Effect of nucleotides on the oligomeric state of human lactoferrin

Lactoferrin (LF) is a multifunctional acute-phase protein involved in nonspecific defense against bacteria, viruses, and cancer diseases and is present in human barrier fluids, blood, and milk. Small-angle X-ray scattering (SAXS) and light scattering (LS) demonstrated for the first time that LF occurs in the form of oligomers, with a high monomer unit number in the solution. The degree of LF oligomerization depends on the LF concentration and the storage period of non-frozen neutral LF solutions. The average inertial radius of scattering particles (R _g) reaches 100–450 Å at LF concentrations comparable with those in human milk, while R _g of LF monomers is 26.7 Å. LF forms complexes with various nucleotides and hydrolyzes them. The addition of ATP or AMP to LF solutions accelerates LF oligomerization and increases R _g to 600–700 Å, regardless of the initial degree of LF oligomerization. According to the different models (sphere, plate, and cylinder) of LF aggregates, its complexes with such R _g presumably contain several tens to thousands of LF monomers. The possible role of oligomeric complexes in multiple biological functions of LF is discussed.     

DNA,oligosaccharides, and mononucleotides stimulate oligomerization of human lactoferrin

Using small‐angle X‐ray scattering (SAXS), light scattering (LS), and soft laser ablation we have shown that lactoferrin (LF) in solution at neutral pH is oligomerized in the absence of salt or at physiological salt concentrations. The level of oligomerization depends on the concentration of LF, KCl or NaCl, and on the duration of the protein storage in solution. At the concentrations comparable with those in human milk (1 ÷ 6 mg/ml), the average radius of gyration (R_g) values of LF can attain 400 ÷ 480 Å´, while fresh solution of previously lyophylized LF demonstrate a lower average R_g (50 ÷ 100 Å´), and R_g value characterizing the LF monomer formed at 1 M NaCl is 26.7 Å´. The addition of oligonucleotides, oligosaccharides, or mononucleotides to LF in the presence or in the absence of KCl with different level of initial oligomerization accelerates the oligomerization rate and increases the R_g values up to ～600 ÷ 700 Å´, which correspond to associates containing ten or more protein molecules. During gel filtration on Sepharose 4B, high‐degree LF oligomers dissociate nearly completely forming different degraded complexes, but in some cases it is possible to reveal small amount of a decamer. A possible role for oligomerization of LF, a highly polyfunctional protein, for its different biological activities is discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Human milk lactoferrin hydrolyzes nucleoside-5'-triphosphates

Lactoferrin (LF) is a main iron-transfering glycoprotein of human barrier body fluids, blood and milk. LF, a protein of the acute phase, is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. LF is an important component of the passive immunity of newborns system. LF, an extremely polyfunctional protein, is the object of intensive investigations. In this work electrophoretically homogeneous LF from human milk was prepared. Affinity chromatography of LF on Blue Sepharose separated the protein into several distinct isoforms with different affinities to this resin. Two of this isoforms possess nucleoside-5'-triphosphate-hydrolyzing activity. Using several methods including in-gel ATPase activity assays, we show that ATP (and others NTP) hydrolysis is an intrinsic property of LF, and that LF is the major ATPase of human milk. It was shown that ATP-hydrolyzing site is located in C-lobe of LF.     

DNA and oligosaccharides stimulate oligomerization of human milk lactoferrin

Lactoferrin (LF) is a Fe³⁺-transferring glycoprotein and is contained in human barrier fluids, blood, and milk. LF is an acute phase protein, is involved in nonspecific defense, and displays a unique set of biological functions. Small-angle X-ray scattering and light scattering experiments demonstrated that DNA and oligosaccharides added to LF with various levels of initial oligomerization increased the oligomerization rate. Almost complete dissociation into monomers was observed when 1 M NaCl was added to LF oligomers obtained in the presence of DNA, oligosaccharides, and nucleotides, previously identified as oligomerization effectors. LF complexes obtained with different oligomerization effectors differed in stability. Incubation with 50 mM MgCl₂ completely destructed LF complexes formed in the presence of ATP and oligosaccharides but only partly destructed AMP- and d(pT)₁₀-dependent complexes, which was followed by the formation of new complexes with a higher salt stability. A possible role of oligomerization in various LF functions is discussed.     

Analysis of oligomeric forms of recombinant human leukocyte interferons

Using SDS-PAAG electrophoresis, gel-permeation HPLC and immunoblotting, it was demonstrated that homogeneous preparations of human leukocyte interferons (alpha-INF)-A, -N and -I1 obtained from the biomass of the corresponding producer strains (Pseudomonas sp.) contained several oligomeric forms produced by way of S-S intermolecular cross-linkage and making up to 10-15%, 4-7% and 2-5% of the total monomeric form content in the protein preparations. Immunologic testing with the use of MAB NK-2 and [125I]NK-2 showed that the oligomeric forms of alpha-INF-A, -N and -I1 were present in the protein preparations at all purification stages and seemed to be formed at early steps of interferon synthesis in the cell. The effects of limited proteolysis as well as of acid, alkaline and thermodenaturation on the aggregation and oligomerization of alpha-INF-A were studied. SDS-PAAG electrophoresis performed in the absence of the reducing agents showed that upon denaturation of 10% TCA, the amount of the oligomeric forms in the preparations of homogeneous and especially partly proteolytic INF was significantly increased. The causes and the putative mechanisms of aggregation and oligomerization of INF are discussed.     

Human Milk Lactoferrin Hydrolyzes Ribonucleoside 5′-Triphosphates

Lactoferrin (LF), one of the most important and polyfunctional factors of nonspecific cell defense against bacteria, viruses, and cancer diseases, is the main iron-transferring protein of human biological fluids, blood, and milk. Electrophoretically homogeneous preparations of LF from human milk were obtained and studied. Using chromatography on Blue Sepharose, LF preparations were separated into several subfractions differing in their affinity for the sorbent, two of which exhibited nucleoside 5-triphosphate-hydrolyzing activity. Using different methods, including in-gel ATPase activity assay, it was demonstrated that hydrolysis of ATP and other nucleotides is an intrinsic property of LF and that LF is the major ATPase of human milk. It is shown that the ATP-hydrolyzing center is located at the C-terminal domain of the LF molecule and that interaction between ATP and this center increases the oligonucleotide-hydrolyzing activity of the DNA-binding domain of this protein.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 513–520.Original Russian Text Copyright © 2005 by Babina, Semenov, Buneva, Nevinsky.     

The evolutionary transition from monomeric to oligomeric proteins: tools, the environment, hypotheses

Recently, renewed interest in the evolution of oligomeric proteins has seen new approaches explored and new hypotheses proposed. The model systems chosen are generally made up of pairs of homologous proteins, each composed of a monomer and a dimeric counterpart, but the question has been also approached by comparing statistically significant structural patterns in sets of monomeric and oligomeric proteins. Here the tools of genetics and chemistry potentially available to the evolution of oligomeric proteins are discussed, as well as the possible effects of environments on the early attempts to oligomerization. Traces of an ancestral monomeric status of oligomers may be detected in the significant presence of polar and charged residues at intersubunit interfaces, and by the recognition that, besides the hydrophobic effect, a 'hydrophilic' effect has also had a role in the construction of these interfaces. The traditional 'mutation' model is described and found to be based on a hierarchy of mutations, crowned by a 'primary' mutation, one that could prime oligomerization by irreversibly altering the structure of an ancestral monomer. The mechanism of oligomerization based on the exchange or 'swap' of structural elements between monomers is discussed. The possibility is also discussed that the main steps in the folding pathway of an oligomeric protein reiterate the main steps in its evolution.     

Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.     

Tryptophan spectroscopy studies and black lipid bilayer analysis indicate that the oligomeric structure of Cry1Ab toxin from Bacillus thuringiensis is the membrane-insertion intermediate

During intoxication, the Cry protoxins must change from insoluble crystals into membrane-inserted toxins, which form ionic pores. Binding of Cry1A toxins to the cadherin receptor promotes the formation of a 250 kDa oligomer. In this work, we analyzed for the first time the structural changes presented by Cry1Ab toxin upon membrane insertion. Trp fluorescence of pure monomeric and oligomeric structures in solution and in a membrane-bound state was analyzed. Cry1Ab has nine Trp residues, seven of them in pore-forming domain I. Trp quenching analysis with iodide indicated that oligomerization caused a 27% reduction in the level of Trp exposed to the solvent. Most of the oligomeric structure (96%) inserts into the membrane as a function of the lipid:protein ratio, in contrast to the monomer (10%). Additionally, the membrane-associated oligomer presented a blue shift of 5 nm in lambda(max) of the emission spectrum, indicating a more hydrophobic environment for some Trp residues. In agreement with this, iodide was unable to quench the Trp of the membrane-bound oligomer, suggesting that a significant part of the protein may be buried in the membrane. Quenching analysis using brominated and spin-labeled phospholipids in the vesicles indicates that most of the Trp residues are located close to the membrane-water interface. Finally, ionic currents in black lipid bilayers revealed that the oligomeric structure has kinetics different from those of the monomer, producing stable channels with a high probability of being open in contrast to the monomer that exhibited unstable opening patterns. These data show that the oligomer, in contrast to the monomer, is able to interact efficiently with phospholipid membranes forming stable pores.     

Very stable high molecular mass multiprotein complex with DNase and amylase activities in human milk

For breastfed infants, human milk is more than a source of nutrients; it furnishes a wide array of proteins, peptides, antibodies, and other components promoting neonatal growth and protecting infants from viral and bacterial infection. It has been proposed that most biological processes are performed by protein complexes. Therefore, identification and characterization of human milk components including protein complexes is important for understanding the function of milk. Using gel filtration, we have purified a stable high molecular mass (~1000 kDa) multiprotein complex (SPC) from 15 preparations of human milk. Light scattering and gel filtration showed that the SPC was stable in the presence of high concentrations of NaCl and MgCl₂ but dissociated efficiently under the conditions that destroy immunocomplexes (2 M MgCl₂, 0.5 M NaCl, and 10 mM DTT). Such a stable complex is unlikely to be a casual associate of different proteins. The relative content of the individual SPCs varied from 6% to 25% of the total milk protein. According to electrophoretic and mass spectrometry analysis, all 15 SPCs contained lactoferrin (LF) and α‐lactalbumin as major proteins, whereas human milk albumin and β‐casein were present in moderate or minor amounts; a different content of IgGs and sIgAs was observed. All SPCs efficiently hydrolyzed Plasmid supercoiled DNA and maltoheptaose. Some freshly prepared SPC preparations contained not only intact LF but also small amounts of its fragments, which appeared in all SPCs during their prolonged storage; the fragments, similar to intact LF, possessed DNase and amylase activities. LF is found in human epithelial secretions, barrier body fluids, and in the secondary granules of leukocytes. LF is a protein of the acute phase response and nonspecific defense against different types of microbial and viral infections. Therefore, LF complexes with other proteins may be important for its functions not only in human milk. Copyright © 2014 John Wiley & Sons, Ltd.     

Time-resolved small-angle X-ray scattering investigation of the folding dynamics of heme oxygenase: implication of the scaling relationship for the submillisecond intermediates of protein folding

Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (Rg) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method. The folding process of HO was found to contain a transient oligomerization; however, the conformation within 10 ms was demonstrated to be monomeric and to possess Rg of 26.1(+/-1.1) A. Together with the corresponding data for proteins with different chain lengths, the seven Rg values demonstrated the scaling relationship to chain length with a scaling exponent of 0.35+/-0.11, which is close to the theoretical value of 1/3 predicted for globules in solutions where monomer-monomer interactions are favored over monomer-solvent interactions (poor solvent). The finding indicated that the initial folding dynamics of proteins bears the signature of the coil-globule transition, and offers a clue to explain the folding mechanisms of proteins with different chain lengths.     

Biophysical analyses of human resistin: oligomer formation suggests novel biological function

Resistin, a small secreted peptide initially identified as a link between obesity and diabetes in mice, was shown to be involved in mediating inflammation in humans. We had shown earlier that recombinant human resistin has a tendency to form aggregates by formation of inter/intramolecular disulfide linkages and that it undergoes a concentration-dependent conformational change in secondary structure from alpha-helical to beta-sheet form. Here we report that this change in secondary structural conformation is due to the increase in the oligomeric form of human resistin as a function of protein concentration. Gel filtration analysis under different conditions further demonstrated that recombinant human resistin exists as a mixture of oligomer and trimer but is converted to a mixture of monomer and oligomer in the presence of 100 mM NaCl. We show that while the trimeric form of human resistin is stable to urea-induced denaturation, it is highly susceptible to NaCl and NaF, indicating the importance of ionic interactions in stabilization of trimer. In addition, urea was able to destabilize the oligomers indicating the involvement of hydrophobic interactions in oligomerization. Ionic as well as hydrophobic interactions stabilize the monomeric human resistin. Our data suggest that human resistin exists predominantly as oligomer and trimer in vitro. The oligomeric form of human resistin shows more potent effect on stimulation of proinflammatory cytokines. Therefore, it is very tempting to propose that the structural conformation of resistin may be involved in maintaining the very fine balance in regulation of macrophage function for successful response to a variety of pathological conditions.     

Conformational changes and protein stability of the pro-apoptotic protein Bax

Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed.     

Factors affecting oligomerization status of UDP-glucose pyrophosphorylase

UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6-9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.     

Oligomerization-dependent changes in the thermodynamic properties of the TPR-MET receptor tyrosine kinase

Although oligomerization of receptor tyrosine kinases (RTKs) is necessary for receptor activation and signaling, a quantitative understanding of how oligomerization mediates these critical processes does not exist. We present a comparative thermodynamic analysis of functionally active dimeric and functionally inactive monomeric soluble analogues of the c-MET RTK, which clearly reveal that oligomerization regulates the binding affinity and binding kinetics of the kinase toward ATP and tyrosine-containing peptide substrates. Thermodynamic binding data for oligomeric c-MET were obtained from the dimeric TPR-MET oncoprotein, a functionally active fusion derivative of the c-MET RTK. This naturally occurring oncoprotein contains the cytoplasmic domain of c-MET fused to a coiled coil dimerization domain from the nuclear pore complex. Comparative data were obtained from a soluble monomeric kinase compromising the c-MET cytoplasmic domain (cytoMET). Significantly, under equilibrium binding conditions, the oligomeric phosphorylated kinase showed a significantly lower dissociation constant (K(d,dimer) = 11 microM) for a tyrosine-containing peptide derived from the C-terminal tail of the c-MET RTK when compared to the phosphorylated monomeric kinase cytoMET (K(d,monomer) = 140 microM). Surprisingly, equilibrium dissociation constants measured for the kinase and ATP were independent of the oligomerization state of the kinase (approximately 10 microM). Stopped-flow analysis of peptide substrate binding showed that the association rate constants (k(2)) differed 2-fold and dissociation rate constants (k(-2)) differed 10-fold when phosphorylated TPR-MET was compared to phosphorylated cytoMET. ATP binding abrogated the differences in k(2) rates observed between the two oligomeric states of the c-MET cytoplasmic domain. These results clearly imply that oligomerization induces important thermodynamic and conformational changes in the substrate binding regions of the c-MET protein and provide quantitative mechanistic insights into the necessary role of oligomerization in RTK activation.     

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.     

Oligomerization and regulated proteolytic processing of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is a recently identified circulating protein expressed primarily in adipose tissue and liver. Also known as peroxisome proliferator-activated receptor (PPAR)-gamma angiopoietin-related, fasting induced adipose factor, and hepatic fibrinogen/angiopoietin-related protein, recombinant Angptl4 causes increase of plasma very low density lipoprotein levels by inhibition of lipoprotein lipase activity. Similar to angiopoietins and other angiopoietin-like proteins, Angptl4 contains an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen-like domain. We report here that Angptl4 is evolutionarily conserved among several mammalian species and that full-length Angptl4 protein is an oligomer containing intermolecular disulfide bonds. Oligomerized Angptl4 undergoes proteolytic processing to release its carboxyl fibrinogen-like domain, which circulates as a monomer. Angptl4's N-terminal coiled-coil domain mediates its oligomerization, which by itself is sufficient to form higher order oligomeric structure. Adenovirus-mediated overexpression of Angptl4 in 293 cells shows that conversion of full-length, oligomerized Angptl4 is mediated by a cell-associated protease activity induced by serum. These findings demonstrate a novel property of angiopoietin-like proteins and suggest that oligomerization and proteolytic processing of Angptl4 may regulate its biological activities in vivo.     

Interaction of lactoferrin with ceruloplasmin

When added to human blood serum, the iron-binding protein lactoferrin (LF) purified from breast milk interacts with ceruloplasmin (CP), a copper-containing oxidase. Selective binding of LF to CP is evidenced by the results of polyacrylamide gel electrophoresis, immunodiffusion, gel filtration, and affinity chromatography. The molar stoichiometry of CP:LF in the complex is 1:2. Near-uv circular dichroism spectra of the complex showed that neither of the two proteins undergoes major structural perturbations when interacting with its counterpart. K(d) for the CP/LF complex was estimated from Scatchard plot as 1.8 x 10(-6) M. The CP/LF complex is found in various fluids of the human body. Upon injection into rat of human LF, the latter is soon revealed within the CP/LF complex of the blood plasma, from where the human protein is substantially cleared within 5 h.     

Differential recognition of epitopes present on monomeric and oligomeric forms of gp160 glycoprotein of human immunodeficiency virus type 1 by human monoclonal antibodies.

The mechanism of infectivity neutralization of human immunodeficiency virus type 1 (HIV-1) by Ig is poorly understood. Three human monoclonal antibodies (mAbs 1b12, 2G12 and 2F5) that are able to neutralize primary isolates of HIV-1 in vitro have been shown to act synergistically. In the present study this synergy was analyzed by measuring the epitope accessibility and binding kinetics for these three mAbs with respect to monomeric and oligomeric env protein gp160 IIIB using surface plasmon resonance. The results indicate that oligomerization of gp160 affects the accessibility of some of the epitopes recognized by the mAbs and provide some insight into the mechanism of synergy between different anti-(HIV-1) mAbs.     

Oligomerization paths of the nucleoprotein of influenza A virus

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.