Human Orosomucoid Research Articles

Genetic polymorphism of cat (Felis catus) plasma orosomucoid.

Genetic polymorphism of orosomucoid (ORM) was observed in 22 breeds of cats (Felis catus) using isoelectric focusing (pH 4.0-6.5) of desialylated plasmas followed by immunoblotting with rabbit antiserum to human ORM. From a total of 943 plasma samples examined, 15 phenotypes were identified and family studies demonstrated an inheritance of five codominant alleles, ORMA, ORMB, ORMC, ORMD, and ORME, at a single locus.

Human genetic variation in the Sierra de Gredos mountain (central Spain): study of several polymorphisms.

The variation in the third component of human complement (C3) and orosomucoid (ORM1) serum proteins, and 6-phosphogluconate dehydrogenase (6PGD), adenosine deaminase (ADA), esterase D (ESD), and acid phosphatase (ACP1) red cell enzymes was examined in two samples from autochthonous populations living on either side of the Sierra de Gredos range in Central Spain. These results support only a moderate differentiation between Gredos subpopulations, which is discussed in relation to other Iberian Peninsula groups. Allele frequencies in Gredos show a remarkable heterogeneity as contrasted with other Iberian populations for most of the markers examined; relatively high C3*S, ORM1*F, ADA*1, ESD*2, and ACP1*C gene frequencies are characteristics of Gredos samples. This differentiation was more marked for the Northern Gredos population and could be related to the geographical peculiarities of this region.

Picosecond Tryptophan Fluorescence of Human Blood Serum Orosomucoid

The state of three tryptophyl residues in human serum orosomucoid was estimated by prediction methods based on parameters characterizing their hydrophobicity either directly, or in terms of buried surfaces of the individual amino acid residues. It is shown that tryptophan 25 is the most buried, while Trp 160 is the most exposed to the solvent. Trp 122 is in this respect in an intermediate state. The fluorescence decay behaviour was determined using a picosecond single photon counting system. The multiwavelength data were analyzed using a global analysis as well as a distribution of lifetimes program. Both procedures yielded the existence of four wavelength independent lifetimes (0.22 ns, 1.0 ns, 2.5 ns, and 8.4 ns). A tentative assignment of the decay associated spectra of the four components to the three individual tryptophans is presented.

Preparation and characterization of biodegradable poly(isobutylcyano acrylate) nanoparticles with the surface modified by the adsorption of proteins

The purpose of this work was to develop and characterize a biodegradable colloidal drug carrier which avoids uptake by the mononuclear phagocyte system. In order to imitate the cell surface, a sialic-acid-rich glycoprotein (human orosomucoid) was adsorbed onto poly(isobutylcyano acrylate) nanoparticles. The adsorption of human serum albumin and asialo-orosomucoid was also tested as a control. The adsorption was found to be dependent on the pH value and reached its maximum at a pH value close to the isoelectric point ( pI) of each protein. The increase in the ionic strength due to the addition of NaCl generally resulted in an increase in the amount adsorbed. Considering the amounts of protein adsorbed (maximum of 4.5 mg m −2), the adsorption was assumed to be of the monolayer type. The adsorption kinetics performed at the pI of each protein showed that the equilibrium was reached within 1 h for albumin and within 8 h for the two glycoproteins. This significant difference suggested that conformational rearrangements could be much slower for the two glycoproteins than for the non-glycosylated albumin. The protein layer was found to be stable at pH 7 when the adsorption was performed beforehand at the pI, i.e. at an acidic pH. Finally, using hydrophobic interaction chromatography, the surface of the coated nanoparticles was found to be much more hydrophilic than the surface of the unmodified nanoparticles.

Heat denaturation of human orosomucoid in water/methanol mixtures

Heat denaturation of orosomucoid in solutions of methanol concentrations ranging from 0 to 70% (v/v) has been studied by using circular dichroism, intrinsic protein fluorescence and thermal difference absorption spectroscopy. Regardless of its high saccharide content (40%), the highly cooperative denaturation transition of orosomucoid is fully reversible in neutral water solution. A two-state model has been successfully applied; the numerical analysis results in thermodynamical parameter values that are in close agreement with previously reported experimental data from calorimetric measurements. However, in solutions containing even minute concentrations of methanol (5%) the heat denaturation is irreversible. After cooling of the denatured protein the refolded molecules exhibit a higher α-helical content than the native one. Possibilities of methanol interaction with native and denatured protein molecule are discussed.

The lipocalin model for human orosomucoid: Support from the chromatography of orosomucoid-monoclonal antibody complexes

The accessibility of the surface of human orosomucoid to monoclonal antibodies (MAbs) was investigated by size-exclusion HPLC to examine the validity of its proposed membership in the lipocalin family. The N-terminus half of the molecule contains all the five glycan chains, and of fifty-six MAbs, only one bound to this piece of the polypeptide backbone, indicating that extensive screening was taking place. The simultaneous binding properties of selected MAbs to the fully glycosylated molecule was examined by size exclusion HPLC. The pattern of inhibition was contiguous, with all regions being overlapped. The epitopes of four MAbs having mutual noncompetitive binding were found to be appropriately spaced when placed on a preliminary three dimensional model of orosomucoid. Together, the results support a structure for orosomucoid composed of an exposed continuous protein surface, and a glycan surface screening the remaining protein. Such a structure is provided by the antiparallel β-barrel motif of the lipocalins.

Thermal Stability of Human Blood Serum Orosomucoid (Acid α1-Glycoprotein) in Water and Water-Ethanol Systems

Thermal stability of human serum orosomucoid (acid α1-glycoprotein) was studied in water and in water - ethanol systems by the differential scanning microcalorimetry and temperature perturbation difference spectrometry. This investigation has led to the following results: (i) the change in heat capacity Cp of isoionic orosomucoid with temperature was irreversible; different values of ∆H were obtained on repeated heating; (ii) in buffered solution at pH 7, hysteresis in temperature perturbation difference spectra was observed within the wavelength range of 250 - 266 nm; (iii) a strong destabilizing effect of ethanol on the orosomucoid molecule was observed in the alkaline region at an alcohol volume fraction of 20%. This effect corresponds to the anomalous behaviour of the water ethanol systems.

Capillary electrophoresis monitoring of the competitive adsorption of albumin onto the orosomucoid-coated polyisobutylcyanoacrylate nanoparticles.

The simultaneous and rapid quantitation of a glycoprotein, human orosomucoid, and a protein, human serum albumin (HSA), by micellar electrokinetic capillary electrophoresis was developed and optimized (pH and sodium dodecyl sulfate concentration) in order to study the competitive adsorption of HSA onto orosomucoid-coated nanoparticles of polyisobutylcyanoacrylate. This method provided satisfactory repeatability and the response was linear for concentrations of orosomucoid and HSA ranging from 0.025 to 0.500 mg/mL (r > 0.994 for both proteins). To study the competitive adsorption of HSA, the amounts of free orosomucoid and free HSA were determined in the supernatant obtained after centrifugation of the orosomucoid-coated nanoparticle suspension incubated with HSA. Furthermore, the quantities of both orosomucoid and HSA absorbed onto nanoparticles were also determined after desorption of these molecules from the nanoparticle surface, using sodium dodecyl sulfate. No analytical interference was observed with this surfactant. The results showed that HSA did not at all absorb onto the orosomucoid-coated nanoparticles and, thus, that this protein did not displace the layer of absorbed orosomucoid from the nanoparticle surface.

Restricted variation in the glycosylation of human α1-acid glycoprotein

The glycosylation patterns of multiglycosylated proteins reflect both the cellular control of glycosyl transferase activity and the local control of the transfer event. Little information is available concerning these mechanisms. Changes in glycosylation occur in the disease state and provide a convenient way of examining the control mechanism(s) operating at individual glycosylation sites. Chromatographic methods have been applied to the human orosomucoid (OMD) isolated from seventeen different pathologies. Based on the distribution of the glycans at the individual sites it is clear that the major protein-glycan interactions that restrict glycosylation in “normal” OMD remain intact in the disease state in the presence of changing glycosyl transferase activity.

Subtyping of alkylated human orosomucoid: evidence for a duplicated gene, ORM1*F2S.

Isoelectric focusing of human orosomucoid (ORM) was studied following different sample treatment. It is shown that: (i) alkylation with iodoacetamide leads to a drastic change in the isoelectric point (pI) of both ORM1 F2 and ORM2 A gene products and greatly improves the discrimination between ORM1 F1 and ORM1 F2; (ii) previous reduction of the molecule with dithiothreitol partially inhibits the pI transitions with resultant artifactual ORM1 F1F2S patterns that correspond in most cases to F2S phenotypes. With the technique now described, the persistence of three ORM1 gene products was found in only one individual and the segregation analysis is consistent with the existence of a rare ORM1*F2S haplotype.

Behaviour of Human Blood Serum Orosomucoid (Acid α1-Glycoprotein) in the Presence of Small Aliphatic Alcohols

The influence of both methanol and ethanol on the stability of orosomucoid molecule was studied over a broad range of alcohol concentrations (volume fraction 0 - 70%) and pH between 7 and 12. The data obtained by optical methods were compared with information from prediction studies which were focused on the structural features and physicochemical parameters of amino acid in the orosomucoid molecule. From this analysis the following conclusions can be drawn: (i) ethanol exerts a more pronounced effect on α-helix formation than methanol does, (ii) at higher pH the electrostatic contribution is the crucial effect in the destabilization of the orosomucoid molecule, (iii) out of 12 phenylalanines in the orosomucoid molecule only 1 - 2 are exposed to the solvent; of the residues which are not subjected to substitution appears to be Phe 141, (iv) its neighbor, Tyr 142, appears to be a key residue, the dissociation of which destabilizes one of two longer helical segments of the orosomucoid molecule, (v) the limits of applicability of the four-derivatives of UV spectra with respect to phenylalanines were examined; analysis of the data is recommended on a more precise mathematical basis, taking into consideration the bandwidth of the spectral peaks.

Effect of Methanol on the Structure of Human Blood Serum Orosomucoid (Acid α1-Glycoprotein)

The effect of methanol (volume fraction 0-70%) on the optical properties of human blood serum orosomucoid (acid α1-glycoprotein) was studied over the pH range 5-12. The changes of these properties were correlated with the concept of three-dimensional arrangement of the orosomucoid molecule as obtained by a prediction study based on refined conformational preferences. From a complex analysis of the data obtained following conclusions on the orosomucoid structure in solution could be made: 1) α-helix content increases at constant pH with the methanol volume fraction exceeding 30%. 2) α-helix content at constant methanol concentration is maximal at pH 9-10. 3) The changes in conformation brought about by the presence of methanol result in the masking of 1 tyrosine and 1-tryptophan residue in 70% alcohol. It has been observed that the complex action of even the lowest alcohol leads to marked changes at the various levels of the three-dimensional organization of the orosomucoid molecule.

Orosomucoid typing of apes (family Pongidae) by isoelectric focusing: Among primates do only humans have two functional orosomucoid loci?

It has been demonstrated that human orosomucoid (ORM) is controlled by more than one functional loci, while Macaca ORM is controlled by one locus. To examine the time when the ORM gene was duplicated in the evolution of primates, plasma samples from 118 apes (family Pongidae) belonging to 4 genera and 12 species were investigated for ORM polymorphism using isoelectric focusing followed by immunoprinting. The band patterns of ORM in the subfamily Ponginae showed quantitatively different products as in humans. A pedigree study of common chimpanzees supported the two-locus model for ORM. Gibbons (subfamily Hylobatinae) displayed highly variable band patterns, but the number of loci was not determined unequivocally. Thus, this study shows that duplication of the ORM gene in primates occurred either before or after the divergence of Hylobatinae and Ponginae, consistent with a previous prediction from the molecular evolutionary rate of ORM.

Orosomucoid (ORM) Typing by Isoelectric Focusing: Evidence for Several New Variants Including ORM1 and ORM2 Silent Alleles

Human orosomucoid (ORM) is composed of two tightly linked loci, ORM1 and ORM2, located on chromosome 9q. Some haplotypes contain duplicated ORM1 or ORM2. The present study using isoelectric focusing has revealed seven ORM haplotypes including new variants and/or silent alleles detected in eight Japanese families. ORM1*5.2-ORM2*15 in combination with common ORM1*1-ORM2*1 produced a five-band pattern. ORM1*5-ORM2*1 was observed in three families. ORM2*9 was coexistent with ORM1*1 or ORM1*2.1. ORM1*Q0 was linked to ORM2*1 or ORM2*14. ORM2*Q0 was suggested to be in association with ORM1*2. The ORM haplotypes with a silent gene must have arisen from unequal crossing-over between the tandemly duplicated genes of ORM.

Orosomucoid in human pregnancy serum diminishes bioavailability of the progesterone antagonist RU 486 in rats

Bioavailability of the progesterone antagonist RU 486 was examined in vivo by measurement of first-pass uptake by hepatic and uterine tissues in rats. Presence of human pregnancy serum or alpha 1-acid glycoprotein (orosomucoid) in injection vehicles diminished uptake of this steroid, which suggests limited bioavailability. These data indicate that elevated human serum orosomucoid levels may account for some failures of RU 486 in early pregnancy termination.

Orosomucoid (ORM) Typing by Isoelectric Focusing: Evidence for Several New Variants Including ORM1 and ORM2 Silent Alleles

Human orosomucoid (ORM) is composed of two tightly linked loci, ORM1 and ORM2, located on chromosome 9q. Some haplotypes contain duplicated ORM1 or ORM2. The present study using isoelectric focusing has revealed seven ORM haplotypes including new variants and/or silent alleles detected in eight Japanese families. ORM1*5·2-ORM2*15 in combination with common ORM1*1-ORM2*1 produced a five-band pattern. ORM1*5-ORM2*1 was observed in three families. ORM2*9 was coexistent with ORM1*1 or ORM1*2·1. ORM1*Q0 was linked to ORM2*1 or ORM2*14. ORM2*Q0 was suggested to be in association with ORM1*2. The ORM haplotypes with a silent gene must have arisen from unequal crossing-over between the tandemly duplicated genes of ORM.

Sequestration electrochemistry: The interaction of chlorpromazine and human orosomucoid

A simple and rapid method is presented for determination of the association constants and stoichiometries describing ligand macromolecule interactions. Based on flow injection analysis and electrochemical detection by amperometry, the only requirements for direct measurements are that the ligand have redox properties and that these properties change upon binding to the macromolecule. Bound ligand may then be measured in the presence of free ligand. Detection limits are of the order of 2 pmol of ligand or less, a level that should provide access to previously unmeasurable systems. For the exemplary system, chlorpromazine and human orosomucoid, K ass 0 was determined as 0.39 × 10 6 m −1 with 0.76 chlorpromazine binding sites of this affinity per orosomucoid molecule.

Orosomucoid as one of the serum components contributing to normal capillary permselectivity in rat skeletal muscle.

The effects of human serum orosomucoid (normal serum concentration 0.7-1.0 g l-1) on capillary permeability were investigated in 12 isolated maximally vasodilated rat hindquarters perfused with bovine serum albumin (50 g l-1) in modified Tyrode. Measurements were made of capillary filtration coefficient (CFC), permeability surface area product (PS) for vitamin B12 and isogravimetric clearance of radiolabelled albumin (Cl alb). The results were compared with those obtained using perfusates without addition of orosomucoid ('albumin group') or perfusates containing horse serum ('serum group'). Clearance of albumin was almost four-fold higher in the albumin than in the serum group, 0.0895 +/- 0.0066 (n = 12) and 0.0252 +/- 0.016 ml min-1 per 100 g (n = 18), respectively, while intermediate Cl alb values were obtained with human orosomucoid in the perfusate (greater than 0.1 g l-1), 0.0436 +/- 0.0034 ml min-1 per 100 g) (n = 8). These changes in Cl alb were not accompanied by any differences in CFC or PS. We conclude that orosomucoid is one of the components in serum (besides albumin) needed for the maintenance of normal permselectivity of the capillary walls of rat skeletal muscle. Alternatively, human orosomucoid is structurally related to other substances exerting this 'serum effect'.

The influence of N-acetylneuraminic acid on the properties of human orosomucoid

Little is known of the relationships that may exist among the three principal functionalities of glycoproteins. Orosomucoids of closely defined N-acetylneuraminic acid content were examined for evidence of influence of N-acetylneuraminic acid content on the physical properties of the glycoprotein. Fluorescence spectroscopy gave no indication of conformational change in the protein core upon desialylation. Small changes in the chromatographic partition coefficient, sigma, and thermal stability, Td, are interpreted to reflect loss of water of hydration and increased glycan stem-protein interaction without a major repositioning of the chains. Ligand-binding measurements indicate no alteration in the hydrophobic binding domain and a possible interaction between chlorpromazine and N-acetylneuraminic acid. All changes seen are progressive and occur through a region where changes in biological activity are not found. It is suggested that the dependence of biological activity on N-acetylneuraminic acid content in orosomucoid reflects, not coupled changes in protein conformation, but a charge-density-related interaction such that, below a contribution of four or five N-acetylneuraminic acid residues, activity is modified.

Efficient hplc separation of N‐p‐nitrophenylglycosylamines derived from complex oligosaccharide mixtures. Human orosomucoid as a model

With human orosomucoid as model compound, a new method was developed to separate neutral oligosaccharides as N-p-nitro-phenylglycosylamines. Asialo orosomucoid was prepared by treatment with neuraminidase and purified by size exclusion HPLC on TSK 2000 SW. Oligosaccharides were isolated by reversed phase HPLC on Hamilton PRP-1 after hydrazinolysis and re-N-acetylation. Glycosylamination was performed with p-nitroaniline in DMSO-formic acid-water, where the whole mixture of oligosaccharide derivatives was isolated by reversed phase HPLC on Hamilton PRP-1 and separated into single glycosylamines on Shandon Hypersil ODS. The purified glycosylamines could be methylated by a new rapid method with sodium hydroxide and methyl iodide in DMSO, isolation and purification were carried out on Hamilton PRP-1 and Spherisorb ODS 2, respectively, as described for the glycosylamines. Preparative scale HPLC separations were performed on analytical columns using repetitive collection mode and automatic sample loading by means of a peristaltic pump operated by the HPLC controller. The purified glycosylamines can be used for sugar analyses or, after permethylation, for methylation analyses or related procedures.