Association of a single nucleotide polymorphism in the SH2D1A intronic region with systemic lupus erythematosus.

SH2D1A, also known as signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), is an adaptor protein. Recently, it was reported that SAP deficient mice were protected from systemic lupus erythematosus (SLE). In this study, we postulated SH2D1A gene to be a candidate susceptibility gene for SLE and analyzed its association with SLE. A case-control association study was conducted on 5 tag single nucleotide polymorphisms (SNPs) in SH2D1A region in 506 Japanese female SLE patients and 330 healthy female controls. The luciferase assay was performed to determine the functional role of the SNP associated with SLE. One SNP in the intron 2, rs2049995, showed association with SLE (p=0.0110, odds ratio (OR) 1.97, 95% confidence interval (CI) 1.16-3.34, under the dominant model). The association of rs2049995 seemed to be stronger in the subset with the age of onset less than 20 years (p=0.0067, OR 2.65, 95% CI 1.28-5.46). Functional evaluation of rs2049995 showed that reporter gene activity was increased 1.9-fold for the susceptible allele compared with the resistant allele. An intronic SNP of SH2D1A is associated with SLE.

Crystallization and preliminary crystallographic data of the alpha-amylase inhibitors, Haim I and Paim I.

Two proteins that act as alpha-amylase inhibitors, Haim I and Paim I, were crystallized and preliminary X-ray diffraction studies on them were carried out. We also sequenced Haim I prepared from Streptomyces griseosporeus YM-25 and confirmed that it is composed of 78 amino acid residues. Crystals of Haim I were grown from ammonium sulfate solution mixed with ethanol by the vapor diffusion technique. The crystals grew as hexagonal bipyramids and diffracted X-rays beyond 2.0 A resolution. They belong to the space group P6(1)22 (or P6(5)22) with unit cell dimensions of a = b = 36.7 A, c = 192.4 A, and contain one molecule per asymmetric unit. Paim I, a protein of 39 amino acid residues produced by Streptomyces corchorusii, was crystallized under similar conditions to Haim I. The crystals diffracted X-rays beyond 2.5 A. They belong to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 65.4 A, c = 96.1 A, and contain three molecules per asymmetric unit.

Structures of sugar chains of hen egg yolk riboflavin-binding protein.

The structures of the sugar chains of hen yolk riboflavin-binding protein (RBP) were established. Asparagine-linked sugar chains of yolk-RBP were liberated by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of the sugar chains were purified by gel-filtration and reversed-phase HPLC. Seven PA-sugar chains were isolated, and the structure of each was determined by composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-mHz 1H-NMR spectroscopy. These analyses showed that the main sugar chains had sialylbiantenna and sialyltriantenna structures. PA-sugar chains of plasma-RBP were also isolated, and the structures of the PA-sugar chains of yolk- and plasma-RBPs were compared as to their elution patterns on anion-exchange chromatography and reversed-phase HPLC. The plasma RBP had almost the same sugar chains as the yolk RBP did, indicating that sugar chains are not modified during incorporation into the oocyte.

Assay of urinary free fucose by fluorescence labeling and high-performance liquid chromatography.

The concentrations of free fucose and other sugars in urine of cancer patients and healthy subjects were analyzed by high-performance liquid chromatography. After the urine samples were dried, we coupled the sugars in the residue with 2-aminopyridine to be detected as fluorescent derivatives. We then analyzed the pyridylamino derivatives of the sugars with an anion-exchange column and borate buffer. The difference between cancer patients and healthy subjects for the mean concentrations of fucose corrected for creatinine was significant (P less than 0.001). We checked the relationship between the concentrations of other sugars and the presence of cancer. This method is highly sensitive, and neither a cleanup procedure before labeling nor purification before injection into the column is needed. Not only fucose but also other sugars can be detected simultaneously, so this method should be useful for studying any changes in sugars in urine in various diseases.

Studies on the active site of human alpha-amylases: examination of the third subsite S3' of the aglycone-binding site by control of substrate binding mode.

Modified maltooligosaccharides, IG-G-G-G-AG-G (IG: 6-deoxy-6-iodo-D-glucopyranose residue, G: D-glucopyranose residue, AG: 6-amino-6-deoxy-D-glucopyranose residue, -: alpha-1,4-glycosidic linkage), IG-G-G-G-AG-M (M: methyl), and IG-G-G-G-AG-phi (phi: phenyl) were prepared by the use of cyclodextrin glucanotransferase in order to examine the third subsite (S3') of the aglycone-binding site of human salivary and pancreatic alpha-amylases. Human alpha-amylases hydrolyzed the modified maltooligosaccharides to IG-G-G and G-AG-G, IG-G-G and G-AG-M, and IG-G-G and G-AG-phi. This implied that G, M, and phi fit into S3'. There was no difference in the rate parameters between the two enzymes. The Km values for the hydrolysis of IG-G-G-G-AG-G by both enzymes were the same as those for IG-G-G-G-AG-M, and twice those for IG-G-G-G-AG-phi. The results showed that S3' of the two enzymes has no affinity for the glucose residue and is not a subsite but a hydrophobic environment.

Investigation of the active site of Bacillus macerans cyclodextrin glucanotransferase by use of modified maltooligosaccharides.

The active site of Bacillus macerans cyclodextrin glucanotransferase (CGTase) was examined by use of derivatives of phenyl alpha-maltopentaoside and phenyl alpha-glucoside as the substrates and acceptors, respectively. The active site of this enzyme is considered to be composed of tandem subsites (S4, S3, S2, S1, S1', S2', etc.) geometrically complementary to several glucose residues, and the alpha-1,4-glycosidic linkage of a substrate is split between S1 and S1'. The features of subsites S3 and S4 of the glycon binding site were estimated from the modes of the enzymatic action on phenyl alpha-maltopentaoside (G-G-G-G-G-phi; G, glucose residue; phi, phenyl residue; -, alpha-1,4-glycosidic bond) and its derivatives in which the CH2OH groups of the non-reducing-end glucose residues were converted to CH2I (IG-G-G-G-G-phi; IG, 6-deoxy-6-iodo-D-glucose residue), CH2NH2 (AG-G-G-G-G-phi; AG, 6-amino-6-deoxy-D-glucose residue), or COOH (CG-G-G-G-G-phi; CG, glucuronic acid residue). p-Nitrophenyl alpha-glucopyranoside (G-P; P, p-nitrophenyl residue) was used as an acceptor. HPLC analysis of the digests revealed that the CG residue of CG-G-G-G-G-phi was excluded from subsite S3, while it was accommodated in subsite S4. The Km and Vmax values for CG-G-G-G-G-phi were remarkably larger and smaller, respectively, than those for any other substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of neutral alpha-mannosidase that is activated by Co2+ from Japanese quail oviduct.

An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.

Reconstitution of new inhibitors through mutual exchange of Ile(64)-Met(84) peptides of soybean trypsin inhibitors, Tia and Tib.

Singly modified soybean trypsin inhibitors (STIs), Tia* [Tia cleaved at Arg(63)-Ile(64)] and Tib* [Tib cleaved at Arg(63)-Ile(64)], were prepared by limited proteolysis with trypsin at pH 3.0. These singly modified inhibitors were further modified to yield doubly modified inhibitors, Tia** and Tib**, by limited proteolysis with subtilisin BPN', which cleaved the Met(84)-Leu(85) bonds of Tia* and Tib*, respectively. The doubly modified inhibitors could be separated into two parts: protein moiety A and peptide moiety a (IRFIAEGHPLSLKFDS-FAVIM) for Tia**, and protein moiety B and peptide moiety b (IRFIAEGNPLRLKFDS-FAVIM) for Tib**. These protein and peptide moieties showed no trypsin inhibitory activities alone. However, the inhibitors can be reconstituted through the mutual exchange of the protein and peptide moieties isolated from STIs. The reconstituted inhibitor which has tyrosine at position 62 and histidine at position 71 shows the highest inhibitory activity. Its Ki value for bovine trypsin is around 10(-10) M, which is almost the same as that of Tia for bovine trypsin. The inhibitor possessing either tyrosine at position 62 or histidine at position 71 exhibits a Ki value of around 10(-9) M, which is between those of Tia and Tib. The inhibitor having phenylalanine and asparagine at positions 62 and 71, respectively, shows the weakest inhibitory activity of around 10(-8) M similar to that of Tib for bovine trypsin.

Examination of aglycone-binding site of human salivary alpha-amylase by means of transglycosylation reactions.

The active site of human salivary alpha-amylase is composed of tandem subsites (S3, S2, S1, S1',S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary alpha-amylase were examined by means of transglycosylation reaction using phenyl alpha-maltoside (GG phi: G-G-phi) and its derivatives (GAG phi: G-AG-phi, GCG phi: G-CG-phi, AGG phi: AG-G-phi, and CGG phi: CG-G-phi) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, 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- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl alpha-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAG phi occurred more effectively than to GG phi. Transfers to GCG phi and CGG phi were less than to GG phi and very little transfer to AGG phi occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus.

We have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonucleotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

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.

Active-site-directed inactivation of Aspergillus oryzae beta-galactosidase with beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene (beta-GalMNT), a specific inhibitor of beta-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae beta-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (beta-galactopyranosylmethyl carbonium or beta-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of beta-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli beta-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed beta-GalMNT faster than the latter did. A. oryzae beta-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of beta-galactopyranosylmethyltriazene. However, E. coli beta-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli beta-galactosidases by beta-GalMNT was an enzyme-activated and active-site-directed irreversible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.

Separation of oligomannose-type sugar chains having one to five mannose residues by high-performance liquid chromatography as their pyridylamino derivatives.

Ten oligomannose-type sugar chains (ManGlcNAc2-Man5GlcNAc2) were prepared from various glycoproteins and fluorescence labeled with 2-aminopyridine. The fluorescent pyridylamino (PA)-sugar chains were first separated into five fractions according to their molecular sizes by HPLC on a TSK gel Amide-80 column. Each fraction was then separated into the component PA-sugar chains by reversed-phase HPLC on a Capcell Pak C18 column according to their chemical structures. The method is useful for studying the substrate specificities of alpha-mannosidases with Man5GlcNAc2-PA as a substrate.

The structure of (xylose)2glucose-O-serine 53 found in the first epidermal growth factor-like domain of bovine blood clotting factor IX.

We reported the presence of a new trisaccharide composed of two xylose and reducing terminal glucose residues linked to serine residues of bovine blood clotting factors VII and IX (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The present paper describes the detailed structural analysis of the trisaccharide. Glycopeptides were prepared from bovine factor IX by digestion with Pronase followed by purification by column chromatography. The trisaccharide was released from the protein by the beta-elimination reaction with hydrazine, and the reducing end of the sugar chain was tagged with 2-aminopyridine. The fluorescent pyridylamino derivative of the trisaccharide was purified by gel filtration and reversed-phase high performance liquid chromatography. The glycopeptides and pyridylamino-trisaccharide thus obtained were subjected to methylation study, 500-MHz 1H nuclear magnetic resonance spectroscopy, and periodate oxidation. Glucose and xylose belong to the D series by high performance liquid chromatography on a chiral column. From the results, the structure of the trisaccharide is proposed as: D-Xyl p alpha 1-3-D-Xyl p alpha 1-3-D-Glcp beta 1-O-Ser-53.

Estimation of elution times on reverse-phase high-performance liquid chromatography of pyridylamino derivatives of sugar chains from glycoproteins.

Addition of a sugar residue to a pyridylamino (PA) sugar chain affects its elution time on reverse-phase HPLC and the contribution of the sugar residue is not influenced by the other sugar residues [S. Hase, S. Natsuka, H. Oku, and T. Ikenaka (1987) Anal. Biochem. 167, 321-326]. The partial relative elution times (Ei) of 22 sugar residues were calculated from the relative elution times (the elution time relative to Man5Glc-NAc2-PA) of three kinds of PA-sugar chain: the oligomannose and N-acetyllactosamine types and sugar chains with a xylose residue. The relative elution time of a PA-sugar chain can be calculated by summing the Ei values of all of the constituent sugar residues. The calculated relative elution times of 44 PA-sugar chains agreed well with the observed values. This method can be used to estimate the relative elution times of PA-sugar chains that are not yet available, and to estimate the structures of sugar chains in limited amounts of glycoproteins by a combination of sugar composition analysis and exoglycosidase digestion.

A new trisaccharide sugar chain linked to a serine residue in the first EGF-like domain of clotting factors VII and IX and protein Z.

Recently, we determined the complete amino acid sequence of bovine factor VII (Takeya, H. et al. (1988) J. Biol. Chem. 263, 14868-14877). In the course of the studies, we found an unknown serine derivative at position 52 in the first epidermal growth factor-like domain of factor VII. A pentapeptide isolated from the S-aminoethylated factor VII contained Ser-52, which could not be identified with a gas-phase sequencer. The same results were also obtained for a pentapeptide containing Ser-53 of factor IX and protein Z. Component sugar analysis revealed that the peptide contained 1 mol of glucose and 2 mol of xylose. This sugar component was also confirmed by high-resolution fast atom bombardment mass spectrometric analysis of the pentapeptide. The trisaccharide was released from the peptides by means of beta-elimination reaction and its reducing end was identified as pyridylamino-glucose. These results indicate the existence of a (Xyl2)Glc-Ser structure in factors VII, IX and protein Z. Similar results were obtained for human factors VII, IX and protein Z. This is the first report of a (Xyl2)-Glc-Ser structure in glycoproteins to our knowledge. The presence of the unique trisaccharide structure in factors VII, IX and protein Z leads us to anticipate its biological role in the tissue factor pathway.