Tschining Chang

Toxicity domain in presynaptically toxic phospholipase A2 of snake venom

About 42 complete amino-acid sequences of phospholipases A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) are known, including those of 13 presynaptically toxic enzymes, but the structural features responsible for the neurotoxicity and distinguishing the toxins from the non-neurotoxic enzymes are far from being clear. In this study, we examined the charged-residue distributions and hydrophobic characteristics based on the sequence data and the predicted tertiary structure and proposed a possible toxicity domain. We found that the presynaptically toxic enzymes have three or four more basic amino-acid residues than the non-neurotoxic enzymes at positions 59, 60, 65, 70-73 and 97 or 98. These residues appear to cluster near the surface region at the N-terminal side. The cationic nature of this basic cluster in the toxin is enhanced by the alpha-amino group of the N-terminus and the dipole moment of helices 96-110 and 1-10. Moreover, these toxic-site residues are usually associated with hydrophobic regions at 1-7, 64-81 and 97-109.

Dynamic Change of Neural Cell Adhesion Molecule Polysialylation on Human Neuroblastoma (IMR-32) and Rat Pheochromocytoma (PC-12) Cells during Growth and Differentiation

Polysialic acid (PSA) is a regulatory epitope of neural cell adhesion molecule (NCAM) in homophilic adhesion of neural cells mediated by NCAM, is also known to be re-expressed in several human tumors, thus serves as an oncodevelopmental antigen. In this study, using a recently developed ultrasensitive chemical method in addition to immunochemical methods, growth stage-dependent and retinoic acid (RA)-induced differentiation-dependent changes of PSA expression in human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells were analyzed both qualitatively and quantitatively. Both IMR-32 and PC-12 cells expressed PSA on NCAM, and the level of PSA expressed per unit weight of cells increased with post-inoculation incubation time. The most prominent feature was seen at the full confluence stage. RA induced neuronal differentiation in both IMR-32 and CP-12 cells that paralleled the change in the PSA level. Chemical analysis revealed the presence of NCAM glycoforms differing in the degree of polymerization (DP) of oligo/polysialyl chains, whose DP was smaller than 40. DP distribution of PSA was different between the cell lines and was changed by the growth stage and the RA treatment. Thus DP analysis of PSA is important in understanding both mechanism and biological significance of its regulated expression.

Developmentally Regulated Expression of a Peptide:N-Glycanase during Germination of Rice Seeds (Oryza sativa) and Its Purification and Characterization

Peptide:N-glycanase (PNGase; EC3.5.1.52) activity was detected in dormant rice seeds (Oryza sativa) and the imbibed rice grains. Time-course studies revealed that the enzyme activity remained almost constant until about 30 h after imbibition in both of endosperm- and embryo tissue-containing areas, and started to increase only in growing germ part, reached a peak at about 3-day stage, followed by a gradual decrease concomitant with a sharp increase in the coleoptile. The specific activity increased about 6-fold at about 3-day stage. PNGase was purified to electrophoretic homogeneity from the extracts of germinated rice seeds at 24 h, and the apparent molecular weight of the purified enzyme, estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), was about 80,000. The purified enzyme was designated PNGase Os to denote its origin. The N-terminal sequence of the 10 residues was determined to be SYNVASVAGL. The purified PNGase Os in SDS-PAGE appeared as a rather broad band, consistent with the presence of multiple glycoforms as indicated by chromatographic behavior on a Sephadex G-75 column. PNGase expressed in coleoptile under anoxia condition was also purified, and both of the purified enzymes were found to exhibit very similar, if not identical, electrophoretic mobility in SDS-PAGE. PNGase Os exhibited a broad pH-activity profile with an optimum of 4–5 and, interestingly, was significantly inactivated by K+ and Na+ at near the physiological concentration, 100 mm. These results are discussed in relation to other work.

Crystal structure of a nucleoside diphosphate kinase from Bacillus halodenitrificans: coexpression of its activity with a Mn-superoxide dismutase.

We found that when grown under anaerobic conditions the moderate halophile, gram-positive bacterium Bacillus halodenitrificans (ATCC 49067) synthesizes large amounts of a polypeptide complex that contains a heme center capable of reversibly bind nitric oxide. This complex, when exposed to air, dissociates and reassociates into two active components, a Mn-containing superoxide dismutase (SOD) and a nucleoside diphosphate kinase (BhNDK). The crystal structure of this latter enzyme has been determined at 2.2A resolution using molecular replacement method, based on the crystal structure of Drosophila melanogaster NDK. The model contains 149 residues of a total 150 residues and 34 water molecules. BhNDK consists of a four-stranded antiparallel beta-sheet, whose surfaces are partially covered by six alpha-helices, and its overall and active site structures are similar to those of homologous enzymes. However, the hexameric packing of BhNDK shows that this enzyme is different from both eukaryotic and gram-negative bacteria. The need for the bacterium to presynthesize both SOD and NDK precursors which are activated during the anaerobic-aerobic transition is discussed.

Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media

KDN (Deaminoneuraminic acid, or deaminated neuraminic acid) is a minor but biosynthetically independent member of the sialic acid. Human occurrence of KDN has already been established, although its level is so little that it is often undetectable by conventional sialic acid analysis. Elevated expression of KDN in fetal cord blood cells and some malignant tumor cells have been reported. However, in mammalian cells and tissues KDN mostly occurs as the free sugar and little occurred conjugated to glycolipids and/or glycoproteins. A positive correlation between the ratio of free KDN/free Neu5Ac in ovarian adenocarcinomas and the stage of malignancy has been noted for diagnostic use. We hypothesized that elevated expression of KDN in mammalian systems may be closely related to elevated activities of enzymes involved in the formation of sialoglycoconjugates and/or aberrant supply of the precursor sugar, mannose, used in the biosynthesis of KDN. In this study we used human ovarian teratocarcinoma cells PA-1 to further analyze KDN expression in human cells. Major findings reported in this paper are, (i) a 30 kDa KDN-glycoprotein immunostainable with monoclonal antibody, mAb.kdn3G, (specific for the KDNα2 → 3Galβ1→ epitope) and sensitive to KDNase was identified in the membrane fraction of the cell: (ii) a 49 kDa KDN-glycoprotein that is not reactive with mAb.kdn3G but is sensitive to KDNase was identified in the soluble fraction: and (iii) PA-1 cells showed unique response to mannose added to the growth medium in that the levels of both free and bound forms of KDN are elevated. This is the first report on the identification of mammalian KDN-glycoproteins by chemical and biochemical methods.

Purification, crystallization and preliminary X-ray crystallographic analysis of rice Bowman-Birk inhibitor from Oryza sativa

Bowman-Birk inhibitors (BBIs) are cysteine-rich proteins with inhibitory activity against proteases that are widely distributed in monocot and dicot species. The expression of rice BBI from Oryza sativa is up-regulated and induced by pathogens or insects during germination of rice seeds. The rice BBI (RBTI) of molecular weight 15 kDa has been crystallized using the hanging-drop vapour-diffusion method. According to the diffraction of rice BBI crystals at a resolution of 2.07 A, the unit cell belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 74.37, b = 96.69, c = 100.36 A. Preliminary analysis indicates four BBI molecules in an asymmetric unit, with a solvent content of 58.29%.

γ-Crystallin genes in carp: cloning and characterization

The carp γ-crystallin gene family was found to be composed of at least three members: γm1, γm2 and γm3. The encoded products are very similar to other known γ-crystallins but with their own peculiarities: (1) they all have a high methionine content: 12.4%, 14% and 8.4% in γm1, γm2 and γm3, respectively; and (2) the amino acid sequences are aberrant in the region before connecting peptides and its corresponding region in motif 4. Their protein structures might remain the same as those of other γ-crystallins since they retain all the conserved amino acid residues essential for maintaining the loops in the protein structures.

Purification, crystallization and preliminary X-ray crystallographic analysis of nucleoside diphosphate kinase from rice.

Nucleoside diphosphate kinase (NDK) catalyses the transfer of the gamma-phosphoryl group from a nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP). NDK is involved in and required for coleoptile elongation in rice. The level of the enzyme changes during seed germination and the early stages of seedling growth. Recombinant rice NDK (rNDK) has been crystallized using the hanging-drop vapour-diffusion method. rNDK crystals diffracted to a resolution of 2.50 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.98, b = 182.26, c = 188.30 A. Preliminary analysis indicates there to be 12 rNDK molecules in each asymmetric unit, with a solvent content of 47.2%.

Purification, crystallization and preliminary X-ray crystallographic analysis of rice lectin from Oryza sativa

Lectins with sugar-binding specificity are widely distributed in higher plants and various other species. The expression of rice lectin from Oryza sativa is up-regulated in the growing coleoptile when anaerobic stress persists. A rice lectin of molecular weight 15.2 kDa has been crystallized using the hanging-drop vapour-diffusion method. From the diffraction of the lectin crystals at 1.93 A resolution, the unit cell belongs to space group P3(1), with unit-cell parameters a = 98.58, b = 98.58, c = 44.72 A. Preliminary analysis indicates that there are two lectin molecules in an asymmetric unit with a large solvent content, 70.1%.

Preliminary crystallographic studies of two C-terminally truncated copper-containing nitrite reductases from Achromobacter cycloclastes: changed crystallizing behaviors caused by residue deletion

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.