Functional properties of glycosylated lysozyme secreted in Pichia pastoris

Various mutant lysozymes having the N-glycosylation signal sequence, R21T (Asn(19)-Tyr(20)-Thr(21)), G49N (Asn(49)- Ser(50)-Thr(51)), R21T/G49N (Asn(19)-Tyr(20)-Thr(21)/Asn(49)-Ser(50)-Thr(51)), were secreted in the Pichia pastoris expression system. The secreted amounts of these mutant glycosylated lysozymes were almost the same as those of wild-type lysozyme (about 30 mg/liter). Glycosylation of the mutant lysozymes was confirmed by SDS-PAGE patterns, Endo-H treatment, TOF-MS analysis and chemical analysis. The composition of the carbohydrate chain attached to the single glycosylated lysozymes, R21T and G49N, was GlcNAc(2)Man(9-11), while that of the double glycosylated lysozyme, R21T/G49N, was GlcNAc(4)Man(27-32). The results of a CD analysis and lytic activity suggested that the conformation of the single glycosylated lysozymes had been conserved, while that of the double glycosylated lysozyme was less stable. The emulsifying properties of the lysozyme when glycosylated were greatly improved, being especially noteworthy in the double glycosylated lysozyme.     

NMR investigations of the N-linked oligosaccharides at individual glycosylation sites of human lutropin

Human lutropin or luteinizing hormone (hLH) is a heterodimeric glycoprotein, composed of two subunits. hLH alpha (N-glycosylated at Asn52 and Asn78) and hLH beta (N-glycosylated at Asn30). The sugar chains were liberated by hydrazinolysis from intact hLH beta and from glycopeptides obtained after tryptic digestion of hLH alpha, subsequently reduced and fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC and identified mainly by one-dimensional (1D) and two-dimensional (2D) 1H-NMR spectroscopy. The results indicate predominantly diantennary. N-acetyllactosamine-type structures at all three glycosylation sites. The oligosaccharides attached to Asn52 (hLH alpha) and Asn30 (hLH beta) show a remarkably similar pattern, with mainly chain-terminating 4-sulphated 2-deoxy-2-N-acetylamino-D-galactose (GalNAc) and a sulphated/sialylated structure as the major single component. However, virtually all N-glycans on the beta subunit bear a fucose residue alpha 1-6-linked to the proximal GlcNAc, whereas those at Asn52 (and Asn78) of the alpha subunit are predominantly non-fucosylated. The oligosaccharides at Asn78 (hLH alpha) are sialylated rather than sulphated and contain the unique sequence NeuAc alpha 2-6 GalNAc beta 1-4GlcNAc beta 1-2 Man alpha 1-3 as part of the majority of mono- and disialylated compounds. The major single constituent at Asn78 has the following structure: [formula, see text]     

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.     

Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase.

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.     

Particular interaction between pyrimethamine derivatives and quadruple mutant type dihydrofolate reductase of Plasmodium falciparum: CoMFA and quantum chemical calculations studies

Comparative molecular field analysis (CoMFA) was performed on twenty-three pyrimethamine (pyr) derivatives active against quadruple mutant type (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) dihydrofolate reductase of Plasmodium falcipaarum (PfDHFR). The represented CoMFA models were evaluated based on the various three different probe atoms, C(sp3) (+1), O(sp3) (-1) and H (+1), resulting in the best model with combined three types of probe atoms. The statistical results were r(2)(cv) = 0.702, S(press) = 0.608, r(2)(nv) = 0.980, s = 0.156, and r(2)(test-set) = 0.698 which can explain steric contribution of about 50%. In addition, an understanding of particular interaction energy between inhibitor and surrounding residues in the binding pocket was performed by using MP2/6-31G(d,p) quantum chemical calculations. The obtained results clearly demonstrate that Asn108 is the cause of pyr resistance with the highest repulsive interaction energy. Therefore, CoMFA and particular interaction energy analyses can be useful for identifying the structural features of potent pyr derivatives active against quadruple mutant type PfDHFR.     

Amino-acid sequence of human alpha 2-antiplasmin

The amino-acid sequence of human alpha 2-antiplasmin was determined by Edman degradation of peptides purified from CNBr, tryptic and chymotryptic digests. Of the total sequence of 452 amino acids of mature alpha 2-antiplasmin, as deduced from the cDNA sequence [Holmes et al. (1987) J. Biol. Chem. 262, 1659-1664], 444 residues were identified by amino-acid sequencing. Two differences were found between the peptide and cDNA analyses (Gly instead of Leu at position 10 and Gly instead of Ser at position 369). alpha 2-Antiplasmin contains two disulfide bridges (Cys64-Cys104 and Cys31-Cys113) and four glucosamine-based carbohydrate chains attached to Asn87, Asn256, Asn270 and Asn277. alpha 2-Antiplasmin is homologous with 12 other proteins belonging to the serine protease inhibitor (serpin) superfamily.     

Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates.

Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661-8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3'-(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666-8670]. The kinetics of the Asn36Ala, Asn98Ala and wild-type catalyzed transesterification of guanosine 3'-(5'-D-ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by D-ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.     

Deamidation of asparagine residues in a recombinant serine hydroxymethyltransferase

Serine hydroxymethyltransferase purified from rabbit liver cytosol has at least two Asn residues (Asn(5) and Asn(220)) that are 67 and 30% deamidated, respectively. Asn(5) is deamidated equally to Asp and isoAsp, while Asn(220) is deamidated only to isoAsp. To determine the effect of these Asn deamidations on enzyme activity and stability a recombinant rabbit liver cytosolic serine hydroxymethyltransferase was expressed in Escherichia coli over a 5-h period. About 90% of the recombinant enzyme could be isolated with the two Asn residues in a nondeamidated form. Compared with the enzyme isolated from liver the recombinant enzyme had a 35% increase in catalytic activity but exhibited no significant changes in either affinity for substrates or stability. Introduction of Asp residues for either Asn(5) or Asn(220) did not significantly alter activity or stability of the mutant forms. In vitro incubation of the recombinant enzyme at 37 degrees C and pH 7.3 resulted in the rapid deamidation of Asn(5) to both Asp and isoAsp with a t(1/2) of 50-70 h, which is comparable to the rate found with small flexible peptides containing the same sequence. The t(1/2) for deamidation of Asn(220) was at least 200 h. This residue may become deamidated only after some unfolding of the enzyme. The rates for deamidation of Asn(5) and Asn(220) are consistent with the structural environment of the two Asn residues in the native enzyme. There are also at least two additional deamidation events that occur during prolonged incubation of the recombinant enzyme.     

N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule

Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn(37), Asn(247), and Asn(319), although Asn(37) is not efficiently glycosylated. To examine N-glycan processing at Asn(247) and Asn(319), full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn(247) sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn(319) in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn(247) and Asn(319). However, Asn(247) had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn(319). Moreover, when comparing the N-glycans at Asn(319) on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn(247) allows additional N-glycan processing to occur at Asn(319), yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined.     

NH2-terminal fragment of rat pro-atrial natriuretic factor in the circulation: identification, radioimmunoassay and half-life

A radioimmunoassay was developed to measure the NH2-terminal counterpart of rat pro-atrial natriuretic factor (pro-ANF) in plasma. Synthetic rat ANF (Asp 11-Ala 37) coupled to bovine serum albumin was used to immunize New Zealand rabbits. The antiserum demonstrated good immunoreactivity towards rat ANF (Asn 1-Arg 98), (Asn 1-Tyr 126), (Asp 11-Ala 37) and even human ANF (Asn 1-Ser 30). The standard curve had an ED80 of 9.5 +/- 2.5 and ED50 of 44.0 +/- 10.5 fmol/tube. Immunoreactive ANF NH2-terminal peptide was measured directly in rat plasma without prior extraction. In fact, extraction of ANF NH2-terminal from plasma by C18 silica gel chromatography revealed inconsistent recovery and a lack of parallelism. Morphine (0.75 mg/100 g), chosen to elicit increased ANF (Ser 99-Tyr 126) secretion, elevated its plasma concentration from 54.1 +/- 3.2 to 190.8 +/- 55.8 fmol/ml after 20 min. At the same time, the immunoreactive NH2-terminal fragment rose from 378 +/- 16 to 1181 +/- 201 fmol/ml. The identity of this immunoreactive material was verified following affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC) of plasma from morphine-treated rats. Molecular sieving and amino acid sequencing demonstrated that it appears to be consistent with or identical to rat ANF (Asn 1-Arg 98). The disappearance rate of ANF (Asn 1-Arg 98) was studied by injecting radioactive material into anesthetized rats. The exponential decay was analyzed by a two-compartment model in which the fast and slow components had a half-life of 2.5 +/- 0.3 and 54.8 +/- 3.9 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine mapping and interaction analysis of a linear rabies virus neutralizing epitope

A novel human antibody AR16, targeting the G5 linear epitope of rabies virus glycoprotein (RVG) was shown to have promising antivirus potency. Using AR16, the minimal binding region within G5 was identified as HDFR (residues 261–264), with key residues HDF (residues 261–263) identified by alanine replacement scanning. The key HDF was highly conserved within phylogroup I Lyssaviruses but not those in phylogroup II. Using computer-aided docking and interaction models, not only the key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, Ile101, and Trp166) of AR16 that participated in the interaction with G5 were identified, the van der Waals forces that mediated the epitope–antibody interaction were also revealed. Seven out of eight presumed key residues (Asp30, Asp31, Tyr32, Trp53, Asn54, Glu99, and Ile101) were located at the variable regions of AR16 heavy chains. A novel mAb cocktail containing AR16 and CR57, has the potential to recognize non-overlapping, non-competing epitopes, and neutralize a broad range of rabies virus.     

Comparison of the monomer structure of the FMN-binding protein from Desulfovibrio vulgaris obtained by NMR and molecular dynamics simulation approaches

Flavin mononucleotide (FMN)-binding proteins (FBPs) play an important role in the electron transport process in bacteria. In this study, the structures of the FBP from Desulfovibrio vulgaris (DvFBP) (Miyazaki F) were compared between those obtained experimentally by nuclear magnetic resonance (NMR) spectroscopy and those derived from molecular dynamics simulations (MDSs). A high-residue root of mean square deviation (RMSD) was observed in residues located at both sides of the wings (Gly22, Glu23, Asp24, Ala59, Arg60, Asp61, Glu62, Gly75, Arg76, Asn77, Gly78 and Pro79), while a low-residue RMSD was found in residues located in a hollow of the structure (Asn12, Glu13, Gly14, Val15, Val16, Asn30, Thr31, Trp32, Asn33, Ser34, Gly69, Ser70, Arg71 and Lys72). Inter-planar angles between the Phe7 and Iso and between the Phe7 and Trp106 residues were remarkably different between the MDS- and NMR-derived DvFBP structures. Distribution of the torsion angles around the covalent bonds in the aliphatic chain of FMN was similar in the MDS- and NMR-derived structures, except for those around the C1′–C2′ and C5′–O5′ bonds. Hydrogen bond formation between IsoO2 and the Gly49 or Gly50 peptide NH was formed in both the NMR- and MDS-derived structures. Overall, the MDS-derived structures were found to be considerably different from the NMR-derived structures, which must be considered when the photoinduced electron transfer in flavoproteins is analysed with MDS-derived structures.     

Two residues in the anticodon recognition domain of the aspartyl-tRNA synthetase from Pseudomonas aeruginosa are individually implicated in the recognition of tRNAAsn

In many organisms, the formation of asparaginyl-tRNA is not done by direct aminoacylation of tRNA(Asn) but by specific tRNA-dependent transamidation of aspartyl-tRNA(Asn). This transamidation pathway involves a nondiscriminating aspartyl-tRNA synthetase (AspRS) that charges both tRNA(Asp) and tRNA(Asn) with aspartic acid. Recently, it has been shown for the first time in an organism (Pseudomonas aeruginosa PAO1) that the transamidation pathway is the only route of synthesis of Asn-tRNA(Asn) but does not participate in Gln-tRNA(Gln) formation. P. aeruginosa PAO1 has a nondiscriminating AspRS. We report here the identification of two residues in the anticodon recognition domain (H31 and G83) which are implicated in the recognition of tRNA(Asn). Sequence comparisons of putative discriminating and nondiscriminating AspRSs (based on the presence or absence of the AdT operon and of AsnRS) revealed that bacterial nondiscriminating AspRSs possess a histidine at position 31 and usually a glycine at position 83, whereas discriminating AspRSs possess a leucine at position 31 and a residue other than a glycine at position 83. Mutagenesis of these residues of P. aeruginosa AspRS from histidine to leucine and from glycine to lysine increased the specificity of tRNA(Asp) charging over that of tRNA(Asn) by 3.5-fold and 4.2-fold, respectively. Thus, we show these residues to be determinants of the relaxed specificity of this nondiscriminating AspRS. Using available crystallographic data, we found that the H31 residue could interact with the central bases of the anticodons of the tRNA(Asp) and tRNA(Asn). Therefore, these two determinants of specificity of P. aeruginosa AspRS could be important for all bacterial AspRSs.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues. Replacement of asparagine residues by serine stabilizes

The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-Asp8, Leu27] hGRF(1-32)NH2, produced by deamidation of the Asn8 residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [beta-Asp8, Leu27] hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while [alpha-Asp8, Leu27] hGRF(1-32)NH2 was calculated to be 25 times less potent than the parent Asn8 peptide. Three additional analogs of [Leu27] hGRF(1-32)NH2 containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37 degrees). Based on disappearance kinetics, [Leu27] hGRF(1-32)NH2 had a half-life of 202 h while the other analogs had the following half-lives: [Leu27, Asn28] hGRF(1-32)NH2 (150 h); [Ser8, Leu27, Asn28] hGRF(1-32)NH2 (746 h); and [Ser8, Leu27] hGRF(1-32)NH2 (1550 h). After 14 days, incubated samples of the Asn8 analogs lost GH releasing potency, while the Ser8 analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.     

Point mutation of alanine (31) to valine prohibits the folding of reduced lysozyme by sulfhydryl-disulfide interchange

In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coli. Since this lysozyme contained two amino acid substitutions (Ala31----Val and Asn106----Ser) in addition to an extra methionine residue at the NH2-terminus, the substituted amino acid residues were converted back to the original ones by means of oligonucleotide-directed site-specific mutagenesis and in vitro recombination. Thus, four kinds of chicken lysozyme [Met-1Val31Ser106-, Met-1Ser106-, Met-1Val31- and Met-1 (wild type)] were expressed in E. coli. From the results of folding experiments of the reduced lysozymes by sulfhydryl-disulfide interchange at pH 8.0 and 38 degrees C, followed by the specific activity measurements of the folded enzymes, the following conclusions can be drawn: (i) an extra methionine residue at the NH2-terminus reduces the folding rate but does not affect the lysozyme activity of the folded enzyme; (ii) the substitution of Asn106 by Ser decreases the activity to 58% of that of intact native lysozyme without changing the folding rate; and (iii) the substitution of Ala31 Val prohibits the correct folding of lysozyme. Since the wild type enzyme (Met-1-lysozyme) was activated in vitro without loss of specific activity, the systems described in this study (mutagenesis, overproduction, purification and folding of inactive mutant lysozymes) may be useful in the study of folding pathways, expression of biological activity and stability of lysozyme.     

Conformation NMR analysis of the spatial structure of Buthus eupeus insectotoxin I5A

1H NMR spectroscopy has been used to collect data related to the spatial structure of insectotoxin I5A Buthus eupeus: pH-dependence of the chemical shifts, deuterium exchange rates of individual amide hydrogens, spin-spin coupling of the H-N-C alpha-H and H-C alpha-C beta-H protons, and nuclear Overhauser effect between distinct protons belonging to amino acid residues remote in the sequence. Molecular conformation in the regions from Asp9 to Cys19 (beta-turn 9-12 and right-hand alpha-helix 12-19) and from Asn23 to Asn34 (antiparallel beta-sheet with the beta-turn 27-30) directly follows from the observed parameters. Pseudoatomic approach of distance geometry algorithm was used to solve the overall folding of the molecule and propose the most probable set of disulfide bridges: Cys2-Cys19, Cys5-Cys31, Cys16-Cys26 and Cys20-Cys33. The spatial structure of insectotoxin I5A B. eupeus demonstrates remarkable similarity with that of a "long" type scorpion neurotoxin V-3 Centruroides sculpturatus.     

Identification and characterization of the human MOG1 gene

Many of the proteins that mediate transport into and out of the nucleus have been structurally and functionally conserved throughout evolution. Here we describe the sequence and characterization of the human MOG1 gene. The MOG1 gene was originally identified in Saccharomyces cerevisiae as a multi-copy suppressor of conditional alleles of the yeast nuclear transport factor, GSP1 (scRan) (Oki and Nishimoto (1998) Proc. Natl. Acad. Sci. USA 95, 15388-15393). A search of the expressed sequence tag database identified a putative human protein that is 29% identical and 47% similar to the yeast protein. Our experiments demonstrate that the human MOG1 message is expressed in a variety of tissue samples. Several experiments indicate that the human MOG1 protein binds to both yeast and human Ran suggesting functional conservation between the yeast and human MOG1 proteins. Furthermore, hMOG1a, like scMOG1, is localized throughout the cell but is concentrated within the nucleus. Consistent with these findings, hMOG1a can partially complement the growth defect present in yeast MOG1 deletion cells. Taken together, our findings suggest that MOG1 is an evolutionarily conserved Ran binding protein that could play a role in regulating nuclear protein trafficking.     

Evaluation of medetomidine/ketamine for short-term immobilization of variable flying foxes (Pteropus hypomelanus)

Four medetomidine/ketamine (M/K) doses (30 microg/kg/3 mg/kg; 40/4; 50/5; 60/6), administered by intramuscular injection, were evaluated for short-term immobilization of adult male variable flying foxes (Pteropus hypomelanus). The highest dose (60 microg/kg/6 mg/kg) produced a significantly faster induction (31 +/- 46 sec) than the lowest dose (30/3) (125 +/- 62 sec). The highest dose levels (50/5, 60/6) produced significantly longer immobilization times (52.5 +/- 25.7 min and 60.6 +/- 20.8 min, respectively) than did the lower doses (30/3, 40/4) (18.8 +/- 8.7 min and 31.0 +/- 14.3 min, respectively). The dose at which 50% of the bats were immobilized for > or = 30 min (ED(50)) was approximately 40 microg/kg/4 mg/kg. This dose produced a mean immobilization time of 31 +/- 14 min, bradypnea and bradycardia. In conclusion, a M/K dose of 50 microg/kg/5 mg/kg is recommended for greater than 30 min of relaxed immobilization in free-living variable flying foxes and is sufficient for safe collection of samples.     

Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies. Alteration of substrate specificity by replacement of Asn149 with Asp

To examine the interaction of human arginase II (EC 3.5.3.1) with substrate and manganese ions, the His120Asn, His145Asn and Asn149Asp mutations were introduced separately. About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively. The K(m) for arginine (1.4-1.6 mM) was not altered and the wild-type and mutant enzymes were essentially inactive on agmatine. In contrast, the Asn149Asp mutant expressed almost undetectable activity on arginine, but significant activity on agmatine. The agmatinase activity of Asn149Asp (K(m) = 2.5 +/- 0.2 mM) was markedly resistant to inhibition by arginine. After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn(2+) and contained < 0.1 Mn(2+).subunit(-1), whereas wild-type and His145Asn enzymes were half active and contained 1.1 +/- 0.1 Mn(2+).subunit(-1) and 1.3 +/- 0.1 Mn(2+).subunit(-1), respectively. Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with K(d) of 1.8 +/- 0.2 x 10(-8) M for the wild-type and His145Asn enzymes and 16.2 +/- 0.5 x 10(-8) m for the His120Asn variant. Upon mutation, the chromatographic behavior, tryptophan fluorescence properties (lambda(max) = 338-339 nm) and sensitivity to thermal inactivation were not altered. The Asn149-->Asp mutation is proposed to generate a conformational change responsible for the altered substrate specificity of arginase II. We also conclude that, in contrast with arginase I, Mn(2+) (A) is the more tightly bound metal ion in arginase II.     

Structure of a cyanobacterial BLUF protein, Tll0078, containing a novel FAD-binding blue light sensor domain

The sensor proteins for blue light using the FAD (BLUF) domain belong to the third family of the photoreceptor proteins using a flavin chromophore, where the other two families are phototropins and cryptochromes. As the first structure of this BLUF domain, we have determined the crystal structure of the Tll0078 protein from Thermosynechococcus elongatus BP-1, which contains a BLUF domain bound to FAD, at 2A resolution. Five Tll0078 monomers are located around the non-crystallographic 5-fold axis to form a pentamer, and two pentamers related by 2-fold non-crystallographic symmetry form a decameric assembly. The monomer consists of two domains, the BLUF domain at the N-terminal region and the C-terminal domain. The overall structure of the BLUF domain consists of a five-stranded mixed beta-sheet with two alpha-helices running parallel with it. The isoalloxazine ring of FAD is accommodated in a pocket formed by several highly conserved amino acid residues in the BLUF domain. Of these, the three apparent key residues (Asn31, Asn32 and Gln50) were substituted with Ala. Mutant proteins of N31A and N32A showed a nearly normal 10nm spectral shift of the flavin upon illumination, while the Q50A mutant did not exhibit such a shift at all. On the basis of the crystal structure, we discussed a possible role of Gln50, which is structurally and functionally linked with the critical Tyr8 (FAD-Gln50-Tyr8 network), with regard to the light-induced spectral shift of the BLUF proteins.