A novel binding site for bile acids on human serum albumin

Binding sites of bile acids on human serum albumin were studied using various probes: dansylsarcosine (site I probe), 7-anilinocoumarin-4-acetic acid (ACAA, site II probe), 5-dimethylaminonaphthelene-1-sulfonamide (DNSA, site III probe), cis-parinaric acid (probe for fatty acid binding site) and bilirubin. Bile acids competitively inhibited the binding of dansylsarcosine to human serum album whereas bile acids enhanced the binding of ACAA, DNSA, cis-parinaric acid and bilirubin. Considering the concentrations of bile acids required to inhibit the binding of dansylsarcosine to human serum albumin, the secondary binding site of bile acids may correspond to site I. Dissociation constants (K_d) of the primary binding sites of lithocholic and chenodeoxycholic acid to human serum albumin were approximately 0.2 and 4 μM, respectively, which was measured by equilibrium dialysis at 37° C. All the bile acids and their sulfates and glucuronides inhibited the binding of chenodeoxycholic acid to human serum albumin. Lithocholic and chenodeoxycholic acid and their sulfates and glucuronides exhibited more inhibition than cholic acid and its conjugates. In conclusion, bile acids may bind to a novel binding site on human serum albumin.     

Impaired drug-binding capacities of in vitro and in vivo glycated albumin

Albumin, the major circulating protein in blood, can undergo increased glycation in diabetes. One of the main properties of this plasma protein is its strong affinity to bind many therapeutic drugs, including warfarin and ketoprofen. In this study, we investigated whether or not there were any significant changes related to in vitro or in vivo glycation in the structural properties and the binding of human albumin to both therapeutic drugs. Structural parameters, including redox state and ketoamine contents of in vitro and in vivo glycated purified albumins, were investigated in parallel with their affinity for warfarin and ketoprofen. High-performance liquid chromatography was used to determine the free drug concentrations and dissociation constants according to the Scatchard method. An alternative method based on fluorescence spectroscopy was also used to assess drug-binding properties. Oxidation and glycation levels were found to be enhanced in albumin purified from diabetic patients or glycated with glucose or methylglyoxal, after determination of their ketoamine, free thiol, amino group and carbonyl contents. In parallel, significant impairments in the binding affinity of in vitro and in vivo glycated albumin, as indicated by the higher dissociation constant values and confirmed by higher free drug fractions, were observed. To a lesser extent, this alteration also significantly affected diabetic albumin affinity, indicated by a lower static quenching in fluorescence spectroscopy. This work provides useful information supporting in vivo diabetic albumin could be the best model of glycation for monitoring diabetic physiopathology and should be valuable to know if glycation of albumin could contribute to variability in drugs response during diabetes.     

Allosteric modulation of zinc speciation by fatty acids

Background

Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another.

Scope of review

Here we review the Zn^2 + and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn^2 +, its effect upon cellular Zn^2 + uptake and its importance in the diagnosis of myocardial ischemia are considered.

Major conclusions

We previously identified the major binding site for Zn^2 + on albumin. Furthermore, we revealed that Zn^2 +-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn^2 +-binding to albumin in blood plasma.

General significance

Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn^2 + to albumin is important for the control of circulatory/cellular Zn^2 + dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin.     

Nature and characteristics of the binding of oestradiol-17beta to a uterine macromolecular fraction

The binding of oestradiol-17β to two proteins, namely serum albumin and a uterus fraction, was studied in vitro. The former protein has a physiological function in the transport of the hormone and the latter is involved in the selective uptake of the steroid by the target organ. The uterus fraction shows a high degree of stereospecificity for the binding of the steroid. Cortisone, oestradiol-17α and testosterone are bound negligibly and progesterone to a much smaller extent than is oestradiol-17β. This property is in contrast with the wide variety of ligands bound by the serum albumin. The temperature and the presence of the steroid influence markedly the binding properties. Oestradiol binding to the uterus fraction is optimum at 37° and at pH7–8·5. It is markedly decreased at pH values above or below this range, suggesting stringent conformational requirements. The tissue `receptor' protein is a macromolecule with a minimum molecular weight of 100000. The protein moiety is essential for the binding function. The probable concentration of the total binding sites for oestradiol in the ovariectomized-rat uterus cytoplasmic fraction as determined in vitro is about 1mμm at a steroid concentration of 50mμm.     

Albumin binding sites for etodolac enantiomers

Non-steroidal anti-inflammatory drugs (NSAIDs) are strongly bound to human serum albumin (HSA), mainly to sites I and II. The aim of this study was to characterize the binding site(s) of etodolac enantiomers under physiological conditions (580 μM HSA) using equilibrium dialysis. The protein binding of etodolac enantiomers, alone or in various ratios, was studied in order to evaluate the potential competition between them. Our results showed that (S)-etodolac was more strongly bound to HSA than (R)-etodolac. The displacement of one enantiomer by its antipode was observed only at high concentrations of the competitor, and was more pronounced for the (S)-form. Displacement studies of the enantiomers by specific probes of sites I and II of albumin, dansylamide, and dansylsarcosine, respectively, showed that (R)-etodolac was slightly displaced by both these probes whereas the free concentration of (S)-etodolac increased markedly in the presence of dansylsarcosine. Moreover, the binding of ligands to sites I and II is usually affected by alkaline pH, by chloride ions, and by fatty acids. For etodolac, the presence of 0.1 and 1 M chloride ions and increasing pH (5.5-9) decreased the binding of both enantiomers. The same result was obtained with addition of octanoic acid. Conversely, the addition of oleic, palmitic, or stearic acid to the protein solution increased the binding of (R)-etodolac, but decreased that of its antipode. All these findings suggest that (R)- and (S)-etodolac interact mainly with site II of HSA, and that the (R)-isomer is also bound to site I under physiological conditions. © 1996 Wiley-Liss, Inc.     

The interaction of secreted proteins Noggin4 and Wnt8 from <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

We demonstrated that the secreted protein Noggin4 from Xenopus laevis was capable of the in vitro binding to the secreted factor Wnt8, one of the ligands of the Wnt/betaCatenin signaling pathway. It was also shown that posttranslational modifications occurring during secretion of these proteins from the embryonic cells were necessary for their effective interaction. Also, we proposed a method for the preparation of physiologically active secreted morphogenic proteins from the intercellular space of the Xenopus laevis embryos.     

Cross-species Binding Analyses of Mouse and Human Neonatal Fc Receptor Show Dramatic Differences in Immunoglobulin G and Albumin Binding

The neonatal Fc receptor (FcRn) regulates the serum half-life of both IgG and albumin through a pH-dependent mechanism that involves salvage from intracellular degradation. Therapeutics and diagnostics built on IgG, Fc, and albumin fusions are frequently evaluated in rodents regarding biodistribution and pharmacokinetics. Thus, it is important to address cross-species ligand reactivity with FcRn, because in vivo testing of such molecules is done in the presence of competing murine ligands, both in wild type (WT) and human FcRn (hFcRn) transgenic mice. Here, binding studies were performed in vitro using enzyme-linked immunosorbent assay and surface plasmon resonance with recombinant soluble forms of human (shFcRn^WT) and mouse (smFcRn^WT) receptors. No binding of albumin from either species was observed at physiological pH to either receptor. At acidic pH, a 100-fold difference in binding affinity was observed. Specifically, smFcRn^WT bound human serum albumin with a K_D of ∼90 μm, whereas shFcRn^WT bound mouse serum albumin with a K_D of 0.8 μm. shFcRn^WT ignored mouse IgG1, and smFcRn^WT bound strongly to human IgG1. The latter pair also interacted at physiological pH with calculated affinity in the micromolar range. In all cases, binding of albumin and IgG from either species to both receptors were additive. Cross-species albumin binding differences could partly be explained by non-conserved amino acids found within the α2-domain of the receptor. Such distinct cross-species FcRn binding differences must be taken into consideration when IgG- and albumin-based therapeutics and diagnostics are evaluated in rodents for their pharmacokinetics.     

Pharmaceutical-grade albumin: impaired drug-binding capacity <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Albumin is the most abundant protein in blood plasma, and due to its ligand binding properties, serves as a circulating depot for endogenous and exogenous (e.g. drugs) compounds. Hence, the unbound drug is the pharmacologically active drug. Commercial human albumin preparations are frequently used during surgery and in critically ill patients. Recent studies have indicated that the use of pharmaceutical-grade albumin is controversial in critically ill patients. In this in vitro study we investigated the drug binding properties of pharmaceutical-grade albumins (Baxter/Immuno, Octapharma, and Pharmacia & Upjohn), native human serum, and commercially available human serum albumin from Sigma Chemical Company.     

Studies of albumin binding to rat liver plasma membranes: Implications for the albumin receptor hypothesis

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted ¹²⁵I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15 000 × g for 15 min), after repeated cycles of washing with buffer and re-centrifugation. ¹²⁵I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of ¹²⁵I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or ‘off-time’, as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase ¹²⁵I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.     

Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases

The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB.     

Intensification of tumor affinity of 99mTc—dl-homocysteine by cooperative use of SH-containing compounds

The enhancement of tumor-specific distribution was investigated on ^99mTc—dl-homocysteine (^99mTc-Hcy), a possible tumor-imaging agent. In vitro HPLC analysis showed that ^99mTc-Hcy did not bind to nonmercaptalbumin but to mercaptalbumin in blood. When glutathione, homocysteine or cysteine, which converts albumin from the mercapto- to nonmercapto-form, was intravenously injected into Tc-Hcy predosed mice bearing Ehrlich solid tumor, the SH-containing compounds did not affect the radioactivity distribution in tumor, but decreased that in blood to less than half that of untreated animals. In vitro gel filtration analysis showed that glutathione released ^99mTc-Hcy from albumin. Free ^99mTc-Hcy produced in blood by the treatment was rapidly excreted into urine. These results suggest that the combination of ^99mTc-Hcy and SH-containing compounds is useful for tumor-imaging.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

A method for measurement of free and bound plasma cyst(e)ine

A method for the measurement of free and bound cyst(e)ine and the sum thereof has been developed. In adult human blood, cyst(e)ine is distributed equally between that which is bound to plasma proteins and that which is free. Cyst(e)ine is bound predominantly to albumin, and this binding is not an in vitro artifact. Cysteine bound to plasma proteins may be displaced by homocysteine which competes for the available sulfhydryl groups of plasma proteins. Rats starved for 8 days had a significant decrease in both plasma free cyst(e)ine and bound cysteine. These data suggest that present methods for the determination of plasma cyst(e)ine under-estimate the quantity of cyst(e)ine in the plasma available for cellular metabolism.     

Cell culture models of fatty acid overload: Problems and solutions

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.     

Fish Oil and Fenofibrate Prevented Phosphorylation-dependent Hepatic Sortilin 1 Degradation in Western Diet-fed Mice

Obesity and diabetes are associated with hepatic triglyceride overproduction and hypertriglyceridemia. Recent studies have found that the cellular trafficking receptor sortilin 1 (Sort1) inhibits hepatic apolipoprotein B secretion and reduces plasma lipid levels in mice, and its hepatic expression was negatively associated with plasma lipids in humans. This study investigated the regulation of hepatic Sort1 under diabetic conditions and by lipid-lowering fish oil and fenofibrate. Results showed that hepatic Sort1 protein, but not mRNA, was markedly lower in Western diet-fed mice. Knockdown of hepatic Sort1 increased plasma triglyceride in mice. Feeding mice a fish oil-enriched diet completely restored hepatic Sort1 levels in Western diet-fed mice. Fenofibrate also restored hepatic Sort1 protein levels in Western diet-fed wild type mice, but not in peroxisome proliferator-activated receptor α (PPARα) knock-out mice. PPARα ligands did not induce Sort1 in hepatocytes in vitro. Instead, fish oil and fenofibrate reduced circulating and hepatic fatty acids in mice, and n-3 polyunsaturated fatty acids prevented palmitate inhibition of Sort1 protein in HepG2 cells. LC/MS/MS analysis revealed that Sort1 phosphorylation at serine 793 was increased in obese mice and in palmitate-treated HepG2 cells. Mutations that abolished phosphorylation at Ser-793 increased Sort1 stability and prevented palmitate inhibition of Sort1 ubiquitination and degradation in HepG2 cells. In summary, therapeutic strategies that prevent posttranslational hepatic Sort1 down-regulation in obesity and diabetes may be beneficial in improving dyslipidemia.     

Disruption of the tryptophan binding site in the human serum albumin dimer

Monomer and dimer fractions of human serum albumin (HSA) obtained from charcoal-treated Fraction V HSA have very similar fluorescence and circular dichroism spectra, but the dimer neither binds l-tryptophan nor reacts rapidly with p-nitrophenyl acetate. The latter reaction presumably occurs in a major binding site of the monomer as many strongly bound ligands including l-tryptophan, small fatty acid anions (e.g., S.-W. M. Koh and G. E. Means, 1979, Arch. Biochem. Biophys.192, 73–79), and several drugs (e.g, N. P. Sollenne and G. E. Means, 1979, Mol. Pharmacol.15, 754–757) all decrease rates of reaction in direct proportion to their concentration. Binding data for those ligands indicate the presence of less than one binding site per molecule of charcoal-treated Fraction V HSA, and thus appear to reflect only its content of albumin monomer. The absence of that binding site in the dimer may reflect its inclusion within the dimer interface.     

The preclinical pharmacology of the high affinity anti-IL-6R Nanobody? ALX-0061 supports its clinical development in rheumatoid arthritis

IntroductionThe pleiotropic cytokine interleukin-6 (IL-6) plays an important role in the pathogenesis of different diseases, including rheumatoid arthritis (RA). ALX-0061 is a bispecific Nanobody® with a high affinity and potency for IL-6 receptor (IL-6R), combined with an extended half-life by targeting human serum albumin. We describe here the relevant aspects of its in vitro and in vivo pharmacology.MethodsALX-0061 is composed of an affinity-matured IL-6R-targeting domain fused to an albumin-binding domain representing a minimized two-domain structure. A panel of different in vitro assays was used to characterize the biological activities of ALX-0061. The pharmacological properties of ALX-0061 were examined in cynomolgus monkeys, using plasma levels of total soluble (s)IL-6R as pharmacodynamic marker. Therapeutic effect was evaluated in a human IL-6-induced acute phase response model in the same species, and in a collagen-induced arthritis (CIA) model in rhesus monkeys, using tocilizumab as positive control.ResultsALX-0061 was designed to confer the desired pharmacological properties. A 200-fold increase of target affinity was obtained through affinity maturation of the parental domain. The high affinity for sIL-6R (0.19 pM) translated to a concentration-dependent and complete neutralization of sIL-6R in vitro. In cynomolgus monkeys, ALX-0061 showed a dose-dependent and complete inhibition of hIL-6-induced inflammatory parameters, including plasma levels of C-reactive protein (CRP), fibrinogen and platelets. An apparent plasma half-life of 6.6 days was observed after a single intravenous administration of 10 mg/kg ALX-0061 in cynomolgus monkeys, similar to the estimated expected half-life of serum albumin. ALX-0061 and tocilizumab demonstrated a marked decrease in serum CRP levels in a non-human primate CIA model. Clinical effect was confirmed in animals with active drug exposure throughout the study duration.ConclusionsALX-0061 represents a minimized bispecific biotherapeutic of 26 kDa, nearly six times smaller than monoclonal antibodies. High in vitro affinity and potency was demonstrated. Albumin binding as a half-life extension technology resulted in describable and expected pharmacokinetics. Strong IL-6R engagement was shown to translate to in vivo effect in non-human primates, demonstrated via biomarker deregulation as well as clinical effect. Presented results on preclinical pharmacological properties of ALX-0061 are supportive of clinical development in RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0651-0) contains supplementary material, which is available to authorized users.     

Multiple Interactions of Unsaturated Fatty Acids with Opiate and Ouabain Binding Sites and β-Adrenergic Sensitive Adenylate Cyclase System

Abstract

The unsaturated fatty acids oleic, linoleic and arachidonic inhibited binding of ligands to the ouabain, opiate, and β-adrenergic plasma membrane receptors. Low concentrations of fatty acids slightly increased the binding of ouabain to its binding sites. The effect of these fatty acids on β-adrenergic sensitive adenylate cyclase was more complex. 0.2–0.3 mM fatty acids increased adenylate cyclase activity, while higher concentrations of arachidonic and linoleic acids, but not oleic acid     

Lipid and Carbohydrate Modifications of α-Galactosylceramide Differently Influence Mouse and Human Type I Natural Killer T Cell Activation

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type I NKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators.     

Spectrophotometric and radiometric studies on the in vivo binding of phenobarbital to hepatic microsomes.

We have examined hepatic microsomes prepared from phenobarbital-treated animals for the presence of the inducer and found that significant concentrations of the drug remain bound even after a number of washings. The bound drug is at least 70% unmetabolized and interferes with the in vitro binding of ethylmorphine and phenobarbital to the type I binding site but is not bound to the type I site since the K_s is 0.2–6.4 μm whereas the K_s for type I binding of phenobarbital is 103 μm. The high affinity site was not observed in microsomes from control animals either spectrophotometrically or radiometrically. The bound drug can be removed by bovine serum albumin to give a reverse pseudo-type I spectrum with a peak at 412 nm rather than the 425 nm observed for the trough of the typical type I spectrum. These data suggest that induction with phenobarbital may alter the spectral properties of cytochrome P-450 and hence care should be taken in comparing spectral data between microsomes from phenobarbital-treated and control animals.