Nonreducing Conditions Research Articles

Fast and Efficient non-reduced Lys-C digest using pressure cycling technology for antibody disulfide mapping by LC–MS

Conventional sample preparation for antibody disulfide mapping often requires relatively long digestion time (from several hours to overnight) and relatively high endoproteinase concentration. These conditions are typically necessitated by the fact that antibody molecules are not sufficiently denatured under non-reduced conditions and chaotropic agents are used during digestion to achieve optimal denaturation. Disulfide scrambling can occur as artifacts of digestion as proteins are incubated for extended periods, often at neutral to slightly alkaline pH conditions. Shortening digestion time and lowering the pH during digestion frequently result in incomplete peptide cleavages or variable recoveries. Here, we report the development of a fast and efficient non-reduced Lys-C digestion method based on pressure cycling technology (PCT) and its application in determining disulfide-linkages in monoclonal antibodies (mAbs). Conditions were optimized to ensure complete digestion of the mAb with minimal sample preparation-related disulfide scrambling. The PCT-based method was able to generate up to 10-fold signal increase for some disulfide peptides in a 1h Lys-C digestion compared to the conventional bench-top digestion method. As a result of the shorter digestion time, disulfide scrambling that is seen as a major assay artifact of the conventional method was reduced to less than 0.05% in tested molecules. The results show that the PCT-based method offers fast digestion in a shorter time for all the mAbs tested.

The Human Sodium-Glucose Cotransporter (hSGLT1) Is a Disulfide-Bridged Homodimer with a Re-Entrant C-Terminal Loop.

Na-coupled cotransporters are proteins that use the trans-membrane electrochemical gradient of Na to activate the transport of a second solute. The sodium-glucose cotransporter 1 (SGLT1) constitutes a well-studied prototype of this transport mechanism but essential molecular characteristics, namely its quaternary structure and the exact arrangement of the C-terminal transmembrane segments, are still debated. After expression in Xenopus oocytes, human SGLT1 molecules (hSGLT1) were labelled on an externally accessible cysteine residue with a thiol-reactive fluorophore (tetramethylrhodamine-C5-maleimide, TMR). Addition of dipicrylamine (DPA, a negatively-charged amphiphatic fluorescence “quencher”) to the fluorescently-labelled oocytes is used to quench the fluorescence originating from hSGLT1 in a voltage-dependent manner. Using this arrangement with a cysteine residue introduced at position 624 in the loop between transmembrane segments 12 and 13, the voltage-dependent fluorescence signal clearly indicated that this portion of the 12–13 loop is located on the external side of the membrane. As the 12–13 loop begins on the intracellular side of the membrane, this suggests that the 12–13 loop is re-entrant. Using fluorescence resonance energy transfer (FRET), we observed that different hSGLT1 molecules are within molecular distances from each other suggesting a multimeric complex arrangement. In agreement with this conclusion, a western blot analysis showed that hSGLT1 migrates as either a monomer or a dimer in reducing and non-reducing conditions, respectively. A systematic mutational study of endogenous cysteine residues in hSGLT1 showed that a disulfide bridge is formed between the C355 residues of two neighbouring hSGLT1 molecules. It is concluded that, 1) hSGLT1 is expressed as a disulfide bridged homodimer via C355 and that 2) a portion of the intracellular 12–13 loop is re-entrant and readily accessible from the extracellular milieu.

Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in chronic rhinosinusitis

Abstract Thymic stromal lymphopoietin (TSLP) is known to be elevated and truncated in nasal polyps (NPs) of chronic rhinosinusitis and might play a significant role in type 2 inflammation in this disease. However, neither the structure nor the role of the truncated products of TSLP has been studied. We investigated the mechanisms of truncation of TSLP in NPs and the function of the truncated products. We incubated recombinant full-length 15.1 KDa human TSLP (methionine + residues 29–159, referred to as M29–159) with NP extracts. SDS-PAGE revealed two major truncated products of TSLP in NP extracts. Edman sequencing and MALDI-TOF MS results indicated that the longer truncation product was TSLP (M29–124) and the smaller product was TSLP (131–159). We found that PCSK (proprotein convertase subtilisin/kexin) family proteases were rate-limiting enzymes in the truncation and generation of TSLP (M29–130) and TSLP (131–159). Carboxypeptidases further digested 6 amino acids from the C-terminus of TSLP (M29–130) to generate TSLP (M29–124) in NPs. Interestingly, recombinant TSLP (M29–124) and TSLP (131–159) did not manifest TSLP activity, but PCSK-digested TSLP had significantly higher activity than full-length TSLP on myeloid dendritic cells and group 2 innate lymphoid cells. Finally, we identified a 14.2 KDa TSLP from incubation of TSLP with NP extracts under non-reducing conditions that was significantly reduced in the presence of DTT. This indicates that the two cleaved products are dimerized with disulfide bonds and the dimerized form, TSLP (29–124 + 131–159), is an active metabolite and may contribute to the amplification of type 2 inflammation in NPs.

Matrix metalloproteinase (MMP)-9: A realiable marker for inflammation in early human trichinellosis

Matrix Metalloproteinases (MMPs) are involved in many physiological and pathological processes. As regards parasitic infections, the role of these proteins has been particularly studied in malaria, neurocysticercosis and angiostrongyloidosis. Recently, we evaluated serum levels of MMP-9 and -2 (gelatinases) in mice experimentally infected with Trichinella spiralis or Trichinella pseudospiralis, which cause different degrees of myositis and we found their significant increase in the former and, at a lesser extent, in the latter, thus suggesting the possibility that these gelatinases, particularly MMP-9, represent a marker of inflammation.Our aim was to evaluate the levels of MMP-9 and 2 in trichinellosis patients, to assess their possible clinical significance.Serum samples from 31 Trichinella britovi-infected individuals (20 males and 11 females), living in Tuscany, Central Italy, were analysed for MMP-9 and MMP-2 serum levels. Patients acquired infection with Trichinella after consuming raw or undercooked meat of wild boar. Their median age was 49±0.33years (range from 7 to 91). Sera was collected before starting anti-inflammatory treatment, aliquoted and stored at −20°C until use. Sera from healthy subjects was considered as controls. The gelatinolytic activity of MMPs was analysed by gelatin zymography on 8% polyacrylamide-SDS gels containing 0.1% porcine gelatin, under non-reducing conditions. Clear bands corresponding to the digested areas were evaluated with an appropriate software. MMP-9 levels were additionally determined in 15 patients using a commercial ELISA kit for human MMP-9. The zymographic analysis of the gels showed the presence in serum samples of gelatinase bands at approximately 125-kDa, 92-kDa and 72-kDa, corresponding to the MMP-9/Neutrophil gelatinase-associated lipocalin (NGAL) complex and proenzyme forms of MMP-9 and MMP-2, respectively. A significant (p<0.01) increase in gelatinolytic activity in patients compared to the control group was observed for pro-MMP-9 in 25 out of 31. The mean increase in activity was 39.25%±16.67%. No significant differences were observed for pro-MMP-2 activity. The MMP-9 levels detected by ELISA showed significant correlation with zymographic data (r2=0.62, p<0.003) and were higher in more affected patients (suffering diarrhea, facial edemas and myalgia). In conclusion, MMP-9 might be considered as a marker of inflammation in T. britovi patients. On the contrary, MMP-2 did not result significantly different in patients, compared to controls.

Hyaluronidase activity in the salivary glands of tabanid flies

Tabanids are haematophagous insects that act as biological and mechanical vectors of various diseases, including viruses, bacteria and parasites. The saliva of these insects contains strong anticoagulant and vasodilatory activities as well as immunoregulatory peptides. Here we demonstrate pronounced hyaluronidase (hyase) activity in ten tabanid species of the genera Chrysops, Haematopota, Hybomitra and Tabanus. Compared to other haematophagous insects, the ability of tabanid hyases to hydrolyze hyaluronic acid (HA) is extremely high, for example the enzyme activity of Hybomitra muehlfeldi was found to be 32-fold higher than the salivary hyase activity of the sand fly Phlebotomus papatasi. Hyases of all ten tabanid species tested also cleaved chondroitin sulfate A, another glycosaminoglycan present in the extracellular matrix of vertebrates. The pH optimum of the enzyme activity was measured in eight tabanid species; the hyase of Haemopota pluvialis was the only one with optimum at pH 4.0, while in the other seven species the activity optimum was at 5.0. SDS PAGE zymography showed the monomeric character of the enzymes in all tabanid species tested. Under non-reducing conditions the activities were visible as single bands with estimated MW between 35 and 52 kDa. The very high hyaluronidase activity in tabanid saliva might be related to their aggressive biting behavior as well as to their high efficiency as mechanical vectors. As they are supposedly involved in the enlargement of feeding hematomas, hyases might contribute to the mechanical transmission of pathogens. Pathogens present in vector mouthparts are co-inoculated into the vertebrate host together with saliva and may benefit from increased tissue permeability and the immunomodulatory activity of the salivary hyase.

Isolation and Functional Characterisation of Antibacterial Peptide from the Mosquito,Aedes(ochlerotatus)caspius(Diptera: Culicidae)

One antimicrobial peptide was isolated and purified to 5.50-fold from the crude lysate of immunised Aedes caspius larvae by (NH4)2SO4 fractionation, ion-exchange chromatography, and reverse-phase high performance liquid chromatography (HPLC). The peptide, named ACAm, was found to be heat stable below 80 °C and exhibited strong antimicrobial activities against Gram-positive and Gram-negative bacteria as well as against peptidoglycans and lipopolysaccharides of bacterial cell walls. The estimated molecular weight of ACAm by sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE), under non-reducing conditions, was 4 kDa. Using isoelectric focusing (IEF), ACAmexhibited pI in basic pH (8.4) belonging to cationic peptides.

Anti-Phospholipase A2 Receptor Autoantibody: A New Biomarker for Primary Membranous Nephropathy.

Primary membranous nephropathy (also known as idiopathic membranous nephropathy, IMN) is an organ specific autoimmune kidney disease characterized by the development of immune complex deposits in the sub-epithelial spaces, podocyte effacement and glomerular capillary wall thickening in the later stages. Clinical studies have demonstrated that over 70% of patients with IMN possess circulating autoimmune antibodies specifically targeting the phospholipase A2 receptor (PLA2R) on the surface of podocytes. The autoantibodies only bind to the extracellular portion of PLA2R under the non-reducing condition, indicating that the epitope in PLA2R is conformational requiring specific disulfide bonds to maintain its structure. We recently have successfully located the dominant epitope in PLA2R to the extreme N-terminus of the receptor. This finding has opened a new direction for understanding the pathogenesis of anti-PLA2R autoantibody induced IMN and offered a strong basis for developing sensitive clinical assays for IMN diagnosis and prognosis, and potentially, new therapeutic approaches for IMN treatment.

Comparable efficiency of different extraction protocols for wheat and rye prolamins

The identification and quantification of cereal storage proteins is of interest of many researchers. Their structural or functional properties are usually affected by the way how they are extracted. The efficiency of extraction process depends on the cereal source and working conditions. Here, we described various commonly used extraction protocols differing in the extraction conditions (pre-extraction of albumins/globulins, sequential extraction of individual protein fractions or co-extraction of gluten proteins, heating or non-heating, reducing or non-reducing conditions). The total protein content of all fractions extracted from commercially available wheat and rye flours was measured by the Bradford method. Tris-Tricine SDS-PAGE was used to determine the molecular weights of wheat gliadins, rye secalins and high-molecular weight glutelins which are the main triggering factors causing celiac disease. Moreover, we were able to distinguish individual subunits (α/β-, γ-, ω-gliadins and 40k-γ-, 75k-γ-, ω-secalins) of wheat/rye prolamins. Generally, modified extraction protocols against classical Osborne procedure were more effective and yields higher protein content in all protein fractions. Bradford measurement led into underestimation of results in three extraction procedures, while all protein fractions were clearly identified on SDS-PAGE gels. Co-extraction of gluten proteins resulted in appearance of both, low-molecular weight fractions (wheat gliadins and rye secalins) as well as high-molecular weight glutelins which means that is not necessary to extract gluten proteins separately. The two of three extraction protocols showed high technical reproducibility with coefficient of variation less than 20%. Carefully optimized extraction protocol can be advantageous for further analyses of cereal prolamins.

Biochemical and functional characterization of BmooSP, a new serine protease from Bothrops moojeni snake venom

In this work, we describe the purification and characterization of a new serine protease enzyme from Bothrops moojeni snake venom (BmooSP). On SDS-PAGE, BmooSP was found to be a single-chain protein with an apparent molecular mass of 36,000 and 32,000 under reduced and non-reduced conditions, respectively. Mass spectrometry analysis showed that the BmooSP is composed by two isoforms with molecular mass of 30,363 and 30,070, respectively. The purified enzyme consists of 277 amino acid residues, disregarding the cysteine and tryptophan residues that have been degraded by acid hydrolysis, and its N-terminal sequence showed similarity with other serine protease enzymes. BmooSP induced blood-clotting in vitro, defibrination in vivo, caseinolytic and fibrin(ogen)olytic activities. The enzyme is stable at high temperatures (up to 100 °C) and shows maximum activity at pH around 7.0. Preliminary results show that BmooSP can induce the formation of a stable fibrin clot for more than 10 days. BmooSP presents medical interest because it can be used as biodegradable fibrin glue and for the treatment and prevention of cardiovascular disorders because of its ability to promote the defibrination in vivo, decreasing blood viscosity and improving blood circulation.

Long-term controlled GDNF over-expression reduces dopamine transporter activity without affecting tyrosine hydroxylase expression in the rat mesostriatal system

The dopamine (DA) transporter (DAT) is a plasma membrane glycoprotein expressed in dopaminergic (DA-) cells that takes back DA into presynaptic neurons after its release. DAT dysfunction has been involved in different neuro-psychiatric disorders including Parkinson's disease (PD). On the other hand, numerous studies support that the glial cell line-derived neurotrophic factor (GDNF) has a protective effect on DA-cells. However, studies in rodents show that prolonged GDNF over-expression may cause a tyrosine hydroxylase (TH, the limiting enzyme in DA synthesis) decline. The evidence of TH down-regulation suggests that another player in DA handling, DAT, may also be regulated by prolonged GDNF over-expression, and the possibility that this effect is induced at GDNF expression levels lower than those inducing TH down-regulation. This issue was investigated here using intrastriatal injections of a tetracycline-inducible adeno-associated viral vector expressing human GDNF cDNA (AAV-tetON-GDNF) in rats, and doxycycline (DOX; 0.01, 0.03, 0.5 and 3mg/ml) in the drinking water during 5weeks. We found that 3mg/ml DOX promotes an increase in striatal GDNF expression of 12× basal GDNF levels and both DA uptake decrease and TH down-regulation in its native and Ser40 phosphorylated forms. However, 0.5mg/ml DOX promotes a GDNF expression increase of 3× basal GDNF levels with DA uptake decrease but not TH down-regulation. The use of western-blot under non-reducing conditions, co-immunoprecipitation and in situ proximity ligation assay revealed that the DA uptake decrease is associated with the formation of DAT dimers and an increase in DAT–α-synuclein interactions, without changes in total DAT levels or its compartmental distribution. In conclusion, at appropriate GDNF transduction levels, DA uptake is regulated through DAT protein–protein interactions without interfering with DA synthesis.

Probing Intersubunit Interfaces in AMPA-subtype Ionotropic Glutamate Receptors.

AMPA subtype ionotropic glutamate receptors (iGluRs) mediate the majority of fast neurotransmission across excitatory synapses in the central nervous system. Each AMPA receptor is composed of four multi-domain subunits that are organized into layers of two amino-terminal domain (ATD) dimers, two ligand-binding domain (LBD) dimers, transmembrane domains and carboxy-terminal domains. We introduced cysteine substitutions at the intersubunit interfaces of AMPA receptor subunit GluA2 and confirmed substituted cysteine crosslink formation by SDS-PAGE. The functional consequence of intersubunit crosslinks was assessed by recording GluA2-mediated currents in reducing and non-reducing conditions. Strong redox-dependent changes in GluA2-mediated currents were observed for cysteine substitutions at the LBD dimer-dimer interface but not at the ATD dimer-dimer interface. We conclude that during gating, LBD dimers undergo significant relative displacement, while ATD dimers either maintain their relative positioning, or their relative displacement has no appreciable effect on AMPA receptor function.

Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties.

Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten.

Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography–mass spectrometry

The number and positions of disulfide linkages in a therapeutic monoclonal antibody (mAb) play a crucial role in forming and stabilizing a correct mAb structure that is critical to its function. Peptide mapping by liquid chromatography–mass spectrometry (LC–MS) analysis of enzymatically digested mAb under nonreducing condition is a powerful method for disulfide linkage characterization to ensure mAb drug function and quality. However, the development of a robust sample preparation method with improved digestion efficiency and minimized disulfide scrambling for disulfide linkage analysis is essential but challenging. In this study, a sample preparation method for analysis of correct disulfide linkages in therapeutic mAbs was developed. Instead of common trypsin digestion, Lys-C plus trypsin was used in this approach to improve digestion efficiency. In addition, lower digestion temperature (25 °C) and lower digestion pH (pH 6.8) were also examined to minimize disulfide scrambling. Our results showed that Lys-C plus trypsin digestion at pH 6.8 and 25 °C is a better sample preparation condition for all therapeutic mAbs tested in this study because of a better digestion efficiency (all expected disulfide linkages can be confidently observed) and minimal disulfide scrambling.

Carboxypeptidase X-1 (CPX-1) is a secreted collagen-binding glycoprotein

Carboxypeptidase X-1 (CPX-1) is an atypical member of the carboxypeptidase (CP) family of proteins involved in a variety of physiological and pathological processes. However, unlike most other family members CPX-1 lacks catalytic activity making its biological function unclear. CPX-1 contains a 160 amino acid discoidin domain (DSD) that serves as a binding domain in other proteins prompting us to investigate a putative functional role for this domain in CPX-1. Sequence alignment confirmed the overarching homology between the DSD of CPX-1 and other DSDs whilst more detailed analysis revealed conservation of the residues known to form the collagen-binding trench within the DSD of the discoidin domain receptors (DDRs) 1 and 2. Biochemical characterisation of transiently expressed human CPX-1 revealed that CPX-1 was secreted in an N-glycosylation-dependent manner as treatment with the N-glycosylation inhibitor tunicamycin inhibited secretion concomitant with a reduction in CPX-1 mobility on Western blot. Using a collagen pull-down assay we found that secreted CPX-1 bound collagen and this appeared independent of N-glycosylation as treatment with PNGaseF did not affect binding. Further analysis under non-reducing and reducing (+DTT) conditions revealed that CPX-1 was secreted in both monomeric and dimeric forms and only the former bound collagen. Finally, mutation of a key residue situated within the putative collagen-binding trench within the DSD of CPX-1 (R192A) significantly reduced secretion and collagen-binding by 40% and 60%, respectively. Collectively these results demonstrate that CPX-1 is a secreted collagen-binding glycoprotein and provide a foundation for future studies investigating the function of CPX-1.

D-ribose-glycation of insulin prevents amyloid aggregation and produces cytotoxic adducts

Insulin is a key hormone regulating glucose homeostasis, intimately associated with glycemia and is exposed to glycation by glucose, reducing sugars and other highly reactive carbonyls, particularly in diabetes. Glycation of insulin has been reported to differentially affect protein structure, stability and aggregation depending on the glycating agent and experimental conditions. Under reducing conditions glycation produces higher insulin oligomerization thus accelerating amyloid formation whereas, in non-reducing conditions, glycation inhibits amyloid formation. To better detail the effect of glycation on insulin malfunction and toxicity, we investigated the effect of another glycating agent, the D-ribose. Recently, ribosylation has received great interest due to its role in protein glycation and its consequential effects such as protein aggregation, oxidative stress and cell death. Moreover, unusual high concentration of D-ribose has been detected in the urine of type II diabetics.Our results show that, using ribose, as glycating agent, the insulin conformation is preserved and does not evolve in amyloid aggregates because of the block of the α-helix to β-sheet transition, which initiates the aggregation process, maintaining the protein in a soluble state. At the same time, ribose-glycated insulin strongly affects the cell viability, starting a death pathway consisting in the activation of caspases 9 and 3/7, intracellular ROS production and activation of the transcription factor NF-kB.

Identification of immunodominant antigens for the laboratory diagnosis of toxocariasis

To identify immunodominant antigens of Toxocara canis recognised by Toxocara-infected sera as recombinant reagents for immunodiagnosis of toxocariasis. Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice. Eleven antigens with immunodiagnostic potential were identified, including two C-type lectins (CTLs) that reacted strongly with the Toxocara-positive serum pool. The first CTL (Tc-CTL-1) is the same as TES-32, previously identified as a major immunodominant component of TES; the second CTL (Tc-CTL-2) is a novel C-type lectin sharing 83% amino acid sequence identity within the functional domain of Tc-CTL-1. The E. coli-expressed recombinant Tc-CTL-1 was strongly recognised by the Toxocara-positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc-CTL-1 was higher when the unreduced protein was used in an enzyme-linked immunosorbent assay (ELISA), dot-blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc-CTL-1- and Tc-CTL-2-based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice. Both Tc-CTL-1 and Tc-CTL-2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens.

Soluble Collectin-12 (CL-12) Is a Pattern Recognition Molecule Initiating Complement Activation via the Alternative Pathway.

Soluble defense collagens including the collectins play important roles in innate immunity. Recently, a new member of the collectin family named collectin-12 (CL-12 or CL-P1) has been identified. CL-12 is highly expressed in umbilical cord vascular endothelial cells as a transmembrane receptor and may recognize certain bacteria and fungi, leading to opsonophagocytosis. However, based on its structural and functional similarities with soluble collectins, we hypothesized the existence of a fluid-phase analog of CL-12 released from cells, which may function as a soluble pattern-recognition molecule. Using recombinant CL-12 full length or CL-12 extracellular domain, we determined the occurrence of soluble CL-12 shed from in vitro cultured cells. Western blot showed that soluble recombinant CL-12 migrated with a band corresponding to ∼ 120 kDa under reducing conditions, whereas under nonreducing conditions it presented multimeric assembly forms. Immunoprecipitation and Western blot analysis of human umbilical cord plasma enabled identification of a natural soluble form of CL-12 having an electrophoretic mobility pattern close to that of shed soluble recombinant CL-12. Soluble CL-12 could recognize Aspergillus fumigatus partially through the carbohydrate-recognition domain in a Ca(2+)-independent manner. This led to activation of the alternative pathway of complement exclusively via association with properdin on A. fumigatus as validated by detection of C3b deposition and formation of the terminal complement complex. These results demonstrate the existence of CL-12 in a soluble form and indicate a novel mechanism by which the alternative pathway of complement may be triggered directly by a soluble pattern-recognition molecule.

Improved pore connectivity by the reduction of cobalt oxide silica membranes

This work investigated the permeation of binary gas mixtures in non-reducing (He/CO2) and reducing (H2/Ar) conditions at temperatures ranging from 200 to 500 °C. A common performance aspect under both non-reducing and reducing conditions was that the He and H2 purity in the permeate stream was independent of temperature for the tested binary gas mixtures, except at very high He/CO2 or H2/Ar concentrations (⩾90/10) in the retentate stream. Under non-reducing conditions, the transport of gases was consistent with molecular sieving properties of silica derived membranes, and He permeance was constant irrespective of the He/CO2 binary concentration tested. An anomalous H2 transport was observed under reduced conditions, as unexpectedly the H2 permeance was higher for gas mixtures instead of single gas. Further tests showed that H2 permeance increased 170% as the gas mixture changed from single H2 gas to H2/Ar gas mixtures. This was attributed to the experimental procedure, as the membranes were partially reduced each day and tested for gas permeation from pure H2 to lower H2 concentration in gas mixtures. Under these partial reducing conditions, H2 slowly reacts with the surface of the dense Co3O4 particle, thus forming a porous CoO region. The increase in H2 permeance was therefore attributed to improved pore connectivity between the silica structure and the porous CoO region.

β-Galactosidase from Ginkgo biloba seeds active against β-galactose-containing N-glycans: purification and characterization.

In this study, we purified an acidic β-galactosidase to homogeneity from Ginkgo biloba seeds (β-Gal'ase Gb-1) with approximately 270-fold purification. A molecular mass of the purified β-Gal'ase Gb-1 was estimated about 35 kDa by gel filtration and 32 kDa by SDS-PAGE under non-reducing condition, respectively. On the other hand, β-Gal'ase Gb-1 produced a single band with a molecular mass of 16 kDa by SDS-PAGE under reducing condition. The N-terminal amino acid sequences of 32 kDa and 16 kDa molecules were the same and identified as H-K-A-N-X-V-T-V-A-F-V-M-T-Q-H-, suggesting that β-Gal'ase Gb-1 may function as a homodimeric structure in vivo. When complex-type N-glycans containing β-galactosyl residues were used as substrates, β-Gal'ase Gb-1 showed substantial activity for β1-4 galactosyl residue and modest activity for β1-3 galactosyl residue with an optimum pH near 5.0. Based on these results, the involvement of β-Gal'ase Gb-1 in the degradation of plant complex-type N-glycans is discussed.

A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.