa major protein in purified human neutral alpha-glucosidase A. Although we have not demonstrated that neutral alpha-glucosidase AB is microsomal nor that it hydrolyzes the natural substrate of glucosidase II, we believe that the genetic evidence is compelling for and the biochemical data consistent with the hypothesis that neutral alpha-glucosidase AB and glucosidase II are synonymous. These and previous results would localize glucosidase II to the long arm of human chromosome II.     

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.     

Acarbose, a pseudooligosaccharide, is transported but not metabolized by the maltose-maltodextrin system of Escherichia coli

The pseudooligosaccharide acarbose is a potent inhibitor of amylases, glucosidases, and cyclodextrin glycosyltransferase and is clinically used for the treatment of so-called type II or insulin-independent diabetes. The compound consists of an unsaturated aminocyclitol, a deoxyhexose, and a maltose. The unsaturated aminocyclitol moiety (also called valienamine) is primarily responsible for the inhibition of glucosidases. Due to its structural similarity to maltotetraose, we have investigated whether acarbose is recognized as a substrate by the maltose/maltodextrin system of Escherichia coli. Acarbose at millimolar concentrations specifically affected the growth of E. coli K-12 on maltose as the sole source of carbon and energy. Uptake of radiolabeled maltose was competitively inhibited by acarbose, with a Ki of 1.1 microM. Maltose-grown cells transported radiolabeled acarbose, indicating that the compound is recognized as a substrate. Studying the interaction of acarbose with purified maltoporin in black lipid membranes revealed that the kinetics of acarbose binding to LamB is asymmetric. The on-rate of acarbose is approximately 30 times lower when the molecule enters the pore from the extracellular side than when it enters from the periplasmic side. Acarbose could not be utilized as a carbon source since the compound alone was not a substrate of amylomaltase (MalQ) and was only poorly attacked by maltodextrin glucosidase (MalZ).     

Cell wall 1,6-beta-glucan synthesis in Saccharomyces cerevisiae depends on ER glucosidases I and II, and the molecular chaperone BiP/Kar2p.

The role of glucose trimming in the endoplasmic reticulum of Saccharomyces cerevisiae was investigated using glucosidase inhibitors and mutant strains devoid of glucosidases I and II. These glucosidases are responsible for removing glucose residues from the N-linked core oligosaccharides attached to newly synthesized polypeptide chains. In mammalian cells they participate together with calnexin, calreticulin and UDP-glucose:glycoprotein glucosyltransferase in the folding and quality control of newly synthesized glycoproteins. In S.cerevisiae, glucosidase II is encoded by the GLS2 gene, and glucosidase I, as suggested here, by the CWH41 gene. Using castanospermine (an alpha-glucosidase inhibitor) and yeast strains defective in glucosidase I, glucosidase II and BiP/Kar2p, it was demonstrated that cell wall synthesis depends on the two glucosidases and BiP/Kar2p. In double mutants with defects in both BiP/Kar2p and either of the glucosidases the phenotype was particularly clear: synthesis of 1,6-beta-glucan_a cell wall component_was reduced; the cell wall displayed abnormal morphology; the cells aggregated; and their growth was severely inhibited. No defects in protein folding or secretion could be detected. We concluded that glucose trimming in S.cerevisiae is necessary for proper cell wall synthesis, and that the glucosidases function synergistically with BiP/Kar2p in this process.     

Purification by affinity chromatography of glucosidase I, an endoplasmic reticulum hydrolase involved in the processing of asparagine-linked oligosaccharides

Trimming glucosidase I and II have been solubilized from crude calf liver microsomes and partially enriched by a fractionated extraction procedure applying different concentrations of nonionic detergent and salt. The pH optimum of both enzymes was found to be close to 6.2, which discriminates them from hydrolases of lysosomal origin acting on p-nitrophenyl glycosides with the highest rate at more acidic pH. Glucosidase I and II and the nonspecific alpha-glucosidase(s) were inhibited by 1-deoxynojirimycin with median inhibitory concentration of 3 microM, 20 microM, 12 microM, respectively. Discrimination between these enzymes was strongly enhanced by N-alkylation of 1-deoxynojirimycin and formed the basis for the design of the affinity ligand. Glucosidase I has been purified to homogeneity by affinity chromatography on AH-Sepharose 4B with N-carboxypentyl-1-deoxynojirimycin as ligand. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a subunit molecular mass of about 85 kDa. The molecular mass of the native enzyme, determined by gel chromatography, was approximately equal to 320-350 kDa, pointing to the association of subunits to a tetramer. Glucosidase I is rather stable when stored at 4 degrees C in the presence of detergent (t 1/2 approximately equal to 20 days) and showed high specificity for the hydrolysis of the terminal (alpha 1,2)-linked glucose residue in the natural substrate Glc3-Man9-(GlcNAc)2.     

Latency of some glycosidases of rat liver lysosomes.

The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited.     

Effects of castanospermine on purified lysosomal alpha-1,4-glucosidase

The indolizidine alkaloid castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) inhibits hydrolysis of maltose, glycogen and isomaltose by purified lysosomal alpha-glucosidase yielding Ki values of 0.095, 0.10 and 0.30 mumol/l, respectively. Castanospermine exhibited high affinity for both the maltose and isomaltose sites. In distinct contrast, the alkaloid exhibited little or no affinity for the site catalyzing hydrolysis of glycogen as indicated by a noncompetitive mode of inhibition. Kinetic data presented in this report indicate castanospermine to be a very potent inhibitor of lysosomal alpha-glucosidase.     

Glycosidase inhibitors as antiviral and/or antitumor agents.

Glycoprotein processing inhibitors prevent the normal processing of N-linked glycoproteins by inhibiting specific glycosidases involved in these reactions. Thus, a number of compounds are now known that inhibit alpha-glucosidase I and alpha-glucosidase II and therefore prevent the removal of glucoses from the high-mannose chains. Some of these compounds are more potent inhibitors of one or the other of these glucosidases. There are also a number of inhibitors that affect one of the processing alpha-mannosidases (i.e. mannosidase I or mannosidase II). These compounds; especially the glucosidase inhibitors, have been valuable tools to help us understand the role of carbohydrate in viral envelope glycoprotein function. Such processing inhibitors have also been used with various tumorigenic cell lines to determine the function of N-linked glycoproteins in cancer.     

Effect of five-membered sugar mimics on mammalian glycogen-degrading enzymes and various glucosidases

We investigated inhibitory activities of five-membered sugar mimics toward glycogen-degrading enzymes and a variety of glucosidases. 1,4-Dideoxy-1,4-imino-D-arabinitol (D-AB1) is known to be a potent inhibitor of glycogen phosphorylase. However, the structural modification of D-AB1, such as its enantiomerization, epimerization at C-2 and/or C-3, introduction of a substituent to C-1, and replacement of the ring nitrogen by sulfur, markedly lowered or abolished its inhibition toward the enzyme. The present work elucidated that d-AB1 was also a good inhibitor of the de-branching enzyme of glycogen, amylo-1,6-glucosidase, with a IC(50) value of 8.4 microM. In the present work, the de-sulfonated derivative of salacinol was isolated from the roots of Salacia oblonga and found to be a potent inhibitor of rat intestinal isomaltase with an IC(50) value of 0.64 microM. On the other hand, salacinol showed a much more potent inhibitory activity toward maltase in Caco-2 cell model system than its de-sulfonated derivative, with an IC(50) value of 0.5 microM, and was further a stronger inhibitor of human lysosomal alpha-glucosidase than the derivative (IC(50)=0.34 microM). This indicates that the sulfate in the side chain plays an important role in the specificity of enzyme inhibition.     

Synthesis of an alpha-fucosidase inhibitor, 5a-carba-beta-L-fucopyranosylamine, and fucose-type alpha- and beta-DL-valienamine unsaturated derivatives

Discovery of a very potent alpha-fucosidase inhibitor 5a-carba-alpha-L-fucopyranosylamine led to preparation of its beta-anomer and the respective unsaturated derivatives, fucose-type alpha- and beta-valienamines, in order to elucidate the structure-activity relationship of carba-aminosugar inhibitors of this kind. Compound was demonstrated to be a potent inhibitor (K(i)=2.0 x 10(-7) M, bovine kidney), possessing ca. one-tenth of the activity of the parent. Interestingly, and were found to be rather weak inhibitors, contrary to the expectations based on the activity relationships between the alpha-glucosidase inhibitors, alpha-glucose-type validamine and valienamine.     

Inhibition of mouse liver sialidase by plant flavonoids

Flavonoids (103 species) were tested for inhibitory activity against mouse liver sialidase using sodium p-nitrophenyl-N-acetyl-alpha-D-neuraminate (PNP-NeuAc) as substrate. Isoscutellarein-8-O-glucuronide from the leaf of Scutellaria baicalensis showed most potent activity (IC50, 40 microM), and this flavone appeared to be a non-competitive inhibitor of the enzyme. This flavone inhibited the lysosomal solubilized sialidase against PNP-NeuAc and sialyllactose effectively, but not microsomal enzyme against gangliosides and colominic acid, whereas, negligible or weak inhibitory activities were observed for influenza virus sialidase, beta-galactosidase, alpha-mannosidase, and alpha-glucosidase tested. These results indicate that this flavone may be useful to elucidate the function of the lysosomal solubilized sialidase.     

Some properties of human liver acid alpha-glucosidase.

1. Albumin activates human liver acid alpha-glucosidase (alpha-D-glucoside hydrolase, EC 3.2.1.20). From the Arrhenius plot, pH-dependence and Lineweaver-Burk plots it can be concluded that this activation is not only due to stabilisation of the enzyme, but also influences the enzymatic activity. It is proposed that for optimal functioning human liver acid alpha-glucosidase needs a protein environment. 2. Glycogen has a competitive inhibitory effect on the hydrolysis of 4-methylumbelliferyl-alpha-D-glucopyranoside, in contrast to maltose which exhibits a non-competitive type of inhibition. It is concluded that two catalytic sites exist, one for glycogen and one for maltose, while both sites influence each other. With glycogen as substrate a break in the Arrhenius plot is found. This is not the case when maltose is used as substrate. 3. The effect of antibody raised against human liver acid alpha-glucosidase on the activity of human liver acid alpha-glucosidase is studied. No corss-reacting material could be demonstrated in the liver of a patient with glycogen storage disease Type II (M. Pompe, acid alpha-glucosidase deficiency).     

A novel amylolytic enzyme from Thermotoga maritima,resembling cyclodextrinase and alpha-glucosidase,that liberates glucose from the reducing end of the substrates

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.     

Altering the substrate chain-length specificity of an alpha-glucosidase

Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 alpha-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase.     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

Comparative study of glycosidase from cattle liver and exoglycanase from Aspergillus awamori]

Anomerities of products were estimated for glucosidases from cattle liver and Aspergillus awamori. It was demonstrated that the enzyme from cattle liver is alpha-glucosidase and that from Asp. awamori is exogluconase. It was demonstrated that alpha-glucosidase hydrolyzes the C1--O bond in the course of reaction. delta-Lactone of gluconic acid is a competitive inhibitor for both enzymes. The secondary kinetic isotope effects for both enzymes were measured. The isotope effect for alpha-glucosidase is equal to 1, for exogluconase 1,1 for glycogen and 1,18 for maltose. Some aspects of mechanisms of both enzymes are discussed in terms of the data obtained.     

N-linked oligosaccharide processing enzyme glucosidase II produces 1,5-anhydrofructose as a side product

alpha-1,4-Glucan lyase cleaves alpha-1,4-linkages of nonreducing termini of alpha-1,4-glucans to produce 1,5-anhydrofructose (1,5-AnFru). The enzymes isolated from fungi and algae show high homology with glycoside hydrolase family 31. Purification of alpha-1,4-glucan lyase from rat liver using DEAE Cellulose chromatography resulted in separation of two enzymatic active fractions, one was bound to the column and the other was in the flow-through. Partial amino acid sequence determined from the lyase, retained on the anion exchange column, were identical with that of the N:-linked oligosaccharide processing enzyme glucosidase II. The lyase showed similar enzymatic properties as the microsomal glucosidase such as inhibition by 1-deoxynojirimycin and castanospermine. On the other hand, glucosidase II purified from rat liver microsomes produced not only glucose but also a small amount of 1,5-AnFru using maltose as substrate. Furthermore, CHO cells overexpressing pig liver glucosidase II showed a 1.5- to 2-fold higher lyase activity compared to the nontransfected CHO cells. Conversely, no lyase activity was detectable either in PHAR2.7, the glucosidase II-deficient mutant from a mouse lymphoma cell line, or in Saccharomyces cerevisiae strain YG427 having the glucosidase II gene disrupted. These data demonstrate that glucosidase II possesses an additional enzymatic activity of releasing 1,5-AnFru from maltose.     

Plantagoside, a novel alpha-mannosidase inhibitor isolated from the seeds of Plantago asiatica, suppresses immune response

A hot-water extract from the seed of Plantago asiatica showed a potent inhibitory activity against jack bean alpha-mannosidase, and a flavanone glucoside, plantagoside, was isolated as the inhibitor. Plantagoside was a specific inhibitor for jack bean alpha-mannosidase (IC50 at 5 microM) and appeared to be a non-competitive inhibitor of the enzyme. Whereas, negligible or weak inhibitory activities were observed for beta-mannosidase, beta-glucosidase, and sialidase tested. Plantagoside also inhibited alpha-mannosidase activities in mouse liver lysosomal and microsomal fractions, and the enzyme inhibitory activity in microsomal fraction was enhanced in the presence of glucosidase inhibitor, castanospermine. Plantagoside suppressed antibody response to sheep red blood cells and concanavalin A induced lymphocyte proliferation which was measured by [3H]thymidine incorporation.     

Further observations on the activation of lysosomal acid α-glucosidase by cortisone derivatives

1. Cortisone acetate activates the acid alpha-glucosidase in rat liver slices and in isolated liver lysosomes. 2. The reaction is steroid specific and moreover does not occur with lysosomal acid phosphatase or beta-galactosidase. 3. After pretreatment of the lysosomes with cortisone, substrate (maltose) binding to the soluble lysosomal acid alpha-glucosidase is not affected, but the steroid does increase the V(max.) value. Membrane-bound enzyme is not activated by cortisone. 4. 4-[(14)C]Cortisone is preferentially bound to the lysosomal membrane and the possible involvement of this structure in the activation phenomenon is discussed.     

Alpha-glucosidase inhibitory activity of Syzygium cumini (Linn.) Skeels seed kernel in vitro and in Goto-Kakizaki (GK) rats

Syzygium cumini seed kernel extracts were evaluated for the inhibition of alpha-glucosidase from mammalian (rat intestine), bacterial (Bacillus stearothermophilus), and yeast (Saccharomyces cerevisiae, baker's yeast). In vitro studies using the mammalian alpha-glucosidase from rat intestine showed the extracts to be more effective in inhibiting maltase when compared to the acarbose control. Since acarbose is inactive against both the bacterial and the yeast enzymes, the extracts were compared to 1-deoxynojirimycin. We found all extracts to be more potent against alpha-glucosidase derived from B. stearothermophilus than that against the enzymes from either baker's yeast or rat intestine. In an in vivo study using Goto-Kakizaki (GK) rats, the acetone extract was found to be a potent inhibitor of alpha-glucosidase hydrolysis of maltose when compared to untreated control animals. Therefore, these results point to the inhibition of alpha-glucosidase as a possible mechanism by which this herb acts as an anti-diabetic agent.