The structure and diversity of alpha1-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.     

Electrophoretic and immunoelectrophoretic study of the enzymatic degradation of human orosomucoid (alpha 1 -acid glycoprotein)

Purified, intact orosomucoid (alpha 1-acid glycoprotein) derived from whole human plasma was incubated with a number of proteolytic and saccharolytic enzymes under a variety of conditions. The unfractionated digests were immediately examined by both agar gel- and immunoelectrophoresis for the presence of antigenically active (precipitating) and/or inactive macrofragments. Despite otherwise clear evidence of rapid degradation by several proteases, no antigenic subunits were detected. Among the glycosidases, almond emulsin produced a suggestion of modification of the carbohydrate moiety of a sialic acid-poor orosomucoid preparation obtained from Cohn Fr. VI, but had no effect on antigenic properties. These results are presented as further evidence for a lack of involvement of the extensive carbohydrate component of orosomucoid in its antigenic reactions, and support previous data implicating the polypeptide chain as solely responsible for its antigenicity.     

Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study

Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.     

Relation between the secondary structure of carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and the fluorescence of the protein

We studied in this work the relation that exists between the secondary structure of the glycans of alpha(1)-acid glycoprotein and the fluorescence of the Trp residues of the protein. We calculated for that the efficiency of quenching and the radiative and non-radiative constants. Our results indicate that the glycans display a spatial structure that is modified upon asialylation. The asialylated conformation is closer to the protein matrix than the sialylated form, inducing by that a decrease in the fluorescence parameters of the Trp residues. In fact, the mean quantum yield of Trp residues in sialylated and asialylated alpha(1)-acid glycoprotein are 0.0645 and 0.0385, respectively. Analysis of the fluorescence emission of alpha(1)-acid glycoprotein as the result of two contributions (surface and hydrophobic domains) indicates that quantum yields of both classes of Trp residues are lower when the protein is in the asialylated form. Also, the mean fluorescence lifetime of Trp residues decreases from 2.285 ns in the sialylated protein to 1.948 ns in the asialylated one. The radiative rate constant k(r) of the Trp residues in the sialylated alpha(1)-acid glycoprotein is higher than that in the asialylated protein. Thus, the carbohydrate residues are closer to the Trp residues in the absence of sialic acid. The modification of the spatial conformation of the glycans upon asialylation is confirmed by the decrease of the fluorescence lifetimes of Calcofluor, a fluorophore that binds to the carbohydrate residues. Finally, thermal intensity quenching of Calcofluor bound to alpha(1)-acid glycoprotein shows that the carbohydrate residues have slower residual motions in the absence of sialic acid residues.     

Thermal stability and ligand-binding properties of human plasma alpha 1-acid glycoprotein (orosomucoid) as determined with fluorescent probes

The fluorescence of 1,8-anilinonaphthalene sulfonate is enhanced and blue-shifted upon binding to alpha 1-acid glycoprotein, a human plasma protein of uncertain function. Fluorescence titrations of delipidated protein indicate at least two classes of binding sites having dissociation constants of 0.33 microM and 12 microM at 25 degrees C in 0.02 M potassium phosphate/0.15 M NaCl, pH 7.4. Exclusion chromatography measurements indicate only 1 binding site per mol protein, suggesting that the heterogeneity is due to differences between protein molecules, the origin of which remains unclear. The fluorescence of a mixture of dye and protein is progressively diminished upon addition of ethanol and other organic solvents whose presence could be detected at concentrations as low as 100 mM. Addition of the adrenergic drug propranolol to a mixture of alpha 1-acid glycoprotein (2.5 microM) and 1,8-anilinonaphthalene sulfonate (4 microM) caused a hyperbolic decrease in dye fluorescence to 30% of the initial value, with half-maximal response near 1 microM propranolol. When the protein-dye mixture was heated, the fluorescence of the dye exhibited a reversible downward transition with midpoint near 65 degrees C, compared to a midpoint of 58.5 degrees C obtained by intrinsic fluorescence in the absence of dye. This stabilization was confirmed with fluorescein-labeled protein, whose fluorescence polarization revealed a melting transition at 58.8 degrees C in the absence of ligands which increased by 5-6 Cdeg in the presence of 1,8-anilinonaphthalene sulfonate or propranolol. The sensitivity of 1,8-anilinonaphthalene sulfonate fluorescence to changes in the conformation and ligand environment of alpha 1-acid glycoprotein should facilitate efforts to understand the structure and function of this acute-phase reactant.     

Analysis of the trypsin hydrolysate of a 40-kDa protein, found in the nucleoprotein of serum. Identification of it as alpha(1)-acidic glycoprotein (orosomucoid)

From the tryptic hydrolysis of 40-kDa protein from the mouse blood serum thirty-four peptides were isolated by HPLC, of which complete and partial amino acid sequence was established for twenty-eight and six, respectively. On the basis of these data the protein is identified as the blood serum alpha 1-acid glycoprotein.     

Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.     

Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues

We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.     

Use of radioactive glucosamine in the perfused rat liver to prepare alpha 1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues.

1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.     

Protein domain of chicken alpha(1)-acid glycoprotein is responsible for chiral recognition

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31-32% identities to rabbit and human alpha(1)-acid glycoproteins (alpha(1)-AGPs). Thus, OGCHI should be the chicken alpha(1)-AGP. Second, the recombinant chicken alpha(1)-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition.     

Dynamics of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White

Dynamics studies on Calcofluor White bound to the carbohydrate residues of sialylated and asialylated alpha 1-acid glycoprotein (orosomucoid) have been performed. The interaction between the fluorophore and the protein was found to occur preferentially with the glycan residues with a dependence on their spatial conformation. In the presence of sialylated alpha 1-acid glycoprotein, excitation at the red edge of the absorption spectrum of calcofluor does not lead to a shift in the fluorescence emission maximum (440 nm) of the fluorophore. Thus, the emission of calcofluor occurs from a relaxed state. This is confirmed by anisotropy studies as a function of temperature (Perrin plot). In the presence of asialylated alpha 1-acid glycoprotein, red-edge excitation spectra show an important shift (8 nm) of the fluorescence emission maximum of the probe. This reveals that emission of calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that Calcofluor molecules are bound tightly to the carbohydrate residues, a result confirmed by anisotropy studies.     

The rearrangement of the human alpha(1)-acid glycoprotein/orosomucoid gene: evidence for tandemly triplicated genes consisting of two AGP1 and one AGP2

The human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is controlled by the two tandemly arranged genes, AGP1 and AGP2. The further duplication of the AGP1 gene has been suggested by a few duplicated ORM1 locus haplotypes including ORM1*F1. S and ORM1*B9. S, detected by isoelectric focusing. To clarify the triplication of the AGP gene, 39 DNA samples from Japanese subjects were studied by the long-range PCR of intergenic regions. The analysis of PCR products showed that the tandemly triplicated genes, AGP1A-AGP1B-AGP2, occurred on about 20% of chromosomes. These composites were divided into ORM1A*F1-ORM1B*S-ORM2*M and ORM1A*B9-ORM1B*S-ORM2*M by allelic variations. Furthermore, the former was classified into a few haplotypes by three synonymous sequence variations, which might have arisen through gene conversion-like events. The recombination breakpoints existed between the 5' flanking region and intron 2 of the AGP1B gene. Thus, it is likely that the rearrangement of the AGP gene has often occurred.     

An N-linked glycoprotein with alpha(2,3)-linked sialic acid is a receptor for BK virus

BK virus (BKV) is a common human polyomavirus infecting >80% of the population worldwide. Infection with BKV is asymptomatic, but reactivation in renal transplant recipients can lead to polyomavirus-associated nephropathy. In this report, we show that enzymatic removal of alpha(2,3)-linked sialic acid from cells inhibited BKV infection. Reconstitution of asialo cells with alpha(2,3)-specific sialyltransferase restored susceptibility to infection. Inhibition of N-linked glycosylation with tunicamycin reduced infection, but inhibition of O-linked glycosylation did not. An O-linked-specific alpha(2,3)-sialyltransferase was unable to restore infection in asialo cells. Taken together, these data indicate that an N-linked glycoprotein containing alpha(2,3)-linked sialic acid is a critical component of the cellular receptor for BKV.     

Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket

Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the F?rster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.     

Dab1 (Disable homolog-1) reelin adaptor protein is overexpressed in the olfactory bulb at early postnatal stages

Dab1 mediates reelin signalling and plays critical roles in early brain development such as the stereotypical positioning of neurons in the brain. The olfactory bulb undergoes a prominent layering reorganization, but shows not apparent differences between wild type and reeler in the layer organization. Therefore, an accurate regional and cellular simultaneous analysis of these molecules becomes essential to clarify the role played by Dab1 upon Reelin effect. The present study reveals a strong and consistent Dab1 mRNA and protein expressions, throughout the olfactory bulb layers in both wild type and reeler mice. In addition, noteworthy is the pattern of Dab1 location within cell nuclei in both strains. Furthermore, a temporal increment of Dab1 expression levels is detected from P0 to P15 in both strains, being the protein quantity higher in reeler than in wild type mice. Altogether, our results revealed that Reln acts directly from projection neurons via the production of different Reln fragments. Changes in the pattern of Dab1 expression could reflect an alternative Reln function in postnatal and adult stages, besides a possible regulation of Dab1 by other molecules distinct to Reln.     

Effect of binding of Calcofluor White on the carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) on the structure and dynamics of the protein moiety. A fluorescence study

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously studied at low and high concentrations of Calcofluor compared to that of the protein. alpha1-Acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. At equimolar concentrations of Calcofluor and alpha1-acid glycoprotein, the fluorophore displays free motions [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94], while at high concentration of Calcofluor, its surrounding microenvironment is rigid, inducing the rigidity of the fluorophore itself [Albani, J. R.; Sillen, A.; Plancke, Y. D.; Coddeville, B.; Engelborghs, Y. Carbohydr. Res. 2000, 327, 333-340]. In the present work, red-edge excitation spectra and steady-state anisotropy studies performed on Trp residues in the presence of Calcofluor, showed that the apparent dynamics of Trp residues are not modified. However, deconvoluting the emission spectra with two different methods into different components, reveals that the structure of the protein matrix has been disrupted in the presence of high Calcofluor concentrations.     

Cloning and developmental regulation of alpha 1 acid glycoprotein in swine

A cDNA clone of porcine alpha 1 acid glycoprotein (alpha 1AGP) has been isolated and sequenced. Sequence homologies between porcine, human, and rat indicate that porcine alpha 1AGP is similar in structure to the rat and human proteins. RNA blots from days 40, 60, 80, and 110 fetal, newborn, and adult livers showed that alpha 1AGP mRNA is relatively abundant throughout fetal development, particularly at the later stages and in the newborn; there is a rapid decline in abundance following birth. From birth to 3 days of age, there is a three- to four-fold decline in abundance, and alpha 1AGP mRNA is approximately 100 times less abundant in the adult liver than in that of perinatal pigs. Southern blots showed that alpha 1AGP is probably a single-copy gene. The isolation of a cloned cDNA for porcine alpha 1AGP provides a tool to investigate the molecular mechanisms involved in the developmental regulation of the gene and to correlate changes in gene expression during development with fetal growth and well being.