Human Orosomucoid Research Articles

Distal Promoter Regions Are Responsible for Differential Regulation of Human Orosomucoid-1 and -2 Gene Expression and Acute Phase Responses

Human orosomucoid (ORM) is a major acute-phase plasma protein, encoded by 2 highly homologous genes, ORM1 and ORM2. Human ORM induction is assumed to be regulated by each proximal promoter region, where putative glucocorticoid responsive elements and CCAAT/enhancer binding protein (C/EBP)β binding sites are located. However, the details of the differential regulation of these genes remain unknown. To explore this, we assessed the role of the distal promoter region of each ORM in HeLa cells. Luciferase-reporter activities of full constructs, containing approximately 1.1 kbp (FULL), and those of deletion constructs, containing up to 188 bp region (DEL) upstream of the transcription start sites of ORM1 and ORM2 were compared under both basal and inducer-treated conditions. For ORM1 and ORM2 DEL constructs, significantly increased activities after dexamethasone (DEX) treatments (alone and combined with interleukin (IL)-1β) were observed. Significantly higher FULL construct activities than DEL construct activities were observed for ORM1 after IL-1β treatment, while those for ORM2 were significantly lower at basal level and after DEX treatments. Upon C/EBPβ overexpression, FULL construct activities were significantly higher than those of DEL constructs at basal level and after IL-1β treatment for ORM1, and at basal level and after inducer-treatments for ORM2. Higher transcription-induction activity in the distal promoter region was evident for ORM1 in the absence of C/EBPβ overexpression, and for ORM2 under C/EBPβ overexpression conditions. These findings suggest that the ORM distal promoter region differentially regulates expression of ORM genes at basal level and in acute phase responses.

Targeted Delivery of Macromolecular Drugs: Asialoglycoprotein Receptor (ASGPR) Expression by Selected Hepatoma Cell Lines used in Antiviral Drug Development

The asialoglycoprotein receptor (ASGPR), an endocytotic cell surface receptor expressed by hepatocytes, is triggered by triantennary binding to galactose residues of macromolecules such as asialoorosomucoid (ASOR). The capacity of this receptor to import large molecules across the cellular plasma membrane makes it an enticing target for receptor-mediated drug delivery to hepatocytes and hepatoma cells via ASGPR-mediated endocytosis. This study describes the preparation and characterization of (125)I-ASOR, and its utility in the assessment of ASGPR expression by HepG2, HepAD38 and Huh5-2 human hepatoma cell lines. ASOR was prepared from human orosomucoid, using acid hydrolysis to remove sialic acid residues, then radioiodinated using iodogen. (125)I-ASOR was purified by gel column chromatography and characterized by SDS-PAGE electrophoresis. The ASOR yield by acid hydrolysis was 75%, with approximately 87 % of the sialic acid residues removed. Electrophoresis and gel chromatography demonstrated substantial differences in (125)I-ASOR quality depending on the method of radioiodination. ASGPR densities per cell were estimated at 76,000 (HepG2), 17,000 (HepAD38) and 3,000 (Huh-5-2). (125)I-ASOR binding to ASGPR on HepG2 cells was confirmed through galactose- and EDTA- challenge studies. It is concluded that (125)I-ASOR is a facilely-prepared, stable assay reagent for ASGPR expression if appropriately prepared, and that HepG2 cells, but not HepAD38 or Huh-5-2 cells, are suitable for studies exploiting the endocytotic ASGPR.

A/G Gln20Arg (exon 1) and G/A Val156Met (exon 5) polymorphisms of the human orosomucoid 1 gene in Mexico

The human orosomucoid 1 gene (ORM1) codes an alpha-1-acid glycoprotein that has been classified as an acute-phase reactive protein, and a major drug-binding serum component, as well as an immunomodulatory protein with genetic polymorphisms. Evaluation of ORM variation through isoelectric focusing and immunobloting has revealed a world-wide distribution of the ORM1 F and ORM1 S alleles. We evaluated and examined the genetic characteristics of two Mexican populations that have different anthropological and cultural antecedents, examining two ORM1 genotypes (exon 1 - A/G (Gln20Arg) and exon 5 G/A (Val156Met)) in 145 individuals, using nested polymerase chain reaction, sequencing, and restricted fragment length polymorphism. Mexican Mestizos had higher frequencies of the exon 1 A allele (P = 0.020) and AA genotype (P = 0.018) and lower frequency of the G allele (P = 0.020) when compared to Teenek Amerindians. When we examined exon 5 G/A (Val156Met) polymorphisms, we found significantly higher frequencies of the G allele (P = 0.0007) and the GG genotype (P = 0.0003) in the Mexican Mestizo population. The Teenek population had a significantly higher frequency of the A allele than has been reported for Chinese and African (P < 0.05) populations, and the G/A genotype was more frequently found in this Mexican population than in Chinese, African and European populations (P < 0.05).

Alterations of human serum and urine orosomucoid concentration in inflammation diseases and leukemia

Alterations of human serum and urine orosomucoid concentration in inflammation diseases and leukemia

The Structure and Diversity of α1-Acid Glycoprotein/Orosomucoid Gene in Africans

Human orosomucoid (ORM), or alpha(1)-acid glycoprotein, is known to be controlled by duplicated and triplicated genes on chromosome 9, encoding ORM1 and ORM2 proteins. In this study, the structure and diversity of the ORM gene were investigated in 16 Sub-Saharan Africans, who originated from widely dispersed locations in Africa. The duplicated ORM1-ORM2 gene was observed in all 16 samples. ORM1*S1(2), characterized by an ORM2 gene-specific sequence in intron 5, was common in Africans. Three Africans showed the duplication of the ORM1 gene. The organization of the triplicated ORM1A-ORM1B-ORM2 gene was established in two Africans. The recombination breakpoints resulting in the ORM1 duplication lay within a small genomic interval around exon 1 of the ORM1B gene. The duplication of the ORM2 gene reported previously was not detected in this population sample. Several single-nucleotide polymorphisms were observed in the ORM2 gene. The rearrangement of the ORM gene is likely to occur often in Africans.

Selective binding of coumarin enantiomers to human α 1-acid glycoprotein genetic variants

Coumarin-type anticoagulants, warfarin, phenprocoumon and acenocoumarol, were tested for their stereoselective binding to the human orosomucoid (ORM; AGP) genetic variants ORM 1 and ORM 2. Direct binding studies with racemic ligands were carried out by the ultrafiltration method; the concentrations of free enantiomers were determined by capillary electrophoresis. The binding of pure enantiomers was investigated with quinaldine red fluorescence displacement measurements. Our results demonstrated that all investigated compounds bind stronger to ORM 1 variant than to ORM 2. ORM 1 and human native AGP preferred the binding of ( S)-enantiomers of warfarin and acenocoumarol, while no enantioselectivity was observed in phenprocoumon binding. Acenocoumarol possessed the highest enantioselectivity in AGP binding due to the weak binding of its ( R)-enantiomer. Furthermore, a new homology model of AGP was built and the models of ORM 1 and ORM 2 suggested that difference in binding to AGP genetic variants is caused by steric factors.

Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph

A 291-kDa lectin (LsL) was purified from the hemolymph of the white shrimp Litopenaeus setiferus by affinity chromatography on glutaraldehyde-fixed stroma from rabbit erythrocytes. LsL is a heterotetramer of two 80-kDa and two 52-kDa subunits, with no covalently-liked carbohydrate, and mainly composed by aspartic and glutamic acids, glycine and alanine, with relatively lower methionine and cysteine contents. Edman degradation indicated that the NH2-terminal of the 80-kDa subunit is composed DASNAQKQHDVNFLL, whereas the NH2-terminal of the 52-kDa subunit is blocked. The peptide mass fingerprint of LsL was predicted from tryptic peptides from each subunit by MALDI-TOF, and revealed that each subunit showed 23 and 22%, respectively, homology with the hemocyanin precursor from Litopenaeus vannamei. Circular dichroism analysis revealed beta sheet and alpha helix contents of 52.7 and 6.1%, respectively. LsL agglutinate at higher titers guinea pig, murine, and rabbit erythrocytes its activity is divalent cation-dependent. N-acetylated sugars, such as GlcNAc, GalNAc, and NeuAc, were the most effective inhibitors of the LsL hemagglutinating activity. Sialylated O-glycosylated proteins, such as bovine submaxillary gland mucin, human IgA, and fetuin, showed stronger inhibitory activity than sialylated N-glycosylated proteins, such as human orosomucoid, IgG, transferrin, and lactoferrin. Desialylation of erythrocytes or inhibitory glycoproteins abolished their capacity to bind LsL, confirming the relevance of sialic acid in LsL-ligand interactions.

H2, the Minor Subunit of the Human Asialoglycoprotein Receptor, Trafficks Intracellularly and Forms Homo-oligomers, but Does Not Bind Asialo-orosomucoid

The functional human hepatic asialoglycoprotein receptor (ASGP-R) is a hetero-oligomer composed of two subunits, designated H1 and H2, which are highly homologous. Despite their extensive homology, the major H1 subunit is stably expressed by itself, whereas in the absence of H1 most of the H2 subunits are degraded in the ER. In this study, we were able to investigate the capability of the minor ASGP-R subunit, H2, to function independently of H1, because it was apparently stabilized by fusing its NH(2) terminus with an epitope tag. We could thus create stable cell lines in hepatoma-derived SK-Hep-1 cells that expressed the H2 subunit alone. H2 was expressed on the cell surface and was internalized, predominantly through the clathrin-coated pit pathway. Since the internal pool of H2 was also able to traffick to the cell surface, we conclude that H2 recycles between the surface and intracellular compartments, similar to the constitutive recycling of hetero-oligomeric ASGP-R complexes. However, the rate of H2 recycling and internalization was approximately 25-33% that of H1. Similar to H1, the H2 polypeptides were also able to self-associate to form homo-oligomers, including trimers and tetramers. However, unlike H1, which can bind the ligand asialo-orosomucoid (ASOR) when overexpressed in COS-7 cells, H2 failed to bind or endocytose ASOR. In summary, the H2 subunit of the human ASGP-R contains functional, although weak, signal(s) for endocytosis and recycling and has the ability to oligomerize. H2 homo-oligomers, however, do not create binding sites for desialylated glycoproteins, such as ASOR, that contain tri- and tetra-antennary N-linked oligosaccharides. Nonetheless, these results raise the intriguing possibility that naturally occurring H2 homo-oligomers may exist in human hepatocytes and have an as yet undiscovered function.

Reflections on glycobiology.

Reflections on glycobiology.

Molecular analysis of the human orosomucoid gene ORM1*Q0köln responsible for incompatibility in a German paternity case.

In a German paternity test, an alleged father was excluded only by reverse homozygosity of ORM1 phenotypes (mother ORM1 S, child ORM1 S and alleged father ORM1 F1) out of the 28 classical and DNA markers investigated. Without the ORM1 system the biostatistical probability of paternity was calculated to exceed 99.999%. The intensity of the immunoprinted bands of the ORM1 protein for the child and alleged father after isoelectric focusing appeared to be reduced to about half. To identify a possible null allele, gene-specific amplification followed by single-strand conformation polymorphism and sequencing analyses were carried out. Deletion of one of the two copies of a 4 bp direct repeat sequence (GTCT) in exon 4 of the consensus sequence of ORM1*F1 was observed in the child and alleged father. Thus, the sharing of a rare mutant gene, ORM1*Q0köln, increased the probability of paternity.

Cleavage of Human Orosomucoid by a Chromium(V) Species: Relevance in Biotoxicity of Chromium

A chromium(V) complex, CrO(salen)+, was generated in situ and its interaction with human orosomucoid (α1-acid glycoprotein) has been evaluated. The chromium(V) species has been found to oxidize the protein rapidly. A second order rate constant of 5 ± 0.4 × 104 M−1 s−1 has been obtained for the redox process. Gel electrophoresis pattern of AGP in the presence of metal ion clearly reveals the decrease in the intensity of the AGP band with the subsequent formation of protein fragments of lower molecular weight. At higher metal ion concentration a continuous smear is observed which indicates the nonselective cleavage of the glycoprotein. Cleavage of AGP is through the direct pathway of oxidation by a highly reactive chromium(V) species.

Localization of disulfide bonds in α1-acid glycoprotein and their effect on the ability of the protein to interact with ethidium bromide

α1-Acid glycoprotein (orosomucoid) was purified from the human and murine blood sera using phenol deproteinization. As opposed to the murine protein, the human orosomucoid bound the fluorescent dye ethidium bromide but lost this ability after treatment with β-mercaptoethanol, which breaks disulfide bonds. Disulfide bonds between the Cys23 and Cys165 residues of the human orosomucoid and between the Cys91 and Cys184 residues of the murine orosomucoid were identified.

Genetic polymorphisms of orosomucoid on the Han population in Nanjing of China

Human orosomucoid (ORM) is a major binding protein for various basic drugs. The genetic polymorphisms of ORM could be responsible for interindividual variation in the plasma binding of basic drugs, which might influence their effects or toxicities. The genetic polymorphisms of ORM on the Han population in Nanjing of China were analyzed by isoelectric focusing (IEF) on polyacrylamide gels following by immunoblotting. After desialylation of sera from 220 unrelated Chinese subjects, the band patterns of ORM showed that the polymorphism of the structural locus ORM1 is controlled by three codominant autosomal alleles, ORM1*F1, ORM1*F2 and ORM1*S, which presented five phenotypes, ORM1 F1, ORM1 S, ORM1 F1F2, ORM1 F1S, and ORM1 F2S. The allele frequencies were: ORM1*F1=0.7068, ORM1*F2=0.0182, ORM1*S=0.2750. The results presented in this paper indicate the ORM1 locus is polymorphic and the ORM2 locus is monomorphic in sera from the Han population in Nanjing of China.

Human Orosomucoid (ORM1) Subtyping: Further Population Genetic Data and Reports on the Feasibility to Type Aged Blood Samples and Stains

Genetic polymorphism of serum orosomucoid (ORM1) was investigated in 1072 unrelated German Caucasians using isoelectric focusing followed by Western blotting and EIA. The estimated allele frequencies were ORM1 *F1 = 0.5690, ORM1 *S = 0.3927, ORM1 *F2 = 0.0368, ORM1 *F2S = 0.0009 and ORM1 *F5 = 0.0005. The method was successfully applied to determine ORM1 phenotypes in aged blood samples and blood stains. The results indicated that the ORM protein is a informative and remarkably robust blood group system.

Separation of human orosomucoid major gene products using immobilized copper affinity chromatography and identification of the metal-interactive residues

The major gene products of human orosomucoid, GP1 and GP2, were purified using immobilized copper affinity chromatography [Cu(II)-IMAC], with 3 mM imidazole as eluent. Both gene products bound to the Cu(II)-IMAC column in the presence of 1 M NaCl, but at different pHs. GP1 was not retained after treatment with diethylpyrocarbonate (DEPC). This modification was characterized using difference absorbance spectrophotometry and mass spectrometry. The latter provided unambiguous assignment of some of the modified residues. No correlation was observed between the modification of histidine/tyrosine and protein retention. Furthermore, removal of the carbethoxy groups of modified histidine and tyrosine by hydroxylamine treatment did not improve the retention. Therefore neither histidine nor tyrosine could be the critical residues in metal recognition. Results from mass spectrometric analysis of retained and unretained fractions of DEPC modified GP 1 indicated that the lysine residues 130/135 and 152 were modified significantly in both fractions, but to a relatively less extent in the retained one. We suggest that the retnetion of GP1 involves several residues including lysines, and that a critical number of these is necessary for retention.

Preparative Purification of Tetraantennary Oligosaccharides from Human Asialyl Orosomucoid

An approach to isolate micromole quantities of tetraantennary oligosaccharides from human orosomucoid is presented. The N-linked oligosaccharides from 500 mg of the glycoprotein were released enzymatically, desialylated, and isolated free of protein using ion exchange chromatography. The pooled oligosaccharides were converted into oligosaccharide glycosylamines by reaction with ammonium bicarbonate then coupled to BOC-tyrosine to prepare tyrosinamide oligosaccharides. These were resolved on semipreparative RP–HPLC to recover micromole quantities of six purified tyrosinamide oligosaccharides. The oligosaccharide structures were elucidated by a combination of high-field proton NMR and matrix-assisted time of flight mass spectrometry and included biantennary, triantennary, monofucosylated triantennary, tetraantennary, monofucosylated tetraantennary, and a tetraantennary containing a single polylactosamine extension. Edman degradation was utilized to reverse the tyrosinamide oligosaccharide derivatization leading to the generation of reducing oligosaccharides. These were used to characterize the elution profile of asialyl orosomucoid oligosaccharides on high pH anion exchange chromatography. This application of tyrosinamide derivatization has allowed for the first time the complete resolution of the complex oligosaccharide mixture from orosomucoid on a semipreparative scale in a single chromatogram and provide the first NMR characterization of polylactosamine tetraantennary oligosaccharide from this substrate. This study demonstrates the broad utility of the tyrosinamide derivatization to develop oligosaccharide libraries useful for probing the biological functions of glycosylation.

Human orosomucoid polymorphism: molecular basis of the three common ORM1 alleles, ORM1*F1, ORM1*F2, and ORM1*S.

The human orosomucoid (ORM) is controlled by two closely linked loci, ORM1 and ORM2, and two tandem genes, AGP1 and AGP2, encoding the proteins produced by the two loci, have been cloned. In this study the molecular basis of ORM1 polymorphism was investigated. For the detection of mutations the products of the six exons of each gene, amplified by the polymerase chain reaction (PCR), were screened by single-strand conformation polymorphism analysis. Subsequently, the exons with an altered migration pattern were gene-specifically amplified by nested PCR. Sequencing of the gene-specific PCR products showed that the three common ORM1 alleles result from A-->G transitions at the codons for amino acid positions 20 in exon 1 and 156 in exon 5 of the AGP1 gene: ORM1*F1 was characterized by CAG (Gln) and GTG (Val), ORM1*F2, by CAG (Gln) and ATG (Met), and ORM1*S, by CGG (Arg) and GTG (Val). The phylogenesis of the genes encoding these three ORM1 alleles is discussed.

Human Orosomucoid: Dissociation of Tyrosines in the Systems Water-Low Aliphatic Alcohol

The influence of methanol, ethanol, propan-1-ol, and propan-2-ol on the dissociation of tyrosines of human serum orosomucoid (acid α1-glycoprotein) was studied. The content of alcohols was chosen so that their solutions had the same value of the relative permittivity. Then, the contribution of electrostatic effects was equal in all cases and the observed differences between aqueous and alcoholic solutions reflected other, nonelectrostatic effects in the studied systems. Analysis of the spectrophotometric titration curve revealed three kinds of tyrosine groups (n1, n2, n3), but only the values pK1 and pK2 could be calculated. The observed differences of pK values in aqueous and mixed media are discussed and compared with the structural changes of the orosomucoid molecule.

Asialoglycoprotein/asialoglycoprotein receptor (AGP-AGPr) interaction is an important mechanism for the uptake of FK506 by hepatocytes.

Hepatic tissue concentrations of FK506 have been correlated with early acute rejection following liver transplantation. Asialoglycoproteins (AGP) reputedly bind FK506 in blood. AGP are removed from the circulation by the liver via the AGP receptor (AGPr), which resides on hepatocytes. This study was undertaken to determine if the AGP-AGPr mechanism enhances the delivery of FK506 to hepatocytes. Human orosomucoid (OM) was used as a representative AGP. asialoOM (aOM) was prepared by desialation of OM. Fresh rat hepatocytes were isolated by collagenase digestion. Tritium labeled FK506 (FK) was used to identify and quantitate FK506. Quantitation of FK in serum and culture media was by direct counting. FK in animal tissues used a method developed in our laboratory for the purpose. AGPr on resting hepatocytes was demonstrated by flow cytometry using FITC-orosomucoid and FITC-BSA controls. AGPr were enhanced by 2 g glucose/L. Two serum FK-binding fractions, 44 kD and 15 kD, were identified by gel filtration. Exogenous OM avidly bound FK and displaced FK activity from the 15 kD fraction. Serum (1%) and the 44 kD fraction enhanced the uptake of FK by hepatocytes, while serum depleted of OM-aOM by affinity chromatography was only 72.5% as effective as control serum; aOM enhanced the uptake of FK by hepatocytes to a degree similar to that of control serum but OM did not significantly affect the uptake of FK. Cold FK506 blocked the uptake and was dose dependent; cold CsA had no effect. Affinity extraction of OM from serum to which FK had previously been added removed 28.4% of FK activity. Following i.v. infusion, the kidney had the highest and liver the lowest tissue concentration of FK at 1 hr and 3 hr. In contrast, after oral administration the liver had the highest concentrations of the other tissues tested. The AGP-AGPr mechanism plays a significant role in the delivery of FK506 to hepatocytes and is likely responsible for the differences in bioavailability observed after oral and i.v. administration. Factors governing the AGP-AGPr mechanism are germane to understanding both the efficacy and toxicity of FK506 and the development optimal therapeutic strategies.

Effect of mannosylated derivatives on HIV-1 infection of macrophages and lymphocytes.

We previously demonstrated that gp120/160 (Env) of HIV-1 interact in a carbohydrate-specific manner with mannosyl/N-acetylglucosaminyl derivatives and that HIV-1LAI infection of monocytic U937 and lymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactive glycopeptides from U937 cells. We report here that the natural glycoproteins bovine fetuin and asialofetuin, human orosomucoid and alpha-fetoprotein, and mannan, which all specifically interact with Env, inhibited infection of primary macrophages by macrophage-tropic HIV-1 strains, whereas dextran had no such effect. This activity was conserved if fetuin, asialofetuin, or orosomucoid were heat-treated, which rules out the role of their three-dimensional structure. Orosomucoid and mannan partially inhibited Env binding to macrophages but not to U937 or CEM cells. This indicates that Env does not bind in the same manner to primary macrophages and to immortalized CD4+ cells, and that orosomucoid and mannan act at CD4-independent stages of virus binding to macrophages. Mannan also inhibited Env binding to surface glycopeptides obtained after trypsin treatment of macrophages. Furthermore, orosomucoid and fetuin interacted with, and they inhibited the binding of a V3 loop B clade consensus peptide to several macrophage membrane proteins, including two 36 and 42 kDa proteins. These data indicate that these glycoproteins interfere with post-binding events during HIV-1 infection of primary macrophages. In contrast, the compounds did not affect infection of U937 or CEM cells by T-cell tropic HIV-1LAI nor infection of peripheral blood lymphocytes by HIV-1LAI or HIV-1(Ba-L). Thus, carbohydrate-specific inhibition of HIV infection depends on the cell type more than on the viral strain, and differences in the glycan structure of cell-type-specific cofactors may be important for HIV entry into cells.