Human Serum Albumin Mutants Research Articles

Probing the Binding Mechanism of Acylated Peptides to Human Serum Albumin

Peptides represent an increasingly important class of pharmaceutical products. During the last decade or so, acylation with fatty acids has demonstrated considerable success in prolonging the circulating half-life of therapeutic peptides by exploiting the ability of fatty acids to reversibly bind to human serum albumin (HSA), thus significantly impacting their pharmacological profiles. Employing methyl-13C-labeled oleic acid or palmitic acid as probe molecules and exploiting HSA mutants designed to probe fatty acid binding, the signals in two-dimensional (2D) nuclear magnetic resonance (NMR) spectra corresponding to high-affinity fatty acid binding sites in HSA were assigned. Subsequently, using a set of selected acylated peptides, competitive displacement experiments by 2D NMR identified a primary fatty acid binding site in HSA utilized in acylated peptide binding. These results represent an important first step toward understanding the structural basis for acylated peptides binding to HSA.

Virtual Screening of Human Serum Albumin Mutants to Optimize the Search for its Forms that Increase Affinity to Amyloid-Β Peptide

A promising approach to the treatment of Alzheimer's disease (AD) is the removal of amyloid-β peptide (Aβ) from the patient's central nervous system by acting on human serum albumin (HSA). HSA carries 90% of Aβ in blood serum and 40-90% of Aβ in the cerebrospinal fluid (CNS). In this work, virtual screening of all possible mutant forms of HSA based on the data of the I-Mutant service made it possible to predict changes in HSA stability and identify the most “sensitive” regions of its polypeptide chain to substitutions. The data obtained will be used to optimize the search for HSA forms with increased affinity to Aβ, as well as to study the mechanisms underlying the modulating effects of HSA ligands on its interaction with Aβ, which can become the basis for the development of new approaches to therapy and prevention of AD.

Albumin domain mutants with enhanced Aβ binding capacity identified by phage display analysis for application in various peripheral Aβ elimination approaches of Alzheimer's disease treatment.

Deposition of amyloid protein, particularly Aβ1-42 , is a major contributor to the onset of Alzheimer's disease (AD). However, almost no deposition of Aβ in the peripheral tissues could be found. Human serum albumin (HSA), the most abundant protein in the blood, has been reported to inhibit amyloid formation through binding Aβ, which is believed to play an important role in the peripheral clearance of Aβ. We identified the Aβ binding site on HSA and developed HSA mutants with high binding capacities for Aβ using a phage display method. HSA fragment 187-385 (Domain II) was found to exhibit the highest binding capacity for Aβ compared with the other two HSA fragments. To elucidate the sequence that forms the binding site for Aβ on Domain II, a random screening of Domain II display phage biopanning was constructed. A number of mutants with higher Aβ binding capacities than the wild type were identified. These mutants exhibited stronger scavenging abilities than the wild type, as revealed via in vitro equilibrium dialysis of Aβ experiments. These findings provide useful basic data for developing a safer alternative therapy than Aβ vaccines and for application in plasma exchange as well as extracorporeal dialysis.

Differential effects of methoxy group on the interaction of curcuminoids with two major ligand binding sites of human serum albumin.

Curcuminoids are a group of compounds with a similar chemical backbone structure but containing different numbers of methoxy groups that have therapeutic potential due to their anti-inflammatory and anti-oxidant properties. They mainly bind to albumin in plasma. These findings influence their body disposition and biological activities. Spectroscopic analysis using site specific probes on human serum albumin (HSA) clearly indicated that curcumin (Cur), demethylcurcumin (Dmc) and bisdemethoxycurcumin (Bdmc) bind to both Site I (sub-site Ia and Ib) and Site II on HSA. At pH 7.4, the binding constants for Site I were relatively comparable between curcuminoids, while the binding constants for Site II at pH 7.4 were increased in order Cur < Dmc < Bdmc. Binding experiments using HSA mutants showed that Trp214 and Arg218 at Site I, and Tyr411 and Arg410 at Site II are involved in the binding of curcuminoids. The molecular docking of all curcuminoids to the Site I pocket showed that curcuminoids stacked with Phe211 and Trp214, and interacted with hydrophobic and aromatic amino acid residues. In contrast, each curcuminoid interacted with Site II in a different manner depending whether a methoxy group was present or absent. A detailed analysis of curcuminoids-albumin interactions would provide valuable information in terms of understanding the pharmacokinetics and the biological activities of this class of compounds.

Haemoglobin wrapped covalently by human serum albumin mutants containing Mn(iii) protoporphyrin IX: an O2complex stable in H2O2solution

We describe the synthesis of a haemoglobin (Hb) wrapped covalently by recombinant human serum albumin mutants [HSA(Y161H)] containing Mn(III) protoporphyrin IX (MnPP), the Hb-[HSA(Y161H)-MnPP]3 cluster, highlighting the formation of its O2-complex stable even in H2O2 solution.

Human Serum Albumin‐Based Peroxidase Having an Iron Protoporphyrin IX in Artificial Heme Pocket

Cooking up enzymes: Iron protoporphyrin IX (heme) is bound within a hydrophobic cavity in subdomain IB of human serum albumin (HSA). Site-specific mutations to introduce proximal and distal histidines into the heme pocket of HSA conferred a peroxidase activity to the prosthetic heme group. The catalytic activity of the recombinant HSA(mutant)-heme complex for guaiacol oxidation is 17-fold higher than that of HSA(wild type)-heme.

Quantitative evaluation of the role of cysteine and methionine residues in the antioxidant activity of human serum albumin using recombinant mutants

The importance of cysteine (Cys) and methionine (Met) residues for the antioxidant activity of human serum albumin (HSA) was investigated using recombinant HSA mutants, in which Cys34 and/or the six Met residues had been mutated to Ala. The scavenging activities of the mutants against five reactive oxygen and nitrogen species were evaluated by a chemiluminescence assay, electron paramagnetic resonance spectroscopy, or a HPLC-flow reactor assay. Our results showed that the contributions of Cys34 and the Met residues to the antioxidant activity of HSA were 61% and 29% against O(2)(•-), 68% and 61% against H(2)O(2), 38% and 6% against HO(•), 36% and 13% against HOCl, and 51% and 1% against (•)NO, respectively. Thus, the findings propose in a direct way that Cys34 plays a more important role than the Met residues in the antioxidant activity of HSA.

Protein-based nanotubes for biomedical applications

This review presents highlights of our latest results of studies directed at developing protein-based smart nanotubes for biomedical applications. These practical biocylinders were prepared using an alternate layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into a nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers of poly-L-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)(3)]. The obtained (PLA/HSA)(3) nanotubes (NTs) can be dispersed in aqueous medium and are hydrated significantly. Several ligands for HSA, such as zinc(II) protoporphyrin IX (ZnPP), were bound to the HSA component in the cylindrical wall. Similar NTs comprising recombinant HSA mutant, which has a strong binding affinity for ZnPP, captured the ligand more tightly. The Fe(3)O(4)-coated NTs can be collected easily by exposure to a magnetic field. The hybrid NTs bearing a single avidin layer as an internal wall captured biotin-labeled nanoparticles into the central channel when their particle size is sufficiently small to enter the pores. The NTs with an antibody surface interior entrapped human hepatitis B virus with size selectivity. It is noteworthy that the infectious Dane particles were encapsulated completely into the hollows. Other HSA-based NTs having an α-glucosidase inner wall hydrolysed a glucopyranoside to yield α-D-glucose. A perspective of the practical use of the protein-based NTs is also described.

Effects of human serum albumin complexed with free fatty acids on cell viability and insulin secretion in the hamster pancreatic β-cell line HIT-T15

Aims The effects of human serum albumin (HSA) complexed with various free fatty acids (FFAs) on ß-cells have not been studied in detail. In this study, we examined the effects of HSA and its mutants on FFA-induced cell viability changes and insulin secretion from the hamster pancreatic insulinoma cell line, HIT-TI5. Main methods Cells were exposed to different FFAs in the presence of HSA or its mutants and/or bovine serum albumin (BSA) for 24 h. Cell viability, apoptosis, insulin secretion, and unbound FFA (FFA u) levels were determined. Key findings In the presence of 0.1 mM HSA, palmitate and stearate induced significant cell death at 0.1 mM or higher, whereas myristate, palmitoleate, oleate, elaidate, linoleate, linoelaidate, and conjugated linoleate showed minimal changes on cell viability. Furthermore, oleate and linoleate were clearly cytoprotective against palmitate-induced cell death. The apoptosis inhibitors, cyclosporin A (csA) and the caspase inhibitor ZVAD-FMK, did not completely prevent FFA-induced cell death, although ZVAD-FMK blocked apoptosis with no differences in the presence of either HSA or BSA. In addition, insulin secretion from the cells was significantly reduced in the presence of HSA/oleate complexes. We also found differential effects of HSA mutants complexed with FFAs on cell viability. Significance In summary, our results showed that saturated FFAs induced more cell death than unsaturated FFAs. Furthermore, modified HSA/FFA interactions caused by mutations of key amino acids involved in the binding of FFA to HSA resulted in changes in cell viability, suggesting a possible role of HSA polymorphism on FFA-induced changes in cellular functions.

Molecular Capture in Protein Nanotubes

We describe molecular capturing properties of protein nanotubes with a controllable ligand binding affinity and size selectivity. These practical biocylinders were prepared using an alternating layer-by-layer (LbL) assembly of protein and oppositely charged poly(amino acid) into the nanoporous polycarbonate (PC) membrane (pore diameter, 400 nm), with subsequent dissolution of the template. The tube wall typically comprises six layers of poly-L-arginine (PLA) and human serum albumin (HSA) [(PLA/HSA)(3)]. Use of high molecular weight PLA (M(w) = ca. 70 000) yielded robust nanotubes, which are available as lyophilized powder. The (PLA/HSA)(3) nanotubes swelled considerably in water, although the outer diameter was almost unaltered. Uranyl ion, 3,3'-diethylthiacarbocyanine iodide, and zinc(II) protoporphyrin IX (ZnPP) were bound to the HSA component in the cylinder wall. Similar nanotubes comprising recombinant HSA mutant [rHSA(His)], which has a strong binding affinity for ZnPP, captured this ligand more tightly. Furthermore, addition of excess myristic acid released ZnPP from the tubes through a ligand replacement reaction. The hybrid nanotubes bearing a single avidin layer as an internal surface captured FITC-biotin efficiently. Biotin-labeled nanoparticles are also incorporated into the tubes when their particle size is sufficiently small to enter the pores. Subsequent TEM observation revealed a line of loaded nanoparticles (100 nm) in the one-dimensional space interior.

Expanding the Genetic Repertoire of the Methylotrophic Yeast Pichia pastoris

To increase the utility of protein mutagenesis with unnatural amino acids, a recombinant expression system in the methylotrophic yeast Pichia pastoris was developed. Aminoacyl-tRNA synthetase/suppressor tRNA (aaRS/tRNA(CUA)) pairs previously evolved in Saccharomyces cerevisiae to be specific for unnatural amino acids were inserted between eukaryotic transcriptional control elements and stably incorporated into the P. pastoris genome. Both the Escherichia coli tyrosyl- and leucyl-RS/tRNA(CUA) pairs were shown to be orthogonal in P. pastoris and used to incorporate eight unnatural amino acids in response to an amber codon with high yields and fidelities. In one example, we show that a recombinant human serum albumin mutant containing a keto amino acid (p-acetylphenylalanine) can be efficiently expressed in this system and selectively conjugated via oxime ligation to a therapeutic peptide mimetic containing an permittivity-(2-(aminooxy)acetyl)-L-lysine residue. Moreover, unnatural amino acid expression in the methylotrophic host was systematically optimized by modulation of aaRS levels to express mutant human serum albumin in excess of 150 mg/L in shake flasks, more than an order of magnitude better than that reported in S. cerevisiae. This methodology should allow the production of high yields of complex proteins containing unnatural amino acids whose expression is not practical in existing systems.

Comparative Analysis of Serum Proteomes to Discover Biomarkers for Ossification of the Posterior Longitudinal Ligament

Serum proteomes from normal subjects and the patients with ossification of the posterior longitudinal ligament (OPLL) were analyzed by using proteomics. To identify novel serologic biomarkers for diagnosing OPLL. OPLL can compress the spinal cord, and special planning is required for surgeries that are done from the front of the cervical spine. However, the definitive serologic biomarkers for OPLL are still unclear. The 2-dimensional electrophoresis patterns of sera from OPLL patients and normal subjects were compared. The differentially expressed spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization quadruple time-of-flight mass spectrometry. Nine spots that were differentially expressed in the sera of OPLL patients were found and were identified. PRO2675, human serum albumin in a complex with myristic acid and tri-iodobenzoic acid, an unknown protein, chain B of the crystal structure of deoxy-human hemoglobin beta6, pro-apolipoprotein, ALB protein, retinol binding protein and chain A of human serum albumin mutant R218h complexed with thyroxine (3,3',5,5', tetraiodo-L-thyronine), were up-regulated in the sera of OPLL patients, whereas alpha1-microglobulin/bikunin precursor was down-regulated. These proteins could be used as diagnostic biomarkers of OPLL.

Fatty acids bound to human serum albumin and its structural variants modulate apolipoprotein B secretion in HepG2 cells

Epidemiologic studies have shown an inverse relationship between human serum albumin (HSA) levels and coronary heart disease (CHD). However, no mechanisms have been identified to explain this relationship. We hypothesized that this relationship is due to differences in binding affinity of fatty acids to HSA and subsequent atherogenic lipoprotein synthesis and secretion from hepatocytes. To test our hypothesis we undertook the current study. Using HepG2 cells, we demonstrated that oleic acid (OA) bound to HSA in a molar ratio of 4:1 and after incubation for 24 h stimulated apolipoprotein B (apoB) secretion. We also tested whether mutant forms of HSA could alter the binding affinity for fatty acids and change the availability of substrate for lipoprotein secretion. Based on the results obtained in this study using 11 HSA mutant proteins complexed with OA, we were able to classify into three major mutant groups based on their effects on apoB secretion. One group in particular (R410Q/Y411W, R410A/Y411A, and W214L/Y411W) showed a significantly diminished effect on apoB secretion when compared to the wild type HSA/OA complex. Furthermore, the amount of free OA internalized in HepG2 cells in the presence of HSA mutant proteins was in good agreement with the effects seen on apoB secretion by the various HSA mutants. This suggests that some mutant forms of HSA might potentially bind fatty acids with a much higher binding affinity and thus deprive fatty acids available for lipoprotein assembly in hepatocytes. In conclusion, our data illustrate that certain HSA polymorphic forms may be protective against the development of CHD and warrants further investigation.

Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy.

Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [(13)C]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA-HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [(13)C]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [(13)C]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites.

O2 and CO Binding Properties of Artificial Hemoproteins Formed by Complexing Iron Protoporphyrin IX with Human Serum Albumin Mutants

The binding properties of O2 and CO to recombinant human serum albumin (rHSA) mutants with a prosthetic heme group have been physicochemically and kinetically characterized. Iron(III) protoporphyrin IX (hemin) is bound in subdomain IB of wild-type rHSA [rHSA(wt)] with weak axial coordination by Tyr-161. The reduced ferrous rHSA(wt)-heme under an Ar atmosphere exists in an unusual mixture of four- and five-coordinate complexes and is immediately autoxidized by O2. To confer O2 binding capability on this naturally occurring hemoprotein, a proximal histidine was introduced into position Ile-142 or Leu-185 by site-directed mutagenesis. A single mutant (I142H) and three double mutants (I142H/Y161L, I142H/Y161F, and Y161L/L185H) were prepared. Both rHSA(I142H/Y161L)-heme and rHSA(I142H/Y161F)-heme formed ferrous five-N-coordinate high-spin complexes with axial ligation of His-142 under an Ar atmosphere. These artificial hemoproteins bind O2 at room temperature. Mutation at the other side of the porphyrin, Y161L/L185H, also allowed O2 binding to the heme. In contrast, the single mutant rHSA(I142H)-heme could not bind O2, suggesting that removal of Y161 is necessary to confer reversible O2 binding. Laser flash photolysis experiments showed that the kinetics of CO recombination with the rHSA(mutant)-heme were biphasic, whereas O2 rebinding exhibited monophasic kinetics. This could be due to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O2 binding affinities of the rHSA(mutant)-heme were significantly lower than those of hemoglobin and myoglobin, principally due to the high O2 dissociation rates. Changing Leu-161 to Phe-161 at the distal side increased the association rates of both O2 and CO, which resulted in enhanced binding affinity.

Analysis of tryptophan fluorescence lifetimes in a series of human serum albumin mutants with substitutions in subdomain 2A.

Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.

Human serum albumin and its structural variants mediate cholesterol efflux from cultured endothelial cells

In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA–cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux.

Site-Directed Mutagenesis Studies of Human Serum Albumin Define Tryptophan at Amino Acid Position 214 as the Principal Site for Nitrosation

The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO₂^–. The pattern of NO release from BSA nitrosated with acidified NO₂^– was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO₂^– indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO₂^–, several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO₂^– showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N₂O₃, the reactive nitrosating species formed in aqueous acidified NO₂^– solutions.

Mutagenesis studies of thyroxine binding to human serum albumin define an important structural characteristic of subdomain 2A.

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant, with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. In our earlier study, site-directed mutagenesis and a yeast protein expression system were used to synthesize FDH HSA and several other HSA mutants. Measurement of the binding of these HSA mutants to thyroxine and several thyroxine analogs using equilibrium dialysis and quenching of tryptophan 214 fluorescence allowed us to propose a preliminary model of thyroxine binding to the 2A subdomain of wild type and FDH HSA. In this study, we have produced several other HSA mutants. By comparing the binding affinity of these mutants for thyroxine and tetraiodothyroacetic acid to the binding affinity of other mutants, we were able to suggest a new model for thyroxine binding to the 2A subdomain of HSA. We found that the substitution of arginine at position 218 with alanine increased the binding affinity for thyroxine by 2 orders of magnitude relative to the binding affinity of wild type HSA for thyroxine. A more accurate understanding of the mechanism of thyroxine binding to HSA has allowed us to define an important structural characteristic of subdomain 2A, one of the two principal binding sites on HSA for small hydrophobic ligands.

Mutations in a Specific Human Serum Albumin Thyroxine Binding Site Define the Structural Basis of Familial Dysalbuminemic Hyperthyroxinemia

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. Site-directed mutagenesis and a yeast protein expression system were used to synthesize wild type HSA and FDH HSA as well as several other HSA mutants. Studies on the binding of thyroxine to these HSA species using equilibrium dialysis and quenching of tryptophan 214 fluorescence suggest that the FDH mutation affects a single thyroxine binding site located in the 2A subdomain of HSA. Site-directed mutagenesis of HSA and thyroxine analogs were used to obtain information about the mechanism of thyroxine binding to both wild type and FDH HSA. These studies suggest that the guanidino group of arginine at amino acid position 218 in wild type HSA is involved in an unfavorable binding interaction with the amino group of thyroxine, whereas histidine at amino acid position 218 in FDH HSA is involved in a favorable binding interaction with thyroxine. Neither arginine at amino acid position 222 nor tryptophan at amino acid position 214 appears to favorably influence the binding of thyroxine to wild type HSA.