Monica Campagnoli

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 ).

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.

Mapping the 5–50‐kDa fraction of human amniotic fluid proteins by 2‐DE and ESI‐MS

This report presents a proteomic analysis and provides a reference map of the 5–50‐kDa components of normal amniotic fluid collected in gestational weeks 16–18. Early amniocentesis samples were pooled and proteins with molecular mass lower than albumin were separated by gel filtration chromatography. The 2‐DE protocol was optimized for the separation of the small proteins and peptides in the fraction of interest. A total of 132 Coomassie blue‐stained protein spots were analyzed, following in‐gel tryptic digestion, by ESI‐MS/MS and 49 different gene products were identified. The treatment with alkaline phosphatase caused the shift of the phosphoisoforms of insulin‐like growth factor‐binding protein‐1 and of the N‐terminal osteopontin fragment. Of the 33 full‐length proteins identified in the 2‐DE profile, 23 had not been previously detected in the amniotic fluid and, of these, 22 are not present in the human plasma proteome under physiological conditions. Fragments of 16 larger proteins were identified and the sequence coverage data revealed that several correspond to autonomous domains that may have biological roles on their own. Several of the detected proteins and peptides appear to be involved in critical regulatory processes associated with placentation and early development, thus representing potential markers of various physiological or pathological conditions.

Molecular diagnosis of analbuminemia: a new case caused by a nonsense mutation in the albumin gene.

Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin (ALB). We report here a new case diagnosed in a 45 years old man of Southwestern Asian origin, living in Switzerland, on the basis of his low ALB concentration (0.9 g/L) in the absence of renal or gastrointestinal protein loss, or liver dysfunction. The clinical diagnosis was confirmed by a mutational analysis of the albumin (ALB) gene, carried out by single-strand conformational polymorphism (SSCP), heteroduplex analysis (HA), and DNA sequencing. This screening of the ALB gene revealed that the proband is homozygous for two mutations: the insertion of a T in a stretch of eight Ts spanning positions c.1289 + 23–c.1289 + 30 of intron 10 and a c.802 G > T transversion in exon 7. Whereas the presence of an additional T in the poly-T tract has no direct deleterious effect, the latter nonsense mutation changes the codon GAA for Glu244 to the stop codon TAA, resulting in a premature termination of the polypeptide chain. The putative protein product would have a length of only 243 amino acid residues instead of the normal 585 found in the mature serum albumin, but no evidence for the presence in serum of such a truncated polypeptide chain could be obtained by two dimensional electrophoresis and western blotting analysis.

Identification of the amniotic fluid insulin‐like growth factor binding protein‐1 phosphorylation sites and propensity to proteolysis of the isoforms

Insulin‐like growth factor binding protein‐1 (IGFBP‐1) is the major secreted protein of human decidual cells during gestation and, as a modulator of insulin‐like growth factors or by independent mechanisms, regulates embryonic implantation and growth. The protein is phosphorylated and this post‐translational modification is regulated in pregnancy and represents an important determinant of its biological activity. We have isolated, from human normal amniotic fluid collected in the weeks 16–18, the intact nonphosphorylated IGFBP‐1 and five electrophoretically distinct phosphoisoforms and have determined their in vivo phosphorylation state. The unmodified protein was the most abundant component and mono‐, bi‐, tri‐ and tetraphosphorylated forms were present in decreasing amounts. The phosphorylation sites of IGFBP‐1 were identified by liquid chromatography–tandem mass spectrometry analysis of the peptides generated with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. Five serines were found to be phosphorylated and, of these, four are localized in the central, weakly conserved, region, at positions 95, 98, 101 and 119, whereas one, Ser169, is in the C‐terminal domain. The post‐translational modification predominantly involves the hydrophilic stretch of amino acids representing a potential PEST sequence (proline, glutamic acid, serine, threonine) and our results show that the phosphorylation state influences the propensity of IGFBP‐1 to proteolysis.

A novel mutation in the albumin gene (c.1A>C) resulting in analbuminemia.

Eur J Clin Invest 2012

Genetic variants showing apparent hot-spots in the human serum albumin gene.

The molecular defects of three different slow-migrating genetic variants of human serum albumin, albumins Kamloops (formerly RIH), Stirling and Amsterdam, previously characterized only by electrophoretic and dye-binding studies, are now reported. Two of them are proalbumin variants: sequential analysis of the purified whole proteins has established the mutation responsible for albumin Kamloops as -1Arg-->Gln, and for albumin Stirling as -2Arg-->His. A Glu-->Lys substitution in position 570 of the mature albumin molecule was determined in albumin Amsterdam by sequential analysis of two abnormal tryptic fragments. The three alloalbumins are caused by single-base changes all of which seem to represent hot-spots in the albumin gene. The possible functional consequences of the presence of a circulating alloalbumin are discussed.

A novel frameshift deletion in the albumin gene causes analbuminemia in a young Turkish woman.

BACKGROUND Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin. The analbuminemic trait was diagnosed in a young Turkish woman on the basis of her clinical symptoms (bilateral lower limb edema) and biochemical findings (minimal albumin amount and variable increases in other protein fractions). METHODS Total DNA from the analbuminemic proband and her parents was PCR-amplified using oligonucleotide primers designed to amplify the 14 exons of the albumin gene (ALB) and the flanking intron regions. The products were screened for mutations by single-strand conformation polymorphism (SSCP) and heteroduplex analyses (HA). RESULTS HA allowed the identification of the mutation site in exon 12. Direct DNA sequencing of this abnormal fragment revealed that the analbuminemic trait was caused by a homozygous CA deletion at nucleotide positions c. 1614-1615 in the codons for Cys538 and Thr539. The subsequent frameshift should give rise to a putative truncated albumin variant in which the sequence Cys(538)-Thr-Leu-Ser has been changed to Cys(538)-Thr-Phe-Stop. The parents were heterozygous for the same mutation. CONCLUSIONS Gel-based mutation detection and DNA sequencing substantiate the clinical diagnosis of congenital analbuminemia in our patient and show that the condition is caused by a novel mutation within the ALB gene. These results contribute to shed light on the molecular basis of this rare condition.

A novel frame‐shift deletion causing analbuminaemia in an Italian paediatric patient

Eur J Clin Invest 2010; 40 (3): 281–284