Perfusion of the human placenta with red blood cells and xanthine oxidase mimics preeclampsia in-vitro.

BACKGROUND AND PURPOSE: Preeclampsia is a major obstetric problem of unknown etiology. The fact that removal of the placenta is the only cure for preeclampsia, has led to the well-established hypothesis, that the placenta is central in the etiology. Gene profiling and proteomics studies have suggested oxidative stress caused by reperfusion and free oxygen radicals as a potential pathophysiological mechanism in preeclampsia. In this study, the dual placental perfusion model was used in order to evaluate the damaging effects of oxidative stress induced by xanthine/xanthine oxides and free hemoglobin. MATERIAL AND METHODS: The dual placenta perfusion model is a well-established in vitro model for functional placental studies. Placentas were perfused with medium containing either xanthine/xanthine oxidase or erythrocytes as a source of free hemoglobin. Concentration of free hemoglobin in the medium was measured by means of ELISA. Whole genome microarray technique and bioinformatics were used to evaluate the gene expression profile in the two groups. RESULTS: Substantial levels of free adult hemoglobin were detected in the perfusions. A total of 58 genes showed altered gene expression, the most altered were hemoglobin alpha, beta and gamma, tissue factor pathway inhibitor 2 and superoxide dismutase 2. Bioinformatics revealed that biological processes related to oxidative stress, anti-apoptosis and iron ion binding were significantly altered. CONCLUSIONS: The results suggest that perfusion with xanthine/xanthine oxidase and free hemoglobin induce changes in gene expression similar to what has been described for the preeclamptic placenta.

An intriguing member of the lipocalin protein family: alpha 1-microglobulin.

The plasma protein alpha 1-microglobulin is a member of the lipocalin protein superfamily. In the last few years, the work on alpha 1-microglobulin has given unexpected and promising new results. Of particular interest are its molecular association with immunoglobulin A and with proteinase inhibitors, and its interactions with the immune system.

Immunoreactive pancreatic colipase, lipase and phospholipase A2 in human plasma and urine from healthy individuals.

A radioimmunoassay for each of the human pancreatic proteins (colipase, lipase and phospholipase A2) is described. Determinations of the mean concentration of each protein in plasma and urine from healthy individuals were carried out with the radioimmunoassays. The values obtained in plasma were 0.5 nM (5.3 micrograms/l), 0.6 nM (32 micrograms/l) and 0.3 nM (4.3 micrograms/l) for colipase, lipase and phospholipase A2, respectively. In urine, the corresponding values were found to be 0.2 nM (2.4 micrograms/l), 0.09 nM (4.4 micrograms/l) and less than 0.017 nM (0.2 micrograms/l). No physical interaction between any of the three proteins and the lipid particles of plasma was demonstrated by centrifugation experiments or gel filtration. Gel filtration of plasma depleted of fat by centrifugation showed the proteins only in their monomeric form. The corresponding porcine proteins displayed a binding to antibodies against the human proteins, but with a lower affinity than the homologous interactions. The binding was weak but could differentiate between the porcine proforms and activated ones, i.e., procolipase and colipase87.

Isolation of plaice (Pleuronectes platessa) alpha1-microglobulin: conservation of structure and chromophore.

A cDNA coding for plaice (Pleuronectes platessa) alpha1-microglobulin (Leaver et al., 1994, Comp. Biochem. Physiol. 108B, 275-281) was expressed and purified from baculovirus-infected insect cells. Specific monoclonal antibodies were then prepared and used to isolate the protein from plaice liver and serum. Mature 28.5 kDa alpha1-microglobulin was found in both liver and serum. The protein consisted of an 184 amino acid peptide with a complex N-glycan in position Asn123, one intrachain disulfide bridge and a yellow-brown chromophore. Physicochemical characterization indicated a globular shape with a frictional ratio of 1.37, electrophoretic charge-heterogeneity and antiparallel beta-sheet structure. A smaller, incompletely glycosylated, yellow-brown alpha1-microglobulin as well as a 45 kDa precursor protein were also found in liver. The chromophore was found to be linked to alpha1-microglobulin intracellularly. Recombinant plaice alpha1-microglobulin isolated from insect cells had the same N-terminal sequence, globular shape and yellow-brown color as mature alpha1-microglobulin, but carried a smaller, fucosylated, non-sialylated N-glycan in the Asn123 position. The concentration of alpha1-microglobulin in plaice serum was 20 mg/l and it was found both as a 28.5 kDa component and as high molecular weight components. Thus, the size, shape, charge and color of plaice alpha1-microglobulin were similar to mammalian alpha1-microglobulin, indicating a high degree of structural conservation between fish and human alpha1-microglobulin. The monoclonal antibodies against plaice alpha1-microglobulin cross-reacted with human alpha1-microglobulin, emphasizing the structural similarity.

Bovine alpha 1-microglobulin/bikunin. Isolation and characterization of liver cDNA and urinary alpha 1-microglobulin.

cDNA coding for alpha 1-microglobulin, an immunoregulatory plasmaprotein, was isolated from bovine liver. The sequence of a total of 1258 nucleotides revealed an open reading frame of 352 amino acids. This included alpha 1-microglobulin, 182 amino acids, and bikunin, the light chain of the plasmaprotein inter-alpha-inhibitor, 147 amino acids. The two proteins were connected by a basic tetrapeptide, R-A-R-R, which conforms to the consensus sequence recognized by endoproteolytic cleavage enzymes. The deduced amino acid sequence showed a high degree of identity with alpha 1-microglobulin and bikunin sequences from other species, and the alpha 1-microglobulin part displayed sequence motifs typical for members of the lipocalin protein superfamily. A single alpha 1-microglobulin/bikunin mRNA with a size of around 1300 nt was found in bovine liver. The mature alpha 1-microglobulin protein was isolated from bovine urine, and partly characterized. It was found to be a globular molecule with an apparent molecular weight of 23,300, containing one N-linked and at least on O-linked oligosaccharide, one intra-chain disulfide bridge and an electrophoretic heterogeniety with a pI-value of 4.1-5.2.

Cleavage of the alpha 1-microglobulin-bikunin precursor is localized to the Golgi apparatus of rat liver cells.

alpha 1-Microglobulin, a plasma protein with immunoregulatory properties, and bikunin, the light chain of the proteinase inhibitors inter-alpha-inhibitor and pre-alpha-inhibitor, are translated as a precursor protein from the same mRNA. The cosynthesis of alpha 1-microglobulin and bikunin is unique compared to other proproteins such as procomplement components and prohormones, since alpha 1-microglobulin and bikunin have no known functional connection. Different forms of intracellular rat liver alpha 1-microglobulin were isolated and characterized by amino acid sequence analysis, lectin binding and glycosidase treatment. Their subcellular distribution was studied by Nycodenz and sucrose gradient centrifugation, pulse-chase experiments, and electrophoresis with subsequent immunoblotting, using pro-C3 and prohaptoglobin as reference proteins. Two alpha 1-microglobulin-bikunin precursors (40 and 42 kDa), containing one and two N-linked oligosaccharides, respectively, were detected in the endoplasmic reticulum. After transport to the Golgi apparatus, the precursors were cleaved, probably C-terminal to the sequence Arg-Ala-Arg-Arg immediately preceding the bikunin part, yielding free sialylated 28 kDa alpha 1-microglobulin, representing the mature protein. The cleavage was almost complete in phosphatidylinositol 4-kinase-enriched membranes, previously identified as a post-Golgi compartment. A fourth intracellular form of alpha 1-microglobulin, 26 kDa, lacked sialic acid. None of the intracellular forms carried the yellow-brown chromophore associated with alpha 1-microglobulin when purified from serum and urine, suggesting that this chromophore becomes linked to the protein after its secretion from the liver cells.

alpha(1)-Microglobulin: a yellow-brown lipocalin.

alpha(1)-Microglobulin, also called protein HC, is a lipocalin with immunosuppressive properties. The protein has been found in a number of vertebrate species including frogs and fish. This review summarizes the present knowledge of its structure, biosynthesis, tissue distribution and immunoregulatory properties. alpha(1)-Microglobulin has a yellow-brown color and is size and charge heterogeneous. This is caused by an array of small chromophore prosthetic groups, attached to amino acid residues at the entrance of the lipocalin pocket. A gene in the lipocalin cluster encodes alpha(1)-microglobulin together with a Kunitz-type proteinase inhibitor, bikunin. The gene is translated into the alpha(1)-microglobulin-bikunin precursor, which is subsequently cleaved and the two proteins secreted to the blood separately. alpha(1)-Microglobulin is found in blood and in connective tissue in most organs. It is most abundant at interfaces between the cells of the body and the environment, such as in lungs, intestine, kidneys and placenta. alpha(1)-Microglobulin inhibits immunological functions of white blood cells in vitro, and its distribution is consistent with an anti-inflammatory and protective role in vivo.

Rat alpha 1-microglobulin: co-expression in liver with the light chain of inter-alpha-trypsin inhibitor.

A 1162 bp rat liver cDNA clone encoding the immunoregulatory plasma protein alpha 1-microglobulin was isolated and sequenced. The open reading frame encoded a 349 amino acid polyprotein, including alpha 1-microglobulin, 182 amino acids, and bikunin, the light chain of the plasma protein inter-alpha-trypsin inhibitor, 145 amino acids. The alpha 1-microglobulin/bikunin mRNA was found only in the liver when different tissues were examined. Free alpha 1-microglobulin and a polyprotein, containing both alpha 1-microglobulin and inter-alpha-trypsin inhibitor epitopes, were found in the microsomal fraction from rat liver homogenates.

Increase of bikunin and alpha1-microglobulin concentrations in urine of rats during pregnancy is due to decreased tubular reabsorption.

Bikunin and alpha1-microglobulin are two plasma proteins of about 25 kDa which are made in the liver from a common precursor. The concentration of bikunin in human urine has been shown to increase several fold during various conditions of stress. The mechanism behind this increase is unknown. We have studied pregnant rats and found that the bikunin and alpha1-microglobulin levels in their urine increased 3-fold towards the end of the pregnancy, whereas those of albumin and orosomucoid did not. There were no significant changes in either the bikunin/alpha1-microglobulin mRNA level or the concentrations of the two proteins in serum. These findings imply that the synthesis and the clearance rates of bikunin and alpha1-microglobulin are normal during pregnancy but that the tubular reabsorption of these proteins is decreased.

Binding properties of protein Arp, a bacterial IgA-receptor.

A cell surface receptor that binds to the Fc region of IgA is expressed by certain strains of group A streptococci. The physico-chemical properties and binding characteristics of this receptor, called protein Arp, were studied. Like bacterial receptors that bind IgG, protein Arp has an elongated shape and no disulfide bonds. The affinity constant of protein Arp for three different molecular forms of IgA was determined, and was found to be more than ten-fold higher for serum IgA than for two complexed forms of IgA: secretory IgA and IgA bound to alpha 1-microglobulin. Cleavage of protein Arp with CNBr resulted in a peptide corresponding to the region located outside the cell wall, except for the N-terminal 52 amino acids. This CNBr-fragment did not bind IgA, which strongly suggests that the IgA-binding region of protein Arp is located in the N-terminal part of the molecule. In addition to the binding of IgA, protein Arp also binds to IgG weakly. The pH-dependence of these two types of binding is different, with maximal binding of IgA at neutral pH (5-7) and maximal binding of IgG at acidic pH (3-5). Both for IgA and IgG, protein Arp shows strong specificity for immunoglobulins of human origin.

Interaction between streptococcal protein Arp and different molecular forms of human immunoglobulin A.

Protein Arp, the IgA-binding protein of the group A Streptococcus, has affinity for the Fc-part of IgA. The binding between protein Arp and several different molecular forms of human IgA was characterized. It was found that protein Arp bound with higher affinity to uncomplexed forms of IgA than to complexed forms (secretory IgA, alpha 1-antitrypsin-IgA and alpha 1-microglobulin-IgA). Thus, the affinity constant was 2.0-5.9 x 10(8) M-1 for the binding to monomeric, dimeric, trimeric, and quadrimeric IgA, and 4.5-5.0 x 10(7) M-1 for binding to the complexed forms. Among the uncomplexed IgA-molecules, the affinity constant was in the same range for J chain-containing forms (dimeric, trimeric and quadrimeric IgA) as for forms without J chain (monomeric and a particular quadrimeric IgA devoid of J chain). Western blotting demonstrated that protein Arp bound exclusively to the alpha-chain of all IgA-forms. Several lines of evidence pointed to a localization of the binding site to the C alpha 3-domain. First, protein Arp did not bind to three N-terminal alpha-chain fragments which lacked a region corresponding to the C alpha 3-domain, including that form a four-chain myeloma IgA, naturally occurring in plasma. Second, the binding to dimeric and tri/quadrimeric IgA was partially blocked by an added secretory component, which has been suggested to bind to the C alpha 2- and C alpha 3-domains of the alpha-chain. Finally, alpha 1-antitrypsin and alpha 1-microglobulin, in the weakly binding IgA-complexes, have been shown to be linked to the C alpha 3-domain via the penultimate amino acid residue of the alpha-chain peptide, supporting the hypothesis of a localization of the binding site of protein Arp to the C alpha 3-domain.

Alpha1-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound.

Alpha1-microglobulin (alpha1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three-dimensional (3D) structure that forms an internal hydrophobic ligand-binding pocket. Alpha1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alpha1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of alpha1m. A 3D model of alpha1m, based on the structure of rat epididymal retinoic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of alpha1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha1m is complex bound to IgA. Only the free alpha1m carried colored groups, whereas alpha1m linked to IgA was uncolored.

The alpha1-microglobulin/bikunin gene: characterization in mouse and evolution.

The 129Sv mouse gene coding for the alpha1-microglobulin/bikunin precursor has been isolated and characterized. The 11kb long gene contains ten exons, including six 5'-exons coding for alpha1-microglobulin and four 3'-exons encoding bikunin. Exon 7 also codes for the tribasic tetrapeptide RARR which connects the alpha1-microglobulin and bikunin parts. The sixth intron, which separates the alpha1-microglobulin and bikunin encoding parts, was compared in the human, mouse and a fish (plaice) gene. The size of this intron varies considerably, 6.5, 3.3 and 0.1kb in man, mouse and plaice, respectively. In all three genes, this intron contains A/T-rich regions, and retroposon elements are found in the first two genes. This indicates that this sixth intron is an unstable region and a hotspot for recombinational events, supporting the concept that the alpha1-microglobulin and bikunin parts of this gene are assembled from two ancestral genes. Finally, the nonsynonymous nucleotide substitution rate of the gene was determined by comparing coding sequences from ten vertebrate species. The results indicate that the alpha1-microglobulin part of the gene has evolved faster than the bikunin part.

Processing and secretion of rat alpha 1-microglobulin-bikunin expressed in eukaryotic cell lines.

The precursor protein alpha 1-microglobulin-bikunin was cleaved to the same degree whether expressed in CHO cells or in mutated CHO cells, RPE.40 cells, suggested to lack a functional form of the intracellular protease furin. Thus, alpha 1-microglobulin-bikunin probably is not cleaved in vivo by furin. However, simultaneous overexpression of the precursor and furin in COS, CHO and RPE.40 cells increased the cleavage, suggesting that compartmentalisation and concentrations of protease and precursor are important for the cleavage, besides the in vitro specificity. Expression of alpha 1-microglobulin and bikunin alone gave different protein patterns of SDS-PAGE as compared to expression of the precursor and subsequent cleavage, suggesting that the precursor protein is important for the post-translational handling of alpha 1-microglobulin and bikunin.

Formation of the alpha 1-microglobulin chromophore in mammalian and insect cells: a novel post-translational mechanism?

alpha 1-Microglobulin is an immunosuppressive plasma protein synthesized by the liver. The isolated protein is yellow-brown, but the hypothetical chromophore has not yet been identified. In this work, it is shown that a human liver cell line, HepG2, grown in a completely synthetic and serum-free medium, secretes alpha 1-microglobulin which is also yellow-brown, suggesting a de novo synthesis of the chromophore by the cells. alpha 1-Microglobulin isolated from the culture medium of insect cells transfected with the gene for rat alpha 1-microglobulin is also yellow-brown, suggesting that the gene carries information about the chromophore. Reduction and alkylation or removal of N- or O-linked carbohydrates by glycosidase treatment did not reduce the colour intensity of the protein. An internal dodecapeptide (amino acid positions 70-81 in human alpha 1-microglobulin) was also yellow-brown. The latter results indicate that the chromophore is linked to the polypeptide. In conclusion, the results suggest that the alpha 1-microglobulin gene carries information activating a post-translational protein modification mechanism which is present in mammalian and insect cells.

Synthesis of alpha 1-microglobulin in cultured rat hepatocytes is stimulated by interleukin-6, leukemia inhibitory factor, dexamethasone and retinoic acid.

The secretion of alpha 1-microglobulin by primary cultures of rat hepatocytes was found to increase upon the addition of interleukin-6 or leukemia inhibitory factor, two mediators of acute phase response. This stimulatory effect was further enhanced by dexamethasone. alpha 1-Microglobulin is synthesized as a precursor also containing bikunin, and the precursor protein is cleaved shortly before secretion. Our results therefore suggest that both alpha 1-microglobulin and bikunin are acute phase reactants in rat hepatocytes. Furthermore, we found that retinoic acid, previously shown to be involved in the regulation of cell differentiation and development, also stimulated alpha 1-microglobulin synthesis. Only free, uncomplexed alpha 1-microglobulin (28,000 Da) was detected in the hepatocyte media, suggesting that the complex between alpha 1-microglobulin and alpha 1-inhibitor 3, found in rat serum, is formed outside the hepatocyte.

Cross-reacting monoclonal anti-alpha 1-microglobulin antibodies produced by multi-species immunization and using protein G for the screening assay.

In order to generate monoclonal antibodies (MAb) directed against the low molecular weight glycoprotein alpha 1-microglobulin, a BALB/c mouse was immunized with a mixture of human, guinea pig, rat and rabbit alpha 1-microglobulin homologues (multi-species immunization) and boosted several times. On day 194, the mouse splenocytes were fused to SP2/0 myeloma cells. The resulting hybridomas were screened for anti-alpha 1-microglobulin activity against the alpha 1-microglobulin mixture or against the individual homologues. For this screening, protein G (the newly described IgG-binding streptococcal protein) was used in a solid-phase radioimmunoassay. The binding of protein G to immobilized antigen-antibody complexes was enhanced by pre-incubation with rabbit anti-mouse immunoglobulin G. The result was a panel of nine established hybridoma lines, all producing unique monoclonal antibodies, of IgG1 or IgG2a class, to alpha 1-microglobulin. The antibodies were not only reactive in solid-phase radioimmunoassay, but they could also immunoprecipitate 125I-labeled soluble alpha 1-microglobulin. Moreover, they reacted specifically with the alpha 1-microglobulin band in Western blots of urinary proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Such monoclonal antibodies are potentially valuable reagents for the further characterization of alpha 1-microglobulin.

Detection and purification of rat and goat immunoglobulin G antibodies using protein G-based solid-phase radioimmunoassays.

Using the newly described streptococcal surface protein, protein G, which has powerful immunoglobulin G binding properties, solid-phase radioimmunoassays were developed for the quantitation of goat and rat immunoglobulin G bound to the plastic surface of microtiter plates. The binding of goat immunoglobulin G to the surface via a specific antigen (guinea pig alpha 1-microglobulin) permitted the determination of antigen-specific antibodies with a detection limit of 50-100 ng. Optimum assay conditions were established and the whole assay procedure could be brought to completion at room temperature in less than a working day. The value of the assays was illustrated by monitoring rat and goat immunoglobulin G antibodies during their purification from whole sera by classical chromatographic procedures.

Purification of antibodies using protein L-binding framework structures in the light chain variable domain.

Protein L from the bacterial species Peptostreptococcus magnus binds specifically to the variable domain of Ig light chains, without interfering with the antigen-binding site. In this work a genetically engineered fragment of protein L, including four of the repeated Ig-binding repeat units, was employed for the purification of Ig from various sources. Thus, IgG, IgM, and IgA were purified from human and mouse serum in a single step using protein L-Sepharose affinity chromatography. Moreover, human and mouse monoclonal IgG, IgM, and IgA, and human IgG Fab fragments, as well as a mouse/human chimeric recombinant antibody, could be purified from cultures of hybridoma cells or antibody-producing bacterial cells, with protein L-Sepharose. This was also the case with a humanized mouse antibody, in which mouse hypervariable antigen-binding regions had been introduced into a protein L-binding kappa subtype III human IgG. These experiments demonstrate that it is possible to engineer antibodies and antibody fragments (Fab, Fv) with protein L-binding framework regions, which can then be utilized in a protein L-based purification protocol.

On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins.

Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for protein L (4.5 and 1.4 x 10(9) M-1) than for protein A (7.7 and 6.7 x 10(8) M-1), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.