Lorenzo Minchiotti

Mutations and polymorphisms of the gene of the major human blood protein, serum albumin†

We have tabulated the 77 currently known mutations of the familiar human blood protein, serum albumin (ALB). A total of 65 mutations result in bisalbuminemia. Physiological and structural effects of these mutations are included where observed. Most of the changes are benign. The majority of them were detected upon clinical electrophoretic studies, as a result of a point mutation of a charged amino acid residue. Three were discovered by their strong binding of thyroxine or triiodothyronine. A total of 12 of the tabulated mutations result in analbuminemia, defined as a serum albumin concentration of <1 g/L. These were generally detected upon finding a low albumin concentration in patients with mild edema, and involve either splicing errors negating translation or premature stop codons producing truncated albumin molecules. A total of nine mutations, five of those with analbuminemia and four resulting in variants modified near the C‐terminal end, cause frameshifts. Allotypes from three of the point mutations become N‐glycosylated and one C‐terminal frameshift mutation shows O‐glycosylation. Hum Mutat 0,1–10, 2008.

Molecular heterogeneity of ferredoxin-NADP+ reductase from spinach leaves

Ferredoxin-NADP+ reductase (NADPH: ferredoxin oxidoreductase, EC 1.6.7.1) from spinach leaves has been purified according to a new procedure. The enzyme shows the presence of five molecular forms as identified by isoelectric focusing, namely a, b, c, d and e with pI values of 6.0, 5.5, 5.2, 5.0 and 4.8, respectively. All the bands are catalytically active and are clearly identifiable after the first steps of the purification procedure. The basic pattern of the ferredoxin-NADP+ reductase forms is the same whether extracted from one or many spinach plants and is not affected by the different purification procedures used. Two distinct classes of molecular weight have been found for the isolated forms b, c and d as measured by sodium dodecyl sulphate electrophoresis, with values of 33 000-34 000 for the first and 36 000-38 000 for the later two forms. Gel electrophoresis in non-denaturing media at different gel concentrations gives the same order of molecular weight values, thus ruling out the possibility that the native enzyme is a dimer, as has been reported by Schneeman, R. and Krogmann, D.W. ((1975) J. Biol. Chem. 250, 4965-4971). No significant kinetic differences were detectable for the isolated forms of ferredoxin-NADP+ reductase.

Human serum albumin isoforms: Genetic and molecular aspects and functional consequences

BACKGROUND At present, 67 different genetic variants of human serum albumin and proalbumin have been molecularly characterized at the protein and/or gene level. SCOPE OF REVIEW This review summarizes present knowledge about genetic and molecular aspects, functional consequences and potential uses of the variants. MAJOR CONCLUSIONS The frequency of bisalbuminemia in the general population is probably about 1:1000, but it can be much higher in isolated populations. Mutations are often due to hypermutable CpG dinucleotides, and in addition to single-amino acid substitutions, glycosylated variants and C-terminally modified alloalbumins have been found. Some mutants show altered stability in vivo and/or in vitro. High-affinity binding of Ni(++) and Cu(++) is blocked, or almost so, by amino acid changes at the N-terminus. In contrast, substitution of Leu90 and Arg242 leads to strong binding of triiodothyronine and l-thyroxine, respectively, resulting in two clinically important syndromes. Variants often have modified plasma half-lives and organ uptakes when studied in mice. GENERAL SIGNIFICANCE Because alloalbumins do not seem to be associated with disease, they can be used as markers of migration and provide a model for study of neutral molecular evolution. They can also give valuable molecular information about albumins binding sites, antioxidant and enzymatic properties, as well as stability. Mutants with increased affinity for endogenous or exogenous ligands could be therapeutically relevant as antidotes, both for in vivo and extracorporeal treatment. Variants with modified biodistribution could be used for drug targeting. In most cases, the desired function can be further elaborated by producing site-directed, recombinant mutants. This article is part of a Special Issue entitled Serum Albumin.

Structure and properties of the C-terminal domain of insulin-like growth factor-binding protein-1 isolated from human amniotic fluid.

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1) regulates the activity of the insulin-like growth factors in early pregnancy and is, thus, thought to play a key role at the fetal-maternal interface. The C-terminal domain of IGFBP-1 and three isoforms of the intact protein were isolated from human amniotic fluid, and sequencing of the four N-terminal polypeptide chains showed them to be highly pure. The addition of both intact IGFBP-1 and its C-terminal fragment to cultured fibroblasts has a similar stimulating effect on cell migration, and therefore, the domain has a biological activity on its own. The three-dimensional structure of the C-terminal domain was determined by x-ray crystallography to 1.8 Å resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe2+ ion in the crystals through the only histidine residue present in the polypeptide chain. Iron (II) decreases the binding of intact IGFBP-1 and the C-terminal domain to IGF-II, suggesting that the metal binding site is close to or part of the surface of interaction of the two molecules.

Structural and biochemical characterization of a new type of lectin isolated from carp eggs

A previously unidentified glycoprotein present in the eggs of the carp ( Cyprinus carpio ) was isolated and structurally characterized. The protein binds to a Sepharose 4B matrix and can be eluted with 0.4 M N -acetylglucosamine. The protein has an apparent molecular mass of 26686.3 Da. On the basis of gel-filtration chromatography, the protein appears to be present in solution as a monomer. The sequence of its 238 amino acids, the position of its four disulphide bridges and the composition of its single N-linked carbohydrate chain were determined. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram-positive and -negative bacteria. These latter interactions are inhibited by N -acetylglucosamine. A database search shows that its amino acid sequence is similar to that of the members of an invertebrate lectin family that includes tachylectin-1. Tachylectin-1 is present in the amoebocytes of the horseshoe crab, Tachypleus tridentatus, and plays a role in the innate defence system of this species. Homologous genes are also present in other fish, having 85% identity with a gene expressed in the oocytes of the crucian carp ( Carassius auratus gibelio ) and 78% identity with a gene in the cDNA library of the zebrafish ( Danio rerio ).

High-affinity binding of warfarin, salicylate and diazepam to natural mutants of human serum albumin modified in the C-terminal end

High-affinity binding of warfarin, salicylate and diazepam to four natural mutants of human serum albumin was studied by equilibrium dialysis at pH 7.4. The mutants Alb Milano Fast and Alb Vanves possess single amino acid substitutions close to the C-terminus, namely 573 Lys-->Glu and 574 Lys-->Asn, respectively. By contrast, Alb Catania and Alb Venezia are chain termination mutants in which several amino acids have been changed or deleted. Binding of warfarin to the variants was lower than binding to normal (wild-type) albumin (Alb A). The association constants were 73% (Alb Milano Fast, Alb Vanves and Alb Catania) or 67% (Alb Venezia) of that determined for Alb A. The results obtained with salicylate were more dependent on the type of mutation. The constants were either comparable to the normal value (Alb Catania) or reduced to 64% (Alb Milano Fast), 71% (Alb Vanves) or 43% (Alb Venezia) of that value. Diazepam binding to the variants was normal, except for binding to Alb Venezia in which case the association constant was reduced to 76% of that calculated for Alb A. The results are in accordance with the view that warfarin, salicylate and diazepam bind to three different high-affinity sites. It is proposed that the sites for warfarin and salicylate are situated rather close to each other in domain II, and that these high-affinity sites are relatively susceptible to conformational changes of the protein. By contrast, the primary diazepam site is placed closer to, or within, domain III of albumin and seems to be less affected by conformational changes in the protein molecule.

Human α-1-microglobulin is covalently bound to kynurenine-derived chromophores

α-1-Microglobulin carries a set of covalently linked chromophores that give it a peculiar yellow-brown color, fluorescence properties, and both charge and size heterogeneity. In this report it is shown that these features are due to the adducts with the tryptophan metabolite, 3-hydroxykynurenine, and its autoxidation products and that the modification is more pronounced in the protein isolated from urine of hemodialyzed patients. The light yellow amniotic fluid α-1-microglobulin acquires the optical properties and charge heterogeneity of the urinary counterpart following incubation with kynurenines. The colored amino acid adducts of urinary and amniotic fluid α-1-microglobulins were separated by chromatography after acid hydrolysis and analyzed by mass spectrometry. Human serum albumin samples, native and treated with 3-hydroxykynurenine in the presence of oxygen, were used as a control. The retention times and mass fragmentation products were compared, and a lysyl adduct with hydroxantommathin was identified in the urinary α-1-microglobulin and in the modified albumin samples. The more extensive modification of the urinary protein appears to be correlated with uremia, a condition in which the catabolism of tryptophan via the kynurenine pathway is increased, and the consequent rise in the concentration of its derivatives is accompanied by the oxidative processes due to the hemodialysis treatment. The oxidative derivatives of 3-hydroxykynurenine, which are known to act as protein cross-linking agents, are the likely cause of the propensity of urinary α-1-microglobulin to form dimers and oligomers. This process, as well as the redox properties of these metabolites, may contribute to the toxic effects of the kynurenine species.

Effect of genetic variation on the fatty acid-binding properties of human serum albumin and proalbumin

In the circulation, non-esterified fatty acids are transported by albumin which also facilitates their removal from donor cells and uptake into receptor cells. We have studied whether genetic variations in the albumin molecule can affect its in vivo fatty acid-binding properties. The fatty acids bound to 25 structurally different variants and to their wildtype counterparts, isolated from heterozygous carriers, were determined gas chromatographically. The variants were proalbumins, albumins with single amino acid substitutions and glycosylated or truncated albumins. In eight cases the total amount bound to the variants was diminished (0.4-0.8-fold), and in seven cases the load was increased to 1.3 or more of normal. Twenty-one fatty acids were quantitated, and for 19 alloalbumins significant deviations from normal were found. Usually, changes in total and individual fatty acid binding were of the same type, but several exceptions to this rule was found. The glycosylated albumin Casebrook showed the largest changes, the total load and the amount of bound palmitate was 8.6 and 14 times, respectively, the normal. The most pronounced changes and the majority of cases of increased binding were caused by molecular changes in domain III. Mutations in domain I, II and the propeptide resulted in smaller effects, if any, and these were often reductions in binding.

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.

Binding and Relaxometric Properties of Heme Complexes with Cyanogen Bromide Fragments of Human Serum Albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.