Two novel aminooligosaccharides isolated from the culture of Streptomyces coelicoflavus ZG0656 as potent inhibitors of alpha-amylase

Two novel aminooligosaccharides were separated from the culture filtrate of Streptomyces coelicoflavus ZG0656. Their chemical structures were determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and 2D nuclear magnetic resonance (NMR) spectroscopy. Because of their acarviosine core structures, the names acarviostatins II23 and II13 were given to the novel compounds. The two acarviostatins were both mixed noncompetitive inhibitors of porcine pancreatic alpha-amylase (PPA), with inhibition constants (K(i)) of 0.009 microM (acarviostatin II23) and 0.010 microM (acarviostatin II13). Therefore, acarviostatin II23 and acarviostatin II13 are, respectively, 231 and 208 times more potent than acarbose.     

Structures of human pancreatic α-amylase in complex with acarviostatins: Implications for drug design against type II diabetes

Human pancreatic α-amylase (HPA) catalyzes the hydrolysis of α-d-(1,4) glycosidic linkages in starch and is one of the major therapeutic targets for type II diabetes. Several acarviostatins isolated from Streptomyces coelicoflavus var. nankaiensis previously showed more potent inhibition of HPA than acarbose, which has been successfully used in clinical therapy. However, the molecular mechanisms by which acarviostatins inhibit HPA remains elusive. Here we determined crystal structures of HPA in complexes with a series of acarviostatin inhibitors (I03, II03, III03, and IV03). Structural analyses showed that acarviostatin I03 undergoes a series of hydrolysis and condensation reactions in the HPA active site, similar to acarbose, while acarviostatins II03, III03, and IV03 likely undergo only hydrolysis reactions. On the basis of structural analysis combined with kinetic assays, we demonstrate that the final modified product with seven sugar rings is best suited for occupying the full active site and shows the most efficient inhibition of HPA. Our high resolution structures reported here identify first time an interaction between an inhibitor and subsite-4 of the HPA active site, which we show makes a significant contribution to the inhibitory effect. Our results provide important information for the design of new drugs for the treatment of type II diabetes or obesity.     

Taxonomy of the Streptomyces strain ZG0656 that produces acarviostatin α-amylase inhibitors and analysis of their effects on blood glucose levels in mammalian systems

Aims:  To clarify the taxonomic status of strain ZG0656 and analyse the effects of its acarviostatin products on blood glucose levels in mammalian systems.
Methods and Results:  Our program to screen for new α-amylase inhibitors led to the isolation of strain ZG0656. The polyphasic taxonomic study revealed that strain ZG0656 represents a novel variation of Streptomyces coelicoflavus , for which we propose the name S. coelicoflavus var. nankaiensis . Four chemically distinct α-amylase inhibitors, acarviostatins I03, II03, III03 and IV03, were isolated from strain ZG0656. Acarviostatins III03 and IV03 are both novel oligomers. All four acarviostatins are mixed noncompetitive porcine pancreas α-amylase inhibitors. Acarviostatin III03 is the most potent α-amylase inhibitor known to date. Moreover, in the in vitro and in vivo experiments, acarviostatins III03 showed significant inhibition of starch hydrolysis and glucose transfer to blood.
Conclusions:  Strain ZG0656 is a novel variation of S. coelicoflavus, whose products are novel effective α-amylase inhibitors. Among the products, acarviostatins III03 could significantly depress blood glucose levels in mammalian systems and be developed towards a possible therapeutic agent for diabetes.
Significance and Impact of the Study:  Acarviostatin III03 is the most potent α-amylase inhibitor known to date. The oligomer will benefit the research on the relationship between α-amylase and various inhibitors and will offer more choices in diabetes treatments.     

Two Novel Potent α‐Amylase Inhibitors from the Family of Acarviostatins Isolated from the Culture of Streptomyces coelicoflavus ZG0656

Two novel aminooligosaccharides were separated from the culture filtrate of Streptomyces coelicoflavus ZG0656. Their chemical structures were determined by acidic hydrolysis, electrospray‐ionization tandem mass spectrometry (ESI‐MS/MS), and NMR spectroscopy. The compounds were named acarviostatins III0(?1) and III23 according to the nomenclature of this group of metabolites. The two novel acarviostatins were both mixed noncompetitive inhibitors of porcine pancreatic α‐amylase (PPA). The inhibition constants (K_i) for acarviostatins III0(?1) and III23 were 0.009 and 0.026 μM , respectively, 151 and 52 times more potent than acarbose.     

alpha-Amylase from developing embryos of the camel tick Hyalomma dromedarii

alpha-Amylase activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of alpha-amylase III to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). alpha-Amylase III with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of alpha-amylase III was 106 kDa for the native enzyme, composed of two subunits of 43 and 66 kDa, respectively. alpha-Amylase had a value of 10 mg starch/ml. Varying alpha-amylase activity was detected when supplied with various substrates. alpha-Amylase III had a temperature optimum at 40 degrees C with heat stability up to 50 degrees C, and a pH optimum of 7.0. The enzyme activity was activated by CaCl2, MgCl2 and NaNO3, but not activated by NaCl, p-CMB, N-ethylmaleimide and iodoacetamide. EDTA and beta-mercaptoethanol strongly inhibited activity.     

Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat

Tendamistat is a strong inhibitory protein of porcine pancreatic alpha-amylase (PPA) with a K(i) value of 0.2 nM. To develop potent alpha-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (K(i) = 0.27 microM) exhibited the strongest inhibitory activity (K(i) = 0.27-1.41 microM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (K(i) = 0.28-1.00 microM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (K(i) = 0.28 microM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the beta-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity.     

Separation and characterization of four different amylases of Entamoeba histolytica. II. Characterization of amylases

Purified E. histolytica amylases III to VI were characterized by their hydrolytic behaviour towards 4-nitrophenyl alpha-malto-oligosaccharides, malto-oligosaccharides, amylose, amylopectin, glycogen and Y-cyclodextrin. The influence of specific inhibitors on the amylase activity of E. histolytica was examined and compared with typical alpha- and beta-amylases. Amylases III and IV showed alpha-glucosidase and glucosyltransferase activity by cleaving terminal non-reducing glucose from pNPG1 (III, IV) and pNPG2 to pNPG7 (III). Both enzymes were able to cleave malto-oligosaccharides and glucopolysaccharides to a large number of malto-oligosaccharides. Also transglucosidation reactions were observed, but maltose was not hydrolysed. Amylase V showed exoamylase-like properties by preferentially cleaving maltose units from the non-reducing end of synthetic and biogenic malto-oligosaccharides by a multiple-attack mechanism. Amylase VI was characterized as an alpha-amylase, showing great similarities with porcine pancreatic alpha-amylase in the hydrolysis pattern of 4-nitrophenyl alpha-malto-oligosaccharides and glucopolysaccharides. With biogenic malto-oligosaccharides amylase VI showed a transglucosidation reaction.     

Acylated phenolic glycosides from Solenostemma argel

From the aerial parts of Solenostemma argel, four new acylated phenolic glycosides sinapyl alcohol 9-O-feruloyl-4-O-alpha-rhamnopyranosyl-(1-->2)-beta-glucopyranoside, solargin I (1), sinapyl alcohol 9-O-caffeoyl-4-O-alpha-rhamnopyranosyl-(1-->2)-beta-glucopyranoside, solargin II (2), sinapyl alcohol 9-O-feruloyl-4-O-alpha-rhamnopyranosyl-(1-->2)-alpha-rhamnopyranosyl-(1-->2)-beta-glucopyranoside, solargin III (3) and sinapyl alcohol 9-O-caffeoyl-4-O-alpha-rhamnopyranosyl-(1-->2)-alpha-rhamnopyranosyl-(1-->2)-beta-glucopyranoside, solargin IV (4) have been isolated. The structures of the isolated compounds were verified by means of MS and NMR spectral analyses.     

Novel polysialogangliosides of skate brain structural determination of tetra, penta and hexasialogangliosides with a NeuAc-GalNAc linkage.

The gangliosides in the brain of a cartilaginous fish, skate (Bathyraja smirnovi), have been isolated and characterized by means of methylation analysis, antibody binding, enzymatic hydrolysis and MALDI-TOF MS. In addition to gangliosides with known structures (GM2, fucosyl-GM1, GD3, GD2, GT3 and GT2), five polysialogangliosides were isolated and characterized as having the following structures. (1) IV3NeuAc, III6NeuAc, II3NeuAc-Gg4Cer; (2) IV3NeuAc2, III6NeuAc, II3NeuAc-Gg4Cer; (3) IV3NeuAc, III6NeuAc, II3NeuAc2-Gg4Cer; (4) IV3NeuAc, III6NeuAc, II3NeuAc3-Gg4Cer; and (5) IV3NeuAc2, III6NeuAc, II3NeuAc3-Gg4Cer. These structures are 'hybrid-type' which comprise combinations of alpha-series and either a, b or c-series structures. Three gangliosides (2), (4) and (5), were novel. The main features of the ganglioside composition of skate brain were an abundance of gangliotriaosyl species, a lack of gangliotetraosyl species (except fucosyl-GM1), and an abundance of hybrid-types. These characteristics closely resemble those in shark brain which we reported previously [Nakamura, K., Tamai, Y. & Kasama, T. (1997) Neurochem. Int. 30, 593-604]. Two of the hybrid-type gangliosides (1) and (4), were examined for their neuritogenic activity toward cultured neuronal cells (Neuro-2A), and were found to have more potent activity than nonhybrid-type gangliosides such as GM1.     

Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus

A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL(-1)) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.     

Polymorphism and inheritance of swine small intestinal receptors mediating adhesion of three serological variants of Escherichia coli-producing K88 pilus antigen

Summary. Brush borders, enterocytes, or both preparations obtained from the small intestine of 345 pedigreed pigs, carrying components of seven breeds, were tested by adhesion assay in vitro with 6–32 enteropathogenic Escherichia coli strains, each expressing one of the three K88 pilus antigens, K88ab, K88ac and K88ad. With few exceptions, all pigs were classified as belonging to one of four adhesion phenotypes: I – corresponding to K88ab(-),ac(-),ad(-); II – K88ab(-),ac(+),ad(+); III – K88ab(+),ac(+),ad(-); and IV – K88ab(+),ac(+),ad(+). The non-adhering phenotype I was found to be the most frequent among the pigs tested, with the exception of one commercial herd, and this phenotype seems to be inherited as a recessive trait. The remaining three phenotypes are adhering, or are susceptible to adherence by one K88 variant, K88ad (phenotype II), by two variants, K88ab,ac (phenotype III), or by all three K88 variants, K88ab,ac,ad (phenotype IV). Phenotype II was found to be at low frequency, whereas III and IV occurred with similar frequencies. While the prevailing phenomenon was the bacterial adhesion to all, or none, of the brush borders, some pigs exhibited both adhering and non-adhering brush borders, a mixed adherence phenotype. Preliminary segregation data, obtained from the F1 generation, seem to indicate that phenotypes III and IV correspond to two haplotypes with genes at two or three closely linked loci respectively. An alternative hypothesis is that the phenotypes [II and IV are expressions of alleles at a single locus, each allele specifying a receptor able to bind two or three different serological types of K88 E. coli.     

Study of the inhibition of four alpha amylases by acarbose and its 4IV-alpha-maltohexaosyl and 4IV-alpha-maltododecaosyl analogues

Acarbose analogues, 4IV-maltohexaosyl acarbose (G6-Aca) and 4IV-maltododecaosyl acarbose (G12-Aca), were prepared by the reaction of cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and acarbose. The inhibition kinetics of acarbose and the two acarbose analogues were studied for four different alpha-amylases: Aspergillus oryzae, Bacillus amyloliquefaciens, human salivary, and porcine pancreatic alpha-amylases. The three inhibitors showed mixed, noncompetitive inhibition, for all four alpha-amylases. The acarbose inhibition constants, Ki, for the four alpha-amylases were 270, 13, 1.27, and 0.80 microM, respectively; the Ki values for G6-Aca were 33, 37, 14, and 7 nM, respectively; and the G12-Aca Ki constants were 59, 81, 18, and 11 nM, respectively. The G6-Aca and G12-Aca analogues are the most potent alpha-amylase inhibitors observed, with Ki values one to three orders of magnitude more potent than acarbose, which itself was one to three orders of magnitude more potent than other known alpha-amylase inhibitors.     

Soluble adenosine diphosphate glucose–α-1,4-glucan α-4-glucosyltransferases from spinach leaves

Multiple forms of ADP-glucose-alpha-1,4-glucan alpha-4-glucosyltransferase were obtained from spinach leaves by gradient elution from a DEAE-cellulose column. In the presence of high concentrations of some salts and bovine serum albumin, unprimed activity was found in one (transglucosylase III) of the four fractions eluted from the column. In addition to having unprimed activity, transglucosylase III had a lower K(m) for ADP-glucose, a much higher K(m) for oyster glycogen, greater heat sensitivity and lower affinity for maltose, maltotriose and amylopectin beta-limit dextrin than fractions I, II and IV. In addition, the kinetics at low concentrations of amylose, amylopectin and rabbit liver glycogen were non-linear for transglucosylase III. The properties of transglucosylases I, II and IV were generally similar to each other. Rates of the unprimed reaction at physiological concentrations of ADP-glucose were greater than those found for the primed reaction of fraction III. The product formed by the unprimed reaction was a glucan containing principally alpha-1,4 linkages with some alpha-1,6 linkages. The primer, maltose, at a concentration of 0.5m inhibited the synthesis of the unprimed product.     

Arrangement of the respiratory chain complexes in Saccharomyces cerevisiae supercomplex III2IV2 revealed by single particle cryo-electron microscopy

Here we present for the first time a three-dimensional cryo-EM map of the Saccharomyces cerevisiae respiratory supercomplex composed of dimeric complex III flanked on each side by one monomeric complex IV. A precise fit of the existing atomic x-ray structures of complex III from yeast and complex IV from bovine heart into the cryo-EM map resulted in a pseudo-atomic model of the three-dimensional structure for the supercomplex. The distance between cytochrome c binding sites of complexes III and IV is about 6 nm, which supports proposed channeling of cytochrome c between the individual complexes. The opposing surfaces of complexes III and IV differ considerably from those reported for the bovine heart supercomplex as determined by cryo-EM. A closer association between the individual complex domains at the aqueous membrane interface and larger spaces between the membrane-embedded domains where lipid molecules may reside are also demonstrated. The supercomplex contains about 50 molecules of cardiolipin (CL) with a fatty acid composition identical to that of the inner membrane CL pool, consistent with CL-dependent stabilization of the supercomplex.     

Novel peptide toxins from the sea anemone Stichodactyla haddoni

Four peptide toxins, SHTX I-III with crab-paralyzing activity and SHTX IV with crab lethality, were isolated from the sea anemone Stichodactyla haddoni and their primary structures elucidated by protein sequencing and cDNA cloning. SHTX I (new toxin, 28 residues), II (analogue of SHTX I, 28 residues) and III (Kunitz-type protease inhibitor, 62 residues) are potassium channel toxins and SHTX IV (48 residues) is a member of the type 2 sea anemone sodium channel toxins. The precursor protein of SHTX IV is composed of a signal peptide, propart and mature peptide, while the propart is missing in that of SHTX III. In addition to these four toxins, an epidermal growth factor-like peptide was detected in S. haddoni by RT-PCR.     

The outermost lysine in the S4 of domain III contributes little to the gating charge in sodium channels

We investigated the contribution the four outermost basic residues (K1, R2, R3, R4) in segment 4 of domain III in the human cardiac Na channel (hH1a, Na(V)1.5) to the total gating charge (Q(max)). Each of the four basic residues were mutated individually to a cysteine. In addition, R2 was also mutated to a glutamate. All mutant channels were transiently expressed with the alpha1 subunit in fused tsA201 cells. We used the relative reduction in Q(max) caused by anthopleurin-A (ApA) toxin, a site-3 toxin known to inhibit the movement of gating charge associated with domain IV, to estimate the size of the contribution from each basic residue. Studies of the toxin's ability to inhibit gating charge in mutant channels showed that R2 contributed 19-20% to the Q(max), R3 contributed 10%, and K1 and R4 made almost no contribution. In contrast to the outermost basic residue in the S4 of Shaker K channels and in the S4 of domain IV in hH1a, the outermost charge (K1) in domain III of Na channels is outside the voltage field.     

Immunoelectron microscopic localization of laminin, type IV collagen, and type III pN-collagen in reticular fibers of human lymph nodes

We studied the ultrastructural distribution of laminin, type IV collagen, and the amino terminal pro-peptide of type III collagen (type III pN-collagen) in normal human lymph nodes. After fixation with freshly prepared 4% paraformaldehyde mixed with 0.1% glutaraldehyde, cryoultramicrotomy proved to preserve the antigenicity of these proteins better than embedding in Lowicryl K4M. Sections were treated with rabbit antibodies against the 7S domain of human type IV collagen, the fragment P1 of human laminin, and the amino terminal pro-peptide of human type III pro-collagen, followed by anti-rabbit IgG conjugated to 10-nm colloidal gold. Laminin and type IV collagen were seen in the basement membrane structures of the blood vessels and in the walls of sinuses. The amorphous material between the collagenous fibers in locations corresponding to reticular fibers also contained laminin and type IV collagen. The amino terminal pro-peptide of type III pro-collagen was present in the collagenous fibers in reticular fibers and in the walls of blood vessels and sinuses. Therefore, a significant number of the type III collagen molecules in these fibers must have retained their amino terminal pro-peptide. These results indicate that the basement membrane proteins laminin and type IV collagen are genuine components of reticular fibers, as suggested earlier by immunohistochemical studies at the light microscopic level.     

Polymorphism and inheritance of swine small intestinal receptors mediating adhesion of three serological variants of Escherichia coli-producing K88 pilus antigen

Brush borders, enterocytes, or both preparations obtained from the small intestine of 345 pedigreed pigs, carrying components of seven breeds, were tested by adhesion assay in vitro with 6-32 enteropathogenic Escherichia coli strains, each expressing one of the three K88 pilus antigens, K88ab, K88ac and K88ad. With few exceptions, all pigs were classified as belonging to one of four adhesion phenotypes: I I--corresponding to K88ab(-),ac(-),ad(-); II--K88ab(-),ac(-),ad(+); III--K88ab(+),ac(+),ad(-); and IV--K88ab(+),ac(+),ad(+). The non-adhering phenotype I was found to be the most frequent among the pigs tested, with the exception of one commercial herd, and this phenotype seems to be inherited as a recessive trait. The remaining three phenotypes are adhering, or are susceptible to adherence by one K88 variant, K88ad (phenotype II), by two variants, K88ab, ac (phenotype III), or by all three K88 variants, K88ab,ac,ad (phenotype IV). Phenotype II was found to be at low frequency, whereas III and IV occurred with similar frequencies. While the prevailing phenomenon was the bacterial adhesion to all, or none, of the brush borders, some pigs exhibited both adhering and non-adhering brush borders, a mixed adherence phenotype. Preliminary segregation data, obtained from the F1 generation, seem to indicate that phenotypes III and IV correspond to two haplotypes with genes at two or three closely linked loci respectively. An alternative hypothesis is that the phenotypes III and IV are expressions of alleles at a single locus, each allele specifying a receptor able to bind two or three different serological types of K88 E. coli.     

Separation of polypeptides which induce a mitogen response of thymocytes from the culture supernatant of a thymic epithelial cell line

To examine thymic hormonal factors, four polypeptide fractions (estimated molecular weight: I, 10 K; II, 7 K; III, 3 K; IV, 2.5 K) were separated from the culture supernatant of a rat thymic epithelial cell line by high-pressure liquid chromatography (HPLC) with a gel-filtration column. The effects of the fractions on response to mitogens of three small-lymphocyte subsets were studied. All fractions enhanced response to concanavalin A (Con A) of the lighter subset containing mainly immature thymocytes, but only fractions II and IV increased response to phytohemagglutinin (PHA) of the heavier subset containing relatively mature thymocytes. When fraction IV was subfractionated by reversed-phase HPLC, the polypeptides that enhanced response to Con A and PHA were separated into hydrophobic and hydrophilic subfractions, respectively. Fraction I was subfractionated by a similar method, and the inducing activity of Con A response was found in a relatively hydrophobic subfraction. These data suggested that the cell line secretes several kinds of bioactive polypeptides that affect the thymocytes at different stage of maturation.     

Structural studies of a Phe256Trp mutant of human salivary alpha-amylase: implications for the role of a conserved water molecule in enzyme activity

In the mechanism of hydrolysis of starch by alpha-amylases, a conserved water molecule bridging two catalytic residues has been implicated. In human salivary alpha-amylase (HSAmy), this water (W641), observed in many alpha-amylase structures, is part of a chain of water molecules. To test the hypothesis that W641 may be involved in the mechanism, Phe256 in the close vicinity was mutated to a Trp residue. X-ray structure of F256W complexed to 2-amino-2-(hydroxyethyl)-1,3-propanediol at 2.1A revealed that the water chain is disrupted. In the F256W structure exhibits a positional shift in His305, characteristic of alpha-amylase complex structures. Kinetic analysis, in comparison with HSAmy, revealed that the mutant exhibited a 70-fold decrease in the specific activity for starch and significantly reduced k(cat) (20-fold) and K(m) (4-fold) for maltoheptaoside. Collectively, these results suggest that W641 and the chain of water molecules may be critical for the alpha-amylase activity.