Chaperone Skp from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> exhibits immunoglobulin G binding ability

A low-molecular-weight cationic protein that can bind human and rabbit immunoglobulins G has been isolated from Yersinia pseudotuberculosis cells. This immunoglobulin binding protein (IBP) interacts with IgG Fc-fragment, the association constant of the resulting complex being 3.1 μM^?1. MALDI-TOF mass spectrometry analysis of IBP revealed its molecular mass of 16.1 kDa, and capillary isoelectrofocusing analysis showed pI value of 9.2. N-Terminal sequence determination by Edman degradation revealed the sequence of the 15 terminal amino acid residues (ADKIAIVNVSSIFQ). Tryptic hydrolysate of IBP was subjected to MALDI-TOF mass spectrometry for proteolytic peptide profiling. Based on the peptide fingerprint, molecular mass, pI, and N-terminal sequence and using bioinformatic resources, IBP was identified as Y. pseudotuberculosis periplasmic chaperone Skp. Using the method of comparative modeling a spatial model of Skp has been built. This model was then used for modeling of Skp complexes with human IgG1 Fc-fragment by means of molecular docking.     

Ice recrystallization inhibition mediated by a nuclear-expressed and -secreted recombinant ice-binding protein in the microalga Chlamydomonas reinhardtii

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a “green” gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a “green” hydrate inhibitor.     

Cloning and expression of a pharmacologically unique bovine peripheral-type benzodiazepine receptor isoquinoline binding protein.

High affinity binding of isoquinolines, such as PK 11195, is a conserved feature of peripheral-type benzodiazepine receptors (PBR) across species. However, species differences in PBR ligand binding have been described based on the affinity for N1-alkyl-1,4-benzodiazepines, such as Ro5-4864. Ro5-4864 binds with high affinity to the rat receptor but has low affinity for the bovine PBR. Photolabeling with an isoquinoline ligand, [3H]PK 14105, identifies a 17-kDa protein, the PBR isoquinoline binding protein (PBR/IBP), in both species. To further elucidate the role of the PBR/IBP in determining PBR benzodiazepine and isoquinoline binding characteristics, the bovine PBR/IBP was cloned and expressed. Using a cDNA encoding a rat PBR/IBP to screen a fetal bovine adrenal cDNA library, a bovine cDNA encoding a polypeptide of 169 residues was cloned. The bovine and rat PBR/IBPs had similar hydropathy profiles exhibiting five potential transmembrane domains. Transfecting the cloned bovine PBR/IBP cDNA into COS-7 cells resulted in an 11-fold increase in the density of high affinity [3H]PK 11195 binding sites which had only low affinity for Ro5-4864. Expression of the bovine PBR/IBP yields a receptor which is pharmacologically distinct from both endogenous COS-7 PBR and the rat PBR based on the affinity for several N1-alkyl-1,4-benzodiazepine ligands. These results suggest the PBR/IBP is the minimal functional component required for PBR ligand binding characteristics and the different protein sequences account for the species differences in PBR benzodiazepine ligand binding.     

A stable engineered human IgG3 antibody with decreased aggregation during antibody expression and low pH stress

Human IgG comprises four subclasses with different biological functions. The IgG3 subclass has a unique character, exhibiting high effector function and Fab arm flexibility. However, it is not used as a therapeutic drug owing to an enhanced susceptibility to proteolysis. Antibody aggregation control is also important for therapeutic antibody development. To date, there have been few reports of IgG3 aggregation during protein expression and the low pH conditions needed for purification and virus inactivation. This study explored the potential of IgG3 antibody for therapeutics using anti‐CD20 IgG3 as a model to investigate aggregate formation. Initially, anti‐CD20 IgG3 antibody showed substantial aggregate formation during expression and low pH treatment. To circumvent this phenomenon, we systematically exchanged IgG3 constant domains with those of IgG1, a stable IgG. IgG3 antibody with the IgG1 CH3 domain exhibited reduced aggregate formation during expression. Differential scanning calorimetric analysis of individual amino acid substitutions revealed that two amino acid mutations in the CH3 domain, N392K and M397V, reduced aggregation and increased CH3 transition temperature. The engineered human IgG3 antibody was further improved by additional mutations of R435H to obtain IgG3KVH to achieve protein A binding and showed similar antigen binding as wild‐type IgG3. IgG3KVH also exhibited high binding activity for FcγRIIIa and C1q. In summary, we have successfully established an engineered human IgG3 antibody with reduced aggregation during bioprocessing, which will contribute to the better design of therapeutic antibodies with high effector function and Fab arm flexibility.     

An extracellular ice-binding glycoprotein from an Arctic psychrophilic yeast

A psychrophilic yeast was isolated from an Arctic pond and its culture supernatant showed ice-binding activity. This isolate, identified as Leucosporidium sp. based on an analysis of the D1/D2 and ITS regions of its ribosomal DNA, produced a secretory ice-binding protein (IBP). Yeast IBP was purified from the culture medium to near homogeneity by the ice affinity method and appeared to be glycosylated with a molecular mass of ∼26 kDa. In addition, the yeast IBP was shown to have thermal hysteresis (TH) and recrystallization inhibition (RI) activities. The full-length cDNA for yeast IBP was determined and was found to encode a 261 amino acid protein with molecular weight of 26.8 kDa that includes an N-terminal signal peptide and one potential N-glycosylation site. The deduced protein showed high sequence identity with other IBPs and hypothetical IBPs from fungi, diatoms, and bacteria, clustering with a class of ice-active proteins.     

Effect of pH on the physico-chemical and immunologic properties of rabbit antibodies

The effects of an incubation at low pH (during 20 h at 37 degrees) on the antibody activity and anticomplementary activity of rabbit IgG have been studied. Modifications have also been examined by physicochemical methods. The properties of rabbit anti-sheep red cell IgG are not modified by incubation at pH values between 7.4 and 4.0 during 20 h at 37 degrees. Below pH 4 a decrease of hemolytic activity is apparent concomitant with an important increase of the agglutinating activity. This phenomenon is due to the formation of polymers from native IgG. At pH values below 3.8 the anticomplementary activity of a nonspecific IgG decreases rapidly. One observes an increase of optical rotation, a finding which is compatible with the appearance of heavier compounds with sedimentation coefficients of 9.5 and 11.5 S, probably dimers of native IgG. The increase of optical rotation is partially reversible when the pH is readjusted to 7.4. The use of starch-gel and immunoelectrophoresis has shown the appearance of compounds with higher mobility which are closely related to a peptide (PEP III') which was isolated from a peptic hydrolysate of rabbit IgG. The decrease of anticomplementary activity of nonspecific IgG seems to be closely related to the liberation of PEP III'.     

Overexpression of the Interferon regulatory factor 4-binding protein in human colorectal cancer and its clinical significance

Background: IFN regulatory factor 4-binding protein (IBP) is a novel type of activator of Rho GTPases. It has been linked with differentiation and apoptosis of lymphocytes, but its function in oncogenesis remains unclear. Here we studied the expression of endogenous IBP in four human colorectal cancer cell lines, normal, adenoma and tumor colorectal tissues. Methods: Molecular (Western blot and RT-PCR), and confocal analyses were used to investigate IBP expression in human colorectal cancer cell lines. Matched normal and tumor tissue sections of 63 patients and 15 adenoma tissue sections were analyzed for IBP expression by immunohistochemistry (IHC). Results: IBP was ubiquitely expressed in human colorectal cancer cell lines. The expression of IBP can be detected at both the mRNA and protein level in SW480, SW620 and HT29 cells. Clinically, IBP were elevated in human colorectal cancer specimens in comparison to normal colorectal tissues. Substantial high expression of IBP was observed in colorectal cancer tissues (67%), whereas corresponding normal tissues and 15 adenoma tissues showed consistently absent immunoreactivity of IBP. Moreover, IBP expression is correlated with the differentiation level of colorectal cancer cells (p < 0.05) and clinical stage of patients (p < 0.01). Conclusions: Our data show, for the first time, a dysregulated expression of IBP in human colorectal cancer, offering new perspectives for its role in cancer development and progression. IBP may be a novel tumor marker and a therapeutic target for colorectal cancer.     

NOVEL ICE‐BINDING PROTEINS FROM A PSYCHROPHILIC ANTARCTIC ALGA (CHLAMYDOMONADACEAE,CHLOROPHYCEAE)1

Many cold‐adapted unicellular plants express ice‐active proteins, but at present, only one type of such proteins has been described, and it shows no resemblance to higher plant antifreezes. Here, we describe four isoforms of a second and very active type of extracellular ice‐binding protein (IBP) from a unicellular chlamydomonad alga collected from an Antarctic intertidal location. The alga is a euryhaline psychrophile that, based on sequences of the alpha tubulin gene and an IBP gene, appears to be the same as a snow alga collected on Petrel Island, Antarctica. The IBPs, which do not resemble any known antifreezes, have strong recrystallization inhibition activity and have an ability to slow the drainage of brine from sea ice. These properties, by maintaining liquid environments, may increase survival of the cells in freezing environments. The IBPs have a repeating TXT motif, which has previously been implicated in ice binding in insect antifreezes and a ryegrass antifreeze.     

Effect of solution pH and ionic strength on the separation of albumin from immunoglobulins (IgG) by selective filtration

Although protein fractionation by selective membrane filtration has numerous potential applications in both the downstream processing of fermentation broths and the purification of plasma proteins, the selectivity for proteins with only moderately different molecular weights has generally been quite poor. We have obtained experimental data for the transport of bovine serum albumin (BSA) and immunoglobulins (IgG) through 100,000 and 300,000 molecular weight cutoff polyethersulfone membranes in a stirred ultrafiltration device at different solution pH and ionic strength. The selectivity was a complex function of the flux due to the simultaneous convective and diffusive solute transport through the membrane and the bulk mass transfer limitations in the stirred cell. Under phsioligical conditions (pH 7.0 and 0.15 M NaCI) the maximum selectivity for the BSA-IgG separation was only about 2.0 due primarily to the effects of protein adsorption. In contrast, BSA-IgG selectivities as high as 50 were obtained with the same membranes when the protein solution was at pH 4.8 and 0.0015 M NaCl. This enhanced selectivity was a direct result of the electrosatatic contributions to both bulk and membrane transport. The membrane selectivity could actually be reversed, with higher passage of the larger IgG molecules, by using a 300,000 molecular weight cutoff membrane at pH 7.4 and an ionic strength of 0.0015 M NaCl. These results clearly demonstrate that the effectiveness of selective protein filtration can be dramatically altered by appropriately controlling electrostatic interactions through changes in pH and/or ionic strength. (c) 1994 John Wiley & Sons, Inc.     

Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding

Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.     

Composition and bioactivity of polysaccharides from Inula britannica flower

In this paper, the composition and biological activities of polysaccharides from Inula britannica flower IBP obtained by water extraction were investigated. The properties and chemical compositions of IBP were analyzed with HPLC and IR methods. The results showed that IBP consisted of two kinds of polysaccharides with the molecular weight of 3500Da, 700Da. IBP consisted of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, arabinose with a molar ratio of 4.1:1:1.4:2.7:14.6:6.3:7.9. The IR spectrum of IBP revealed the typical characteristics of polysaccharides and protein. IBP was administered orally at three doses [100, 200 and 400mg/kg body weight] for 14 days to the diabetic mice induced by alloxan. The body weight, plasma glucose, serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and liver glycogen were evaluated in normal and alloxan-induced diabetic mice. IBP could dose-dependently significantly increase the body weight of diabetic mice, and reverse the decrease of plasma glucose, glycogen and the decrease of blood lipid of diabetic mice as compared to those in control group. These results indicated that IBP could be developed to a potential anti-diabetic drug in the future.     

Influence of pH and ionic strength on the adsorption, leaching and activity of myoglobin immobilized onto ordered mesoporous silicates

Myoglobin has been immobilized onto different ordered mesoporous silicates. The effect of the pH on the adsorption, leaching and activity was studied. The results showed that the maximum amount of protein was adsorbed at a pH 6.5, just below the protein isoelectric point (7–7.3). There was no effect of increasing ionic strength on the adsorption profile at different pH values. The adsorption is rationalized in terms of local electrostatic forces acting between the enzyme and the silica surface as well as hydrophobic interactions close to the protein isoelectric point, whereas at low pH the global charges give rise to protein–protein repulsion and at high pH enzyme–silica repulsion. Higher amounts of immobilized myoglobin were leached at a pH 4, while lower amounts were leached at pH 6.5. The catalytic activity of myoglobin immobilized onto SBA-15 showed optimal activity at a pH 6.5 in comparison to a pH of 5 for the free form.     

Influence of culture conditions and virulence plasmids on expression of immunoglobulin-binding proteins of Yersinia pseudotuberculosis

The influence of culture conditions and plasmids on immunoglobulin (Ig)-binding activity of two isogenic strains of Yersinia pseudotuberculosis (plasmid-free strain 48(-)82(-) and strain 48(+)82(+) bearing plasmids pYV48 and pVM82) was studied. The highest activity was observed in the bacteria grown on glucose-containing liquid medium in the stationary growth phase. The Ig-binding activity of the bacteria cultured on the liquid medium at pH 6.0 was about 1.5-fold higher than that of the bacteria grown at pH 7.2. Expression of the Ig-binding proteins (IBPs) was most influenced by temperature of cultivation. The IBP biosynthesis was activated in the bacteria grown at 4 degrees C and markedly decreased in those grown at 37 degrees C. The Ig-binding activity of lysates from the bacteria was caused by proteins with molecular weights of 7-20 kD. The activities of the plasmid-free and plasmid-bearing Y. pseudotuberculosis strains (48(-)82(-) and 48(+)82(+), respectively) were analyzed, and the plasmids were shown to have no effect on the IBP expression and biosynthesis, which seemed to be determined by chromosomal genes.     

l-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions

Liquid intravenous immunoglobulin (IVIG) products offer improved convenience in preparation but often lack sufficient stability to allow room temperature storage. Furthermore, clinical tolerability may be affected due to formation of idiotype/anti-idiotype IgG dimers and/or aggregates. Here we report on the development of a 10% IVIG formulation with optimized stability achieved by the use of l-proline. The stability of concentrated liquid IVIG was strongly pH dependent. Aggregate formation, yellowish discoloration of the solution and loss of anti-hepatitis B surface antigen (HBs) antibody activity was minimal at intermediate pH (pH 4.8–5.3). Fragmentation of IgG was highest at low pH (pH 4.1). Idiotype/anti-idiotype IgG dimer formation was highest at neutral pH and was reduced with decreasing pH. The presence of l-proline further improved stability by inhibiting protein aggregation, reducing loss of anti-HBs antibody activity and decreasing coloring, particularly compared with glycine formulations. The IgG dimer content was up to 30% lower in solutions containing l-proline compared with those containing glycine or other stabilizers. In conclusion, a weakly acidic pH of approximately 5 and l-proline as stabilizer are optimal conditions for long-term stability of a liquid IVIG. l-proline, an amphiphilic, naturally occurring amino acid, is superior to glycine in restricting IgG dimer formation.     

IBP-mediated suppression of autophagy promotes growth and metastasis of breast cancer cells via activating mTORC2/Akt/FOXO3a signaling pathway

Interferon regulatory factor-4 binding protein (IBP) is a novel upstream activator of Rho GTPases. Our previous studies have shown that ectopic expression of IBP was correlated with malignant behaviors of human breast cancer cells, and invasive human breast cancer had high expression of IBP that promoted the proliferation of these cells. However, it remains unknown whether autophagy inhibition contributes to IBP-mediated tumorigenesis. In this study, we for the first time, reported that upregulation of IBP expression significantly suppressed the autophagy of breast cancer cells, and downregulation of IBP expression markedly induced autophagy of these cells. Further investigation revealed that IBP effectively counteracted autophagy by directly activating mammalian target of rapamycin complex 2 (mTORC2) and upregulating phosphorylation of Akt on ser473 and FOXO3a on Thr32. Moreover, IBP-mediated suppression of autophagy was dependent on mTORC2/Akt/FOXO3a signaling pathway. Finally, our results demonstrated that IBP-mediated breast cancer cell growth in vitro and in vivo was strongly correlated with suppression of mTORC2-dependent autophagy. These findings suggest that the anti-autophagic property of IBP has an important role in IBP-mediated tumorigenesis, and IBP may serve as an attractive target for treatment of breast cancer.     

Immunoglobulin classes of anti-Sendai virus antibody detected by ELISA in infected nude mouse serum

Sendai virus-infected nude mouse sera obtained on the seventh day after infection or later, in which anti-Sendai virus antibodies were undetectable by hemagglutination-inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase-conjugated anti-mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti-Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti-Sendai virus antibody activity of fractions from Sephadex G-200 gel filtration was detected in the IgM fraction when anti-mouse mu chain-specific antiserum was used and in both IgG and IgM fractions when heavy and light chain-specific anti-mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A-Sepharose affinity chromatography of Sendai virus-infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti-Sendai virus anti-bodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.     

The peripheral-type benzodiazepine receptor and tumorigenicity: isoquinoline binding protein (IBP) antisense knockdown in the C6 glioma cell line

Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.     

Immunoglobulin binding by the regular surface array of Aeromonas salmonicida

The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.     

Comparison of the inactivation of IgM and IgG complement fixation sites by acid and base.

Rabbit IgM antibodies to denatured mammalian or T6 bacteriophage DNA or poly(A)-poly(U) irreversibly lost complement-(C) fixation reactivity on exposure to low pH and reneutralization, with a halving of the complement-fixation titer occurring after treatment at about pH 3. The titers of IgG antibodies to denatured phage DNA, to poly(A)-poly(U), or to hemocyanin were halved only after exposure to pH 2. Inactivation by acid was enhanced by low protein concentrations, incubation at higher temperatures, and by slow reneutralization; under all these conditions it was more extensive with IgM than with IgG. Inactivation of IgM C-fixation activity at pH 2.5 and room temperature was a first order reaction, with a half-time of about 20 min. Both classes retained antigen-binding activity after exposure to pH 2. In the alkaline range, full C-fixation reactivity was retained by both classes after reneutralization from pH 11.5, some loss occurred at pH 12, and total irreversible inactivation occurred by pH 12.5. In the latter case, antigen-binding activity was also lost. The C-fixation inactivation curves in the alkaline range were similar for IgG and IgM antibodies.