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.

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.

Analbuminemia in a Slovak Romany (gypsy) family: case report and mutational analysis.

BACKGROUND: Analbuminemia is a rare autosomal recessive disorder manifested by the absence, or severe reduction, of circulating serum albumin. Here we report three new cases of hereditary analbuminemia, fortuitously detected in three Slovak Romany children, members of the same family, and define the molecular defect that causes the analbuminemic trait. METHODS: Total DNA, extracted from peripheral blood samples from six members of the family, was PCR-amplified using oligonucleotide primers designed to amplify the 14 exons of the human albumin gene and the flanking intron regions. The products were screened for mutations by single-strand conformation polymorphism (SSCP) and heteroduplex analyses (HA). HA allowed the identification of the abnormal fragment, which was then sequenced. RESULTS: In the 3 patients the analbuminemic trait was caused by the same mutation, an AT deletion at nucleotides 2430-31, the 91 th and 92 th bases of exon 3. This defect, previously identified as Kayseri mutation, produces a frameshift leading to a premature stop, two codons downstream. The predicted translation product would consist of 54 amino acid residues. The parents were found to be heterozygous for the mutation. CONCLUSIONS: Our results confirm that the combination of SSCP and HA represents a powerful tool to study the molecular defects causing analbuminemia in humans.

Analbuminemia in a Swiss family is caused by a C --> T transition at nucleotide 4446 of the albumin gene.

OBJECTIVE: To define the molecular defect that causes analbuminemia in an apparently healthy boy, son of non-consanguineous Swiss parents. DESIGN AND METHODS: Total DNA, extracted from peripheral blood samples from the proband and from both parents, was PCR-amplified using oligonucleotide primers designed to amplify the 14 exons of the human albumin gene and the flanking intron regions. The products were screened for mutations by single-strand conformation polymorphism (SSCP) and heteroduplex analyses (HA) either directly or after digestion with restriction enzymes. The combination of these methods identified the abnormal fragment, which was then sequenced. RESULTS: DNA sequence analysis identified in the homozygous proband a C --> T transition at nucleotide 4446. The mutation changes the codon CGA for Arg 114 to a stop codon TGA, resulting in premature termination and is therefore responsible for the analbuminemic trait. The same mutation has been previously reported to cause analbuminemia in an American female. The putative protein product would have a length of 113 residues. The parents were found to be heterozygous for the mutation. CONCLUSIONS: Gel-based mutation detection and DNA sequencing confirmed the diagnosis of congenital analbuminemia in the proband. Our results show that the combination of SSCP and HA represents a powerful tool to study the molecular defects causing analbuminemia in humans.

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 Angstroms resolution. The fragment folds as a thyroglobulin type I domain and was found to bind the Fe(2+) 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.

Catalytic activity, stability, unfolding, and degradation pathways of engineered and reconstituted myoglobins.

The structural and functional consequences of engineering a positively charged Lys residue and replacing the natural heme with a heme-L-His derivative in the active site of sperm whale myoglobin (Mb) have been investigated. The main structural change caused by the distal T67K mutation appears to be mobilization of the propionate-7 group. Reconstitution of wild-type and T67K Mb with heme-L-His relaxes the protein fragment around the heme because it involves the loss of the interaction of one of the propionate groups which stabilize heme binding to the protein. This modification increases the accessibility of exogenous ligands or substrates to the active site. The catalytic activity of the reconstituted proteins in peroxidase-type reactions is thus significantly increased, particularly with T67K Mb. The T67K mutation slightly reduces the thermodynamic stability and the chemical stability of Mb during catalysis, but somewhat more marked effects are observed by cofactor reconstitution. Hydrogen peroxide, in fact, induces pseudo-peroxidase activity but also promotes oxidative damage of the protein. The mechanism of protein degradation involves two pathways, which depend on the evolution of radical species generated on protein residues by the Mb active species and on the reactivity of phenoxy radicals produced during turnover. Both protein oligomers and heme-protein cross-links have been detected upon inactivation.

Human alpha-1-microglobulin is covalently bound to kynurenine-derived chromophores.

Alpha-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 alpha-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 alpha-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 alpha-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 alpha-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.