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PubMed1994

The role of calcium and N-linked glycans in the oligomerization and carbohydrate binding properties of human immunodeficiency virus external envelope glycoprotein.

Envelope glycoproteins of human immunodeficiency virus (gp120 and gp41) occur as oligomers. Here, we show by gel filtration analysis that gp120 oligomerization in vitro is calcium- and temperature-dependent. Recombinant gp120 (rgp120) species were recovered as monomers at 20 degrees C in the absence of calcium, but as tetramers at 37 degrees C in 10 mM CaCl2. Under the latter condition, N-glycanase-deglycosylated rgp120 formed hexamers. Relative to intact rgp120, which has been reported to display carbohydrate-binding properties for N-acetyl-beta-D-glucosaminyl and mannosyl residues, deglycosylation enhanced rgp120 specific binding to mannose-divinylsulfone-agarose, para-aminophenyl-beta-D-GlcNAc-agarose and fetuin-agarose matrices. Taken together, these results rule out the role of homologous lectin-carbohydrate interactions via N-linked glycans in the rgp120 oligomerization, even though its lectin properties may also be calcium-dependent. Deglycosylation may unmask domains of rgp120 polypeptide backbone that independently play a role either in rgp120 lectin activity or in calcium-dependent oligomerization.

Calcium↗
PubMed1990

Oligomerizations of deoxyadenosine bis-phosphates and of their 3'-5', 3'-3', and 5'-5' dimers: effects of a pyrophosphate-linked, poly(T) analog.

A pyrophosphate-linked polynucleotide analog based on thymidine 3',5' bis-phosphate (pTp) catalyzes the oligomerization of activated dimers of pdAp in the presence of MgCl2. Although no catalysis of the oligomerization of the activated monomer (ImpdAplm) was observed in the presence of MgCl2, there was a significant stimulation of oligomerization by the template in the presence of MnCl2.

Deoxyadenine Nucleotides↗
PubMed1991

Attempted nonenzymatic template-directed oligomerizations on a polyadenylic acid template: implications for the nature of the first genetic material.

Previous attempts to produce nonenzymatic template-directed oligomerizations of activated pyrimidines on polypurine templates have been unsuccessful. The only efficient reactions are those where the template is composed primarily of pyrimidines, especially cytosine. Because molecular evolution requires that a synthesized daughter polynucleotide be capable of acting as a template for the synthesis of the original polynucleotide, the one-way replication achieved thus far is inadequate to initiate an evolving system. Several uracil analogs were used in this investigation in order to search for possible replacements for uracil. The monomers used in this investigation were the imidazolides of UMP, xanthosine 5'-monophosphate, the bis-monophosphates of the acyclic nucleosides of uracil, and 2,4-quinazolinedione. The concentrations of various salts, buffers, pH, and temperature were among the different variables investigated in attempts to find conditions that would permit template-directed oligomerizations. Although the different monomers in this study demonstrated varying abilities to form very short oligomers, we were unable to detect any enhancement of this oligomerization that could be attributed to the poly(A) template. Although special conditions might be found that would allow purine-rich templates to work, these reactions cannot be considered robust. The results of our experiments suggest that pyrimidines were not part of the original replicating system on the primitive Earth. It has already been shown that ribose is an unlikely component of the first replicating systems, and we now suggest that phosphate was absent as well. This is due to the low solubility of phosphate in the present ocean (3 x 10(-6) M), as well as the difficulty of prebiotic activation of phosphates.

Evolution, Molecular↗
PubMed2004

Purinergic-receptor oligomerization: implications for neural functions in the central nervous system.

It is becoming clear that the functions of G protein-coupled receptors (GPCRs), the largest family of plasma membrane-localized receptors, are regulated by direct oligomeric formation between GPCRs, as either homo- or hetero-oligomers. This review article explores the mechanistic implications of GPCR dimerization, especially among purinergic receptors, adenosine receptors and P2 receptors, which play critical roles in the regulation of neurotransmission in the central nervous system. Briefly, adenosine receptors are able to form a heteromeric complex with P2 receptors that generates an adenosine receptor with P2 receptor-like agonistic pharmacology. This mechanism may be used to fine-tune purinergic inhibition locally at sites where there is a particular oligomerization structure between purinergic receptors, and to explain the undefined adenosine-like purinergic functions of adenine nucleotides. Purinergic receptors also form oligomers with GPCRs of other families present in the brain, such as dopamine receptors and metabotropic glutamate receptors, to alter the functional properties. The effect of GPCR oligomerization on receptor functions is thus considered as an important system in the central nervous system.

Animals↗
PubMed2005

Functionally independent subunits in the oligomeric structure of the GABA cotransporter rGAT1.

We have combined structural and functional approaches to investigate the role of oligomerization in the operation of the GABA transporter rGAT1. Xenopus laevis oocytes were induced to express, either separately or simultaneously, the wild-type form of rGAT1 and a mutated (Y140W) form, unable to translocate GABA and to generate transport currents, although its intramembrane charge movement properties are only slightly affected. These characteristics, together with the insensitivity of Y140W to the blocking action of SKF89976A, were used to study the possible functional interaction of the two forms in an heteromeric structure. The electrophysiological data from oocytes coexpressing wild-type and Y140W rGAT1 were consistent with a completely independent activity of the two forms. Oligomerization was also studied by fluorescence resonance energy transfer (FRET) in tsA201 cells expressing the transporters fused with cyan and yellow fluorescent proteins (ECFP and EYFP). All combinations tested (WT-ECFP/WTEYFP, Y140W-ECFP/Y140W-EYFP and WT-ECFP/ Y140W-EYFP) were able to give rise to FRET, confirming the formation of homo- as well as heterooligomers. We conclude that, although rGAT1 undergoes structural oligomerization, each monomer operates independently.

Animals↗
PubMed2007

Permeability changes of Manduca sexta midgut brush border membranes induced by oligomeric structures of different cry toxins.

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K(+) permeability. A fluorometric assay performed with 3,3'-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K(+)-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.

Animals↗
PubMed1999

Diphtheria toxin forms pores of different sizes depending on its concentration in membranes: probable relationship to oligomerization.

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed.

Dimerization↗
PubMed1997

Interaction of detergents with bovine lens alpha-crystallin: evidence for an oligomeric structure based on amphiphilic interactions.

We have studied the quaternary structure of alpha-crystallin in the presence of increasing concentrations of amphiphilic and neutral detergents using gel filtration, light-scattering, boundary and equilibrium sedimentation. We observed a continuous reduction of the molar mass of the polymeric alpha-crystallin on increasing the concentration of sodium dodecyl sulphate from 0.1 mM to 5 mM, ending up with the monomeric peptides. Dodecyltrimethylammonium bromide also disrupts the oligomeric structure of alpha-crystallin but the interaction appears to be cooperative: in the sharp transition region (for a 1 mg/ml protein solution) from 3 to 8 mM of the detergent, only the native protein and a mixture of monomeric and dimeric peptide-DTAB complexes can be observed. Concomitant studies of the circular dichroism in the far UV revealed a substantial decrease of the beta-sheet and increase of the alpha-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent alpha A and alpha B peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the alpha A and alpha B peptides and the formation of the oligomeric alpha-crystallin structure: the binding of the amphiphilic detergent reduces the beta-sheet content, induces the formation of alpha-helix secondary structure and reduces the tendency of the peptide to form large aggregates. The different mechanisms for reducing the oligomeric size by anionic and cationic detergents with identical apolar parts stresses the importance of charge interactions. Our findings support some aspects of the micelle model of alpha-crystallin and can be related to its chaperone activity.

Animals↗
PubMed2006

Effect of oxidation of alphaA- and alphaB-crystallins on their structure, oligomerization and chaperone function.

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H(2)O(2) on the structure, oligomerization, and chaperone function of alphaA- and alphaB-crystallins. Recombinant alphaA-and alphaB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H(2)O(2) and 0.1 mM FeCl(3) at 37 ( composite function)C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized alphaA- and alphaB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in beta-sheet or alpha-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of alphaA- and alphaB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.

Animals↗
PubMed1983

Factors affecting the oligomeric structure of yeast external invertase.

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.

Extracellular Space↗
PubMed1983

Oligomerization of malate synthase during glyoxysome biosynthesis.

The octameric malate synthase, found in glyoxysomes of plants, is synthesized as monomeric precursor in the cytoplasm. The precursor form does not possess a different subunit molecular weight than the mature organellar enzyme, but differs from the organellar protein by not oligomerizing and aggregating. This was shown by synthesis in a cell-free reticulocyte lysate system programmed with cucumber poly A+-mRNA followed by immunoprecipitation of the radiolabeled translation products. The precursor form of malate synthase was also detected in vivo in the cytosol of pulse-labeled cucumber cotyledons after immunoprecipitation of the radiolabeled polypeptide. At low salt concentrations, mature malate synthase can be converted into aggregated forms. However, the precursor form obtained either by in vitro translation or by extraction from the cytosol after short pulses of radioactive methionine, could neither be oligomerized into the octameric form nor aggregated into the 100-S form. Processing of malate synthase, assumed to be a requisite for oligomerization, took place rapidly in the glyoxysomes, but proceeded only slowly in the cytosol. This was demonstrated both by the uptake of in vitro-translated malate synthase into glyoxysomes, and by analysis of newly synthesized malate synthase detectable in glyoxysomes in vivo. In both cases the octamer was by far the predominant form.

Cell Compartmentation↗
PubMed1992

Detergents affect insulin binding, tyrosine kinase activity and oligomeric structure of partially purified insulin receptors.

Insulin receptor activities, i.e., insulin binding and tyrosine kinase activation depend on the lipid environment of the receptor. As detergent may disrupt or interfere with this environment, we investigated the effect of various common detergents on insulin receptor properties. Experiments were carried out (i) on solubilized and partially purified insulin receptor and (ii) on the receptor reconstituted into phosphatidylcholine vesicles. The detergents tested, Triton X-100, octyl-beta-D-glucopyranoside, octyl-beta-D-thioglucopyranoside, 3[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid (Chaps), and Na deoxycholate affected the insulin receptor properties differently when compared with the control receptor in the absence of detergent. On the partially purified insulin receptor, Na deoxycholate inhibited both insulin receptor activities; octyl-beta-D-glucopyranoside and octyl-beta-D-thioglucopyranoside decreased insulin binding and kinase activation as their concentration increased, particularly above their respective critical micellar concentration (CMC). Triton X-100 was the only detergent which allowed an increase of insulin binding and kinase activation throughout the whole range of concentrations assayed. Reconstitution of the receptor into phosphatidylcholine vesicles protected the receptor from the direct effects of the detergents, for both the stimulation observed with Triton X-100 and the inhibition produced by the other detergents. In order to determine the effect of detergents on the oligomeric forms of the soluble insulin receptor, we investigated a new rapid sucrose gradient centrifugation technique. Insulin receptors were detected on the gradient by 125I insulin binding. For low concentrations of detergent, i.e., near the CMC, octylglucoside, Chaps, and Triton X-100 favored the (alpha 2 beta 2)2 oligomeric form of the receptor. Higher concentrations of Triton X-100 did not modify the polymeric state of the receptor. In contrast, octylglucoside and Chaps induced an increase in the sedimentation coefficient of the receptor which appeared as (alpha 2 beta 2)3 and (alpha 2 beta 2)4 forms. These alterations in the oligomerization status of the insulin receptor may explain the deleterious effects observed with both Chaps and octylglucoside at higher concentrations.

Centrifugation, Density Gradient↗
PubMed1995

Correct oligomerization is a prerequisite for insertion of the central molecular domain of staphylococcal alpha-toxin into the lipid bilayer.

Staphylococcal alpha-toxin is a primarily hydrophilic molecule that binds as a monomer to target membranes and then aggregates to form amphiphilic oligomers that represent water-filled transmembrane channels. Current evidence indicates that a region located in the center of the molecule inserts deeply into the bilayer. In the present study, we sought to determine whether membrane insertion was triggered by the oligomerization process, and whether insertion correlated with pore formation. Double mutants of alpha-toxin were prepared in which His-35 was replaced by Arg, and cysteine residues were introduced at positions 69, 130 and 186. Substitution of His-35 with Arg rendered the toxin molecules incapable of proper oligomerization, so that they remained in nonlytic form after binding to membranes. The sulfhydryl groups were labelled with the polarity-sensitive fluorescent dye acrylodan. Functionally intact, single mutant toxins containing only the cysteine residues were utilized as controls. Measurements of the fluorescence emission spectrum of acrylodan were performed for the active and inactive alpha-toxin mutants in free solution and in membrane-bound form. The collective results demonstrate that proper oligomerization is required for membrane insertion of the central region in the alpha-toxin molecule, and that lack of insertion correlates with absence of pore formation.

2-Naphthylamine↗
PubMed1995

Homologous ELISA for detection of oligomeric human TNF: properties of the assay.

In order to quantify oligomeric human tumor necrosis factor-alpha (TNF), we have developed a sensitive homologous enzyme-linked immunosorbent assay (Hm-ELISA) using the same monoclonal antibody (MoAb) for both solid and liquid phase. Different anti-TNF MoAb have been compared in terms of their efficacy in the Hm-ELISA, affinity, neutralization capacity and epitope specificity. The data suggest, that effectiveness in the Hm-ELISA may represent a novel characteristic of MoAb. Of the MoAbs tested, 5 N was capable of recognizing oligomeric TNF in the Hm-ELISA with a detection limit of 15 pg/ml. Furthermore, using Hm-ELISA against human TNF, interleukin-8 (IL-8) and lymphotoxin, we have demonstrated that these cytokines are oligomeric in physiological solutions, but are converted into monomeric forms in the presence of the non-ionic detergent Tween 20. High salt buffer was employed to abrogate a nonspecific false positive reaction in the Hm-ELISA found in nearly half of the plasma samples obtained from healthy subjects. Finally, a good correlation between the Hm-ELISA and the L929 bioassay was observed for natural and recombinant TNF measured in human plasma.

Animals↗
PubMed1986

Kinetics of nuclear transport and oligomerization of simian virus 40 large T antigen.

The kinetics of nuclear transport and of oligomerization of simian virus 40 (SV40) large T antigen in lytically infected cells were investigated by pulse-chase experiments, cell fractionation, and sedimentation analyses in sucrose gradients. After synthesis, large T was rapidly translocated to the nucleus. Within 10 min, half of the pulse-labeled molecules had entered the nucleus and after an additional 30 min, nuclear accumulation of large T reached a constant plateau of about 95%. Within that time, the majority of large T was in monomeric form suggesting that nuclear transport takes place in this state. In the nucleus, conversion to tetramers proceeded slowly and steadily. By 60 min half of the molecules had formed tetramers and by 6 hr a steady-state ratio between tetramers and monomers of 4:1 was observed. A small fraction of large T remaining in the cytoplasm oligomerized considerably faster than large T in the nuclear fraction. This phenomenon of accelerated oligomerization was also observed with a mutant of large T defective for nuclear transport. Perhaps, the nuclear envelope is a barrier for the complex forms of large T which prevents premature oligomers in the cytoplasm from entering the nucleus and oligomers in the nucleus from migrating back to the cytoplasm.

Animals↗
PubMed2006

Oligomerization of nitrophorins.

Rhodnius prolixus is a blood-sucking insect that uses a mixture of nitrophorin (NP) proteins to deliver nitric oxide (NO) from the insect saliva to the hosts via a ferric heme coordinated to the protein, causing vasodilatation and anticoagulation to support their feeding. R. prolixus NPs 1-4 are very similar proteins ( approximately 20 kDa) with different NO affinities for stepwise NO release triggered by pH increase and histamine binding in hosts. Ultra-high-resolution X-ray structures of native and mutant NPs and their kinetic analysis already have revealed the fundamental steps of NO binding and release. In this study, we found that NPs can exist in multiple oligomerization states at higher concentrations. The oligomers are characterized by a combination of multiple biophysical methods. The intrinsic features of the oligomerization revealed here led us to propose that this intensive, moderately pH- and ligand-dependent oligomerization of NPs has physiological implications in the facilitation of the efficient storage and release of the highly reactive NO in the insect saliva and the victim, respectively.

Animals↗
PubMed2006

Effect of temperature and pH on the secondary structure and processes of oligomerization of 19 kDa alpha-zein.

Highly hydrophobic protein Z19 zein shows a tendency towards oligomerization. The role of temperature and pH on the oligomerization process was studied monitoring the secondary structure content and the appearance of aggregates by Circular Dichroism Spectroscopy (CD) and Dinamic Light Scattering (DLS). Z19 zein suffers irreversible thermal denaturalization, as demonstrated by far-UV CD measurements. DLS data indicate that this denaturalization is accompanied by oligomerization processes which are strongly dependent on temperature. The aggregates that appear when the sample is heated maintain a certain amount of their native structure. Oligomers, showing high stability to temperature changes and other denaturing conditions with molecular weights of 45, 66 kDa and higher, were detected by SDS-PAGE. The secondary structure strongly depends on pH. Thus, at pH above pI (6.8), all the protein structure is in alpha helix. The formation of disulfide bonds plays an important role in the aggregation process, since most of the sulfhydryls in the protein (97.52%) form disulfide bonds and only 2.47% of them are free and superficially exposed. The sensitivity towards thermal denaturalization is also affected by pH rises.

Circular Dichroism↗
PubMed2004

Heat-induced oligomerization of gp96 occurs via a site distinct from substrate binding and is regulated by ATP.

Gp96 (GRP94) is a dimeric glycoprotein and is the endoplasmic reticulum representative of the hsp90 family of molecular chaperones. In addition to the protein substrates it chaperones, gp96 binds weakly to both peptides and ATP, and has been shown to self-assemble into discrete oligomers upon heat shock at 50 degrees C, although physiological roles for these phenomena have not been well established. Our studies indicate that gp96 homooligomerizes irreversibly in vitro at temperatures as low as 42 degrees C and could involve pre-dissociation of dimers to monomers. Oligomerization is inhibited significantly by ATP; hydrolysis is not required, since ADP, ATP-gamma-S, and NECA inhibit self-assembly equally well. Peptide ligands do not competitively inhibit gp96 self-assembly and, in fact, bind to all oligomeric species, including the dimer. Together, these findings suggest that (1) heat-enhanced chaperone activity does not reside in oligomers per se, and (2) the regions of gp96 involved in peptide binding and oligomerization are distinct.

Adenosine Triphosphate↗