Cloning and characterization of a third type of human alpha-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes.     

Fluorometric rate assay of alpha-amylase using an intramolecularly-quenched fluorescent substrate (FG5P)

The complete hydrolysis of a fluorogenic derivative of rho-nitrophenyl alpha-maltopentaoside, FG5P, by human salivary alpha-amylase, resulted in a 5-fold increase in fluorescence. This is due to disruption of the intramolecular quenching of the fluorescence of the 2-pyridylamino residue by the rho-nitrophenyl residue by separation of the two residues. This change of fluorescence accompanying the cleavage of the glucosidic bond was exploited to develop a fluorometric rate assay of alpha-amylase in human serum.     

Differentiation of human non-salivary, non-pancreatic alpha-amylase from salivary and pancreatic alpha-amylases by use of FG5P

Human non-salivary, non-pancreatic alpha-amylase (yHXA) is the gene product of a newly found human alpha-amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl alpha-maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary alpha-amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P) and to FG4(FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the alpha-amylase assay.     

Preparation of a new fluorogenic substrate of alpha-amylases and a simple alpha-amylase assay by HPLC

A new substrate of alpha-amylases, O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-O-alpha-D-glucopyranosyl-(1 leads to 4)-D-glucopyranose, was prepared using dextrin as a starting material. Compared with other substrates so far reported, the fluorogenic substrate is unique in that it is resistant to exo-alpha-glucosidases due to the blocking group introduced into the non-reducing end glucose residue. The product of alpha-amylase digestion was rapidly separated from the substrate and was detected very sensitively by HPLC and a fluorescence detector. This method for alpha-amylase assay was also applied for determination of alpha-amylase in human serum.     

On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1.

Cathepsin B1 from bovine spleen exhibited its greatest rates of hydrolysis on peptide β-naphthylamide (βNA) derivatives containing paired basic residues, i.e., Cbz-Arg-Arg-βNA, t-Boc-Lys-Lys-βNA, and t-Boc-Lys-Arg-βNA. Internal peptide bonds were not attacked. At its pH 6.5 optimum, cathepsin B1 hydrolyzed Cbz-Arg-Arg-βNA (K_m 0.18 mM) 64 times faster than Bz-DL-Arg-βNA (K_m 3.3 mM or 1.6 mM for the L isomer) and was therefore chosen to replace the latter as a more soluble and sensitive substrate for the assay of cathepsin B1. Although cathepsin B2 had no action on the β-naphthylamide substrates, it did manifest carboxypeptidase activity by attacking COOH-terminal residues exposed by the action of cathepsin B1. At its pH 5.0 optimum, cathepsin B2 behaved as a SH-dependent, non-specific carboxypeptidase by releasing COOH-terminal amino acids from a variety of Cbz-Gly-X substrates and polypeptides such as glucagon, Val-Leu-Ser-Glu-Gly, and penta-lysine.     

UDP-N-acetyl-D-galactosamine as a donor substrate for the glycosyltransferase encoded by the B gene at the human blood group ABO locus

The properties of the enzyme in the serum of blood group B individuals that catalyses the transfer of small amounts of N-acetyl-D-galactosamine to H-active precursor structures were compared with those of the blood group B gene-associated alpha-(1----3)-D-galactosyltransferase and with the blood group A gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases in the serum of blood group A1 and A2 individuals. The biosynthetic products formed by the enzyme in B serum were identical with the A-active structures synthesised by the A1 and A2 gene-associated alpha-(1----3)-N-acetyl-D-galactosaminyltransferases but the enzyme differed from the A1 and A2 transferases in its apparent Km for UDP-N-acetyl-D-galactosamine, its heat susceptibility, its failure to bind to Sepharose 4B, and its adsorption to H-active sites on group O red cell ghosts under conditions which bind the B transferase but fail to adsorb the A1 and A2 transferases. The correlation between the levels of alpha-(1----3)-D-galactosyltransferase and alpha-(1----3)-N-acetyl-D-galactosaminyltransferase activities in all the group B serum samples tested, the maintenance of the same ratio of activities after successive cycles of binding to group O red cell ghosts, the retention of the ability to convert blood group O to A-active cells after treatment of the serum with Sepharose 4B, and the failure to detect any comparable activity in group O serum samples tested under the same conditions indicated that the enzyme in group B serum that utilises UDP-N-acetyl-D-galactosamine to make blood group A-active structures is the B gene-associated alpha-(1----3)-D-galactosyltransferase.     

Expression of a novel human sialidase encoded by the NEU2 gene

Sialidases (E.C.3.2.1.18) belong to a group of glycohydrolytic enzymes, widely distributed in nature, which remove sialic acid residues from glycoproteins and glycolipids. All of the sialidase so far characterized at the molecular level share an Asp block, repeated three to five times in the primary structure, and an F/YRIP sequence motif which is part of the active site. Using a sequence homology-based approach, we previously identified a human gene, named NEU2, mapping to chromosome 2q37. NEU2 encoded protein is a polypeptide of 380 amino acids with two Asp block consensuses and the YRIP sequence in the amino terminal part of the primary structure. Here we demonstrate that NEU2 encodes a functional sialidase. NEU2 was expressed in COS7 cells, giving rise to a dramatic increase in the sialidase activity measured in cell extracts with the artificial substrate 4-MU-NANA. Using a rabbit polyclonal antiserum, on Western blots a protein band with a molecular weight of about 42 kDa was detectable, and its cytosolic localization was demonstrated with cell fractionation experiments. These results were confirmed using immunohistochemical techniques. NEU2 expression in E.coli cells allowed purification of the recombinant protein. As already observed in the enzyme expressed in COS7 cells, NEU2 pH optimum corresponds to 5.6 and the polypeptide showed a K(m)for 4-MU-NANA of 0.07 mM. In addition, based on the detectable similarities between the NEU2 amino acid sequence and bacterial sialidases, a prediction of the three-dimensional structure of the enzyme was carried out using a protein homology modeling approach.     

Rational design of a SHP-2 targeted,fluorogenic peptide substrate

Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH₂ and optimized kinetic parameters (k_cat = 0.059 ± 0.008 s⁻¹, K_m = 220 ± 50 µM, k_cat/K_m of 270 M⁻¹s⁻¹). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a k_cat value of 0.0038 ± 0.0003 s⁻¹, a K_m value of 1100 ± 100 µM and a k_cat/K_m of 3 M⁻¹s⁻¹. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide.     

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate.

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications.     

Measurement of cyclomaltodextrin glucanotransferase activity by high-performance liquid chromatography using a fluorogenic substrate

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.     

Antibacterial potential of hGlyrichin encoded by a human gene

Emerging multidrug‐resistant (MDR) bacteria are an enormous threat to human life because of their resistance to currently available antibiotics. The genes encoding antibacterial peptides have been studied extensively and are excellent candidates for a new generation of antibiotic drugs to fight MDR bacteria. In contrast to traditional antibiotics, antibacterial peptides, which do not cause drug resistance, have an unparalleled advantage. However, because most antibacterial peptides originate in species other than humans, the hetero‐immunological rejection of antibacterial peptides is a key disadvantage that limits their clinical application. In this study, we identify hGlyrichin as a potential human antibacterial polypeptide. The hGlyrichin polypeptide kills a variety of bacteria including the MDR bacteria methicillin‐resistant Staphylococcus aureus, MDR Pseudomonas aeruginosa, and MDR tubercle bacillus. A 19 amino acid peptide (pCM19) at positions 42–60 of hGlyrichin is crucial for its antibacterial activity. The hGlyrichin polypeptide kills bacteria through the destruction of the bacterial membrane. In addition, all peptides that are homologous to hGlyrichin have antibacterial activity and can penetrate the bacterial membrane. Importantly, hGlyrichin does not cause hemolytic side effects in vitro or in vivo. Therefore, based on the virtues of hGlyrichin, i.e., the absence of hetero‐immunological rejection and hemolytic side effects and the unambiguous efficacy of killing pathogenic MDR bacteria, we propose hGlyrichin as a potential human antibacterial polypeptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Analysis of human cytochrome P450 2C8 substrate specificity using a substrate pharmacophore and site-directed mutants

The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar K(m) led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 A from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3-4-fold increase in the K(m) of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6alpha-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with k(cat)/K(m) values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B'.     

Nucleotide sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene: comparison with the B. amyloliquefaciens gene.

The DNA sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene is reported. Comparison of the inferred amino acid sequence of the B. licheniformis alpha-amylase gene with that of the Bacillus amyloliquefaciens gene shows that whereas the amino acid sequences of the mature proteins have considerable homology, the sequences for the signal peptides are distinct.     

Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymatic assay for alpha-amylases

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside, FG5P, was prepared, taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase on a mixture of O-6-deoxy-6-[(2-pyridyl)-amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose and p-nitrophenyl alpha-glucoside. The maltopentaose derivative is resistant to alpha-glucosidase and is suitable as a substrate for the alpha-amylase assay coupled with alpha-glucosidase in which the activity of alpha-amylase is determined by measuring the amount of p-nitrophenol liberated by alpha-glucosidase from p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside produced by the action of alpha-amylase. This alpha-amylase assay method was applied for determination of alpha-amylases in human serum.     

Detection of P2X purinergic receptors on human B lymphocytes

B lymphocytes are known to synthesise the P2X7 subtype of the P2X purinergic receptor family; however, the identification of the other six P2X subtypes on these cells has been limited by the absence of specific antibodies. In this study, we used a panel of anti-P2X polyclonal antibodies and confocal microscopy to examine the presence of each P2X receptor on human B lymphocytes. We observed that P2X1, P2X2, P2X4 and P2X7 subtypes, but not P2X3, P2X5 and P2X6 subtypes, are present on B lymphocytes.