Flavonoid binding to human serum albumin

Dietary flavonoid may have beneficial effects in the prevention of chronic diseases. However, flavonoid bioavailability is often poor probably due to their interaction with plasma proteins. Here, the affinity of daidzein and daidzein metabolites as well as of genistein, naringenin, and quercetin for human serum albumin (HSA) has been assessed in the absence and presence of oleate. Values of the dissociation equilibrium constant (K) for binding of flavonoids and related metabolites to Sudlow’s site I range between 3.3 × 10⁻⁶ and 3.9 × 10⁻⁵ M, at pH 7.0 and 20.0 °C, indicating that these flavonoids are mainly bound to HSA in vivo. Values of K increase (i.e., the flavonoid affinity decreases) in the presence of saturating amounts of oleate by about two folds. Present data indicate a novel role of fatty acids as allosteric inhibitors of flavonoid bioavailability, and appear to be relevant in rationalizing the interference between dietary compounds, food supplements, and drugs.     

Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXalpha

Bilirubin, an insoluble yellow-orange pigment derived from heme catabolism, accumulates to toxic levels in individuals with impaired or immature liver function. The resulting jaundice may be managed with phototherapy to isomerize the biosynthetic 4Z,15Z-bilirubin-IXα to more soluble and excretable isomers, such as 4Z,15E-bilirubin. Bilirubin and its configurational isomers are transported to the liver by human serum albumin (HSA) but their precise binding location(s) on the protein have yet to be determined. To investigate the molecular details of their interaction, we co-crystallised bilirubin with HSA. Strikingly, the crystal structure—determined to 2.42 Å resolution—revealed the 4Z,15E-bilirubin-IXα isomer bound to an L-shaped pocket in sub-domain IB. We also determined the co-crystal structure of HSA complexed with fusidic acid, an antibiotic that competitively displaces bilirubin from the protein, and showed that it binds to the same pocket. These results provide the first crystal structure of a natural bilirubin pigment bound to serum albumin, challenge some of the present conceptions about HSA-bilirubin interactions, and provide a sound structural framework for finally resolving the long-standing question of where 4Z,15Z-bilirubin-IXα binds to the protein.     

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

11-(Dansylamino) undecanoic acid (DAUDA) is a dansyl-type fluorophore and has widely used as a probe to determine the binding site for human serum albumin (HSA). Here, we reported that structure of HSA-Myristate-DAUDA ternary complex and identified clearly the presence of two DAUDA molecules at fatty acid (FA) binding site 6 and 7 of HSA, thus showing these two sites are weak FA binding sites. This result also show that DAUDA is an appropriate probe for FA site 6 and 7 on HSA as previous studied, but not a good probe of FA binding site 1 that is likely bilirubin binding site on HSA.     

Understanding the effects of two bound glucose in Sudlow site I on structure and function of human serum albumin: theoretical studies

Human serum albumin (HSA) is the most abundant protein found in blood serum. It carries essential metabolites and many drugs. The glycation of HSA causes abnormal biological effects. Importantly, glycated HSA (GHSA) is of interest as a biomarker for diabetes. Recently, the first HSA structure with bound pyranose (GLC) and open-chain (GLO) glucose at Sudlow site I has been crystallised. We therefore employed molecular dynamics (MD) simulations and ONIOM calculations to study the dynamic nature of two bound glucose in a pre-glycated HSA (pGHSA) and observe how those sugars alter a protein structure comparing to wild type (Apo) and fatty acid-bound HSA (FA). Our analyses show that the overall structural stability of pGHSA is similar to Apo and FA, except Sudlow site II. Having glucose induces large protein flexibility at Sudlow site II. Besides, the presence of glucose causes W214 to reorient resulting in a change in W214 microenvironment. Considering sugars, both sugars are exposed to water, but GLO is more solvent-accessible. ONIOM results show that glucose binding is favoured for HSA (?115.04 kcal/mol) and GLO (?85.10 kcal/mol) is more preferable for Sudlow site I over GLC (?29.94 kcal/mol). GLO can strongly react with K195 and K199, whereas K195 and K199 provide slightly repulsive forces for GLC. This can confirm that an open-chain GLO is more favourable inside a pocket.     

Fipronil recognition by the FA1 site of human serum albumin

Fipronil is a broad‐spectrum pesticide widely used in agriculture, horticulture, and forestry. Because fipronil can cause a variety of toxic effects in animals and humans, its use is authorized as a pesticide in veterinary medicinal products for pets, but not for the treatment of livestock animals whose products are intended for consumption. Recently, however, the presence of fipronil residues has been detected in the eggs and meat of layer hens from farms located in different European countries. Given the relevance of fipronil toxicity for human health, it is important to gain information concerning its fate in the human body, including its binding mode to human serum albumin (HSA), the most abundant protein in plasma. Here, the inhibition of heme‐Fe(III) binding to the fatty acid site 1 (FA1) of HSA by fipronil is reported. Docking simulations support functional data, indicating that the FA1 site is the preferential cleft for fipronil recognition by HSA. The affinity of fipronil for HSA (K_f = 1.9 × 10^?6 M, at pH 7.3, and 20.0°C) may be relevant in vivo. Indeed, HSA could play a pivotal role in fipronil transport and scavenging, thus reducing the pesticide‐free plasmatic levels, with consequent reduced systemic toxicity. In turn, fipronil binding to the FA1 site of HSA could impair the recognition of endogenous and exogenous molecules.     

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500?µg/L) and the half-life of IGF-1 in blood circulation is only 4.5?min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100?mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.     

Expression of human serum albumin in the milk of transgenic mice

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml⁻¹) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml⁻¹. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.     

Novel AGLP-1 albumin fusion protein as a long-lasting agent for type 2 diabetes

Glucagon like peptide-1 (GLP-1) regulates glucose mediated-insulin secretion, nutrient accumulation, and β-cell growth. Despite the potential therapeutic usage for type 2 diabetes (T2D), GLP-1 has a short half-life in vivo (t_1/2 ＜2 min). In an attempt to prolong half-life, GLP-1 fusion proteins were genetically engineered: GLP-1 human serum albumin fusion (GLP-1/HSA), AGLP-1/HSA which has an additional alanine at the N-terminus of GLP-1, and AGLP-1-L/HSA, in which a peptide linker is inserted between AGLP-1 and HSA. Recombinant fusion proteins secreted from the Chinese Hamster Ovary-K1 (CHO-K1) cell line were purified with high purity (＞96%). AGLP-1 fusion protein was resistant against the dipeptidyl peptidase-IV (DPP-IV). The fusion proteins activated cAMP-mediated signaling in rat insulinoma INS-1 cells. Furthermore, a C57BL/6N mice pharmacodynamics study exhibited that AGLP-1-L/HSA effectively reduced blood glucose level compared to AGLP-1/HSA. [BMB Reports 2013; 46(12): 606-610]     

A study on human serum albumin influence on glycation of fibrinogen

Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [¹³C₆] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen.     

Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.     

Kinetics of fatty acid binding ability of glycated human serum albumin

Kinetics of fatty acid binding ability of glycated human serum albumin (HSA) were investigated by fluorescent displacement technique with 1-anilino-8-naphtharene sulphonic acid (ANS method), and photometric detection of nonesterified-fatty-acid (NEFA method). Changing of binding affinities of glycated HSA toward oleic acid, linoleic acid, lauric acid, and caproic acid, were not observed by the ANS method. However, decreases of binding capacities after 55 days glycation were confirmed by the NEFA method in comparison to control HSA. The decrease in binding affinities was: oleic acid (84%), linoleic acid (84%), lauric acid (87%), and caproic acid (90%), respectively. The decreases were consistent with decrease of the intact lysine residues in glycated HSA. The present observation indicates that HSA promptly loses its binding ability to fatty acid as soon as the lysine residues at fatty acid binding sites are glycated.     

Fatty acid binding to serum albumin: Molecular simulation approaches

Background

Binding affinity for human serum albumin (HSA) is one of the most important factors affecting the distribution and free blood concentration of many ligands. The effect of fatty acids (FAs) on HSA-ligand binding has long been studied. Since the elucidation of the 3-dimensional structure of HSA, molecular simulation approaches have been applied to studies of the structure–function relationship of HSA–FA binding.

Scope of review

We review current insights into the effects of FA binding on HSA, focusing on the biophysical insights obtained using molecular simulation approaches such as docking, molecular dynamics (MD), and binding free energy calculations.

Major conclusions

Possible conformational changes on binding of FA molecules to HSA have been observed through MD simulations. High- and low-affinity FA-binding sites on HSA have been identified based on binding free energy calculations. The relationship between the warfarin binding affinity of HSA and FA molecules has been clarified based on the results of simulations of multi-site FA binding that cannot be experimentally observed.

General significance

Molecular simulation approaches have great potentials to provide detailed biophysical insights into HSA as well as the effects of the binding of FAs or other ligands to HSA. This article is part of a Special Issue entitled Serum Albumin.     

Chain length-dependent binding of fatty acid anions to human serum albumin studied by site-directed mutagenesis

Human serum albumin is the most abundant protein in the circulatory system, and one of its principal functions is to transport fatty acids. Binding of octanoate, decanoate, laurate and myristate was studied by a rate-of-dialysis technique. The primary association constants increased, but not linearly, with chain length. The number of high-affinity sites also increased with chain length; octanoate and decanoate bind to one such site, whereas laurate and myristate most probably bind to two sites. Albumin is composed of three homologous helical domains (I-III), which can be subdivided into two subdomains (A and B). For getting information about the positions of the high-affinity sites we produced 13 recombinant isoforms mutated in four different subdomains. Results obtained with these albumins are in accordance with the following model: octanoate and decanoate bind to a single site in subdomain IIIA, laurate binds to sites in subdomains IIIA and IIIB, whereas myristate binds in subdomains IB and IIIB. The results also showed that primary fatty acid binding is sensitive to amino acid substitutions in other parts of the protein. This is in contrast to the effect of amino acid substitutions of genetic albumin variants (alloalbumins). Usually these substitutions, which are situated at the surface of the protein, have no effect on fatty acid binding. Binding of fatty acid anions to different high-affinity sites and the sensitivity of these sites to amino acid substitutions elsewhere in the protein (and perhaps also to other types of modifications) are important factors that could effect simultaneous binding of other ligands, e.g. in patients treated with albumin-binding drugs.     

hUC-MSCs下调HIF-1α及VEGF表达对NAFLD大鼠肝脏损伤的作用

目的探讨脐带间充质干细胞（hUC-MSCs）介导HIF-1α/VEGF通路对非酒精性脂肪性肝病（NAFLD）大鼠肝脏损伤的作用。 方法SD大鼠随机分为正常对照组（NC组）、模型对照组（MC组）和MC+hUC-MSCs组。每组鼠数为7只,分别喂食不同饲料和治疗8周。检测大鼠谷丙转氨酶（ALT）、谷草转氨酶（AST）和胰岛素抵抗指数（HOMA-IR）。光镜观察大鼠肝脏组织病理改变,计算NAFLD活动度积分（NAS）;Western Blot法检测大鼠肝脏组织HIF-?1α和VEGF蛋白表达。组间比较采用单因素方差分析、相关分析选用pearson。 结果（1）治疗末,NC组ALT、AST和HOMA-IR分别为（41.1±5.9）U/L,（51.7±5.2）U/L,（1.93±0.22）?U/?L低于MC组（153.9±7.1）U/L,（169.8±15.9）U/L,（23.20±2.63）U/L差异具有统计学意义（P?< 0.05）;与MC组比较,MC+hUC-MSCs组大鼠ALT、AST和HOMA-IR降低分别为（90.7±8.1）?U/?L,（110.0±13.1）U/L,（8.43±1.39）U/L差异具有统计学意义（P?< 0.05）。（2）光镜下NC组肝细胞形态正常;MC组肝细胞呈现脂肪变性,较多细胞核变形,肝小叶排列不齐伴炎症细胞浸润;以上肝组织病理改变在MC+hUC-MSCs组明显改善。与NC组比较,MC组大鼠NAS积分增高;与MC组比较,MC+hUC-MSCs组大鼠NAS积分降低[（0.42 ±0.23）分vs （9.15±0.41）?分、（5.15±0.29）分]。（3）Western Blot法检测肝脏组织HIF-1α和VEGF蛋白表达改变：与NC组比较,MC组大鼠HIF-1α和VEGF蛋白表达均增高（P均< 0.05）;与MC组比较,MC+hUC-?MSCs组大鼠HIF-1α和VEGF蛋白表达均降低（P均< 0.05）。单因素相关分析显示大鼠肝脏组织HIF-?1α表达与HOMA-IR指数呈正相关（P均< 0.05）。而且,大鼠肝脏组织NAS评分与肝脏组织HIF-?1α、VEGF表达亦呈正相关（r值分别为0.901、0.874,P均< 0.05）。 结论?hUC-?MSCs对高糖高脂饲料喂养诱导的NAFLD大鼠受损肝脏功能具有改善作用,其机制与其下调HIF-?1α/?VEGF通路相关。     

Structural basis of the drug-binding specificity of human serum albumin

Human serum albumin (HSA) is an abundant plasma protein that binds a remarkably wide range of drugs, thereby restricting their free, active concentrations. The problem of overcoming the binding affinity of lead compounds for HSA represents a major challenge in drug development. Crystallographic analysis of 17 different complexes of HSA with a wide variety of drugs and small-molecule toxins reveals the precise architecture of the two primary drug-binding sites on the protein, identifying residues that are key determinants of binding specificity and illuminating the capacity of both pockets for flexible accommodation. Numerous secondary binding sites for drugs distributed across the protein have also been identified. The binding of fatty acids, the primary physiological ligand for the protein, is shown to alter the polarity and increase the volume of drug site 1. These results clarify the interpretation of accumulated drug binding data and provide a valuable template for design efforts to modulate the interaction with HSA.     

Recombinant Human Albumin in Cell Culture: Evaluation of Growth-Promoting Potential for NRK and SCC-9 Cells In Vitro

Serum-derived albumin has for a long time been used in cell culture media, but the exact role of albumin and/or impurities bound to albumin has not been precisely defined. In this study, recombinant human albumin was evaluated for its growth-promoting activity on two cell lines, NRK and SCC-9. For NRK cells, the recombinant human albumin was found to exert an inhibitory effect. The fact that fatty acid free HSA was also inhibitory while HSA fraction V was stimulatory suggested a role for fatty acids or some other bound moieties in growth stimulation by HSA fraction V. Addition of oleic acid, cholesterol, phosphatidylcholine, phosphatidylserine or a combination of these lipids, however, did not significantly improve the growth stimulating activity of either fatty acid free HSA or the recombinant human albumin. For SCC-9 cells, both recombinant human albumin and fatty acid free HSA showed slight stimulation (although they were not as active as HSA fraction V), suggesting that in some cell systems, the albumin molecule per se may promote cell growth and survival. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mutational analysis of the interaction between albumin-binding domain from streptococcal protein G and human serum albumin

Streptococcal protein G (SpG) is a bacterial cell surface receptor exhibiting affinity to both human immunoglobulin (IgG) and human serum albumin (HSA). Interestingly, the serum albumin and immunoglobulin-binding activities have been shown to reside at functionally and structurally separated receptor domains. The binding domain of the HSA-binding part has been shown to be a 46-residue triple alpha-helical structure, but the binding site to HSA has not yet been determined. Here, we have investigated the precise binding region of this bacterial receptor by protein engineering applying an alanine-scanning procedure followed by binding studies by surface plasmon resonance (SPR). The secondary structure as well as the HSA binding of the resulting albumin-binding domain (ABD) variants were analyzed using circular dichroism (CD) and affinity blotting. The analysis shows that the HSA binding involves residues mainly in the second alpha-helix.