Alpha-1-acid glycoprotein

Alpha-1-acid glycoprotein (AGP) or orosomucoid (ORM) is a 41-43-kDa glycoprotein with a pI of 2.8-3.8. The peptide moiety is a single chain of 183 amino acids (human) or 187 amino acids (rat) with two and one disulfide bridges in humans and rats,respectively. The carbohydrate content represents 45% of the molecular weight attached in the form of five to six highly sialylated complex-type-N-linked glycans. AGP is one of the major acute phase proteins in humans, rats, mice and other species. As most acute phase proteins, its serum concentration increases in response to systemic tissue injury, inflammation or infection, and these changes in serum protein concentrations have been correlated with increases in hepatic synthesis. Expression of the AGP gene is controlled by a combination of the major regulatory mediators, i.e. glucocorticoids and a cytokine network involving mainly interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF alpha), interleukin-6 and IL-6 related cytokines. It is now well established that the acute phase response may take place in extra-hepatic cell types, and may be regulated by inflammatory mediators as observed in hepatocytes. The biological function of AGP remains unknown; however,a number of activities of possible physiological significance, such as various immunomodulating effects, have been described. AGP also has the ability to bind and to carry numerous basic and neutral lipophilic drugs from endogenous (steroid hormones) and exogenous origin; one to seven binding sites have been described. AGP can also bind acidic drugs such as phenobarbital. The immunomodulatory as well as the binding activities of AGP have been shown to be mostly dependent on carbohydrate composition. Finally, the use of AGP transgenic animals enabled to address in vivo, functionality of responsive elements and tissue specificity, as well as the effects of drugs that bind to AGP and will be an useful tool to determine the physiological role of AGP.     

One-step isolation of alpha 1-acid glycoprotein.

alpha 1-Acid glycoprotein could be isolated by a one-step extraction method from human sera and plasma. Protein recovered in the water phase after extraction with phenol at 70 degrees C for 20 min was verified as human alpha 1-acid glycoprotein when it was compared with the reference standard human alpha 1-acid glycoprotein by Ouchterlony double immunodiffusion, sodium dodecylsulfate-polyacrylamide gel electrophoresis, Western blot analysis, and periodic acid-Schiff stain. The present isolation procedure is simple and fast, and can extract about 81% of the total alpha 1-acid glycoprotein in the sera and plasma, as determined by radial immunodiffusion.     

Crystallization of alpha 1-acid glycoprotein

A possible link between cellular cyclic AMP content and Na+K+ATPase activity was investigated in homogenates of rat kidney. Enzyme kinetics of Mg2+ and Na+K+ATPase were run in the presence of cyclic AMP, dibutyryl cAMP and compounds expected to elevate cyclic AMP levels such as forskolin, a potent adenylate cyclase activator, IBMX, an inhibitor of phosphodiesterases, and the beta-agonist isoproterenol. Medullary Na+K+ATPase is strongly inhibited by cyclic AMP whereas cortical Na+K+ATPase was stimulated in the same conditions. The correlation between ATPase activity and cellular cyclic AMP content supports the concept of a possible regulation of the enzyme by cyclic AMP.     

Topography of human plasma alpha1-acid glycoprotein.

Human plasma alpha1-acid glycoprotein, whose linear amino acid sequence has recently been elucidated (Schmid et al. (1973), Biochemistry 12, 2711), was further investigated with regard to its topography. Nitration of this protein and subsequent elucidation of the structures of the peptides containing modified tyrosine indicated that residues 27, 37, 78, 115, 127, and 157 are free, 50 and 91 are in an intermediate state, and 65, 74, 110, and 142 are buried. CD measurements between pH 10 and 12 demonstrated that the buried tyrosines are strongly hydrogen bonded and are probably responsible to a considerable extent for the stability of this protein. Of the three tryptophans of this protein, residue 122 proved to be partially reactive with Koshland reagent while the other two (25 and 160) were found to be unreactive. The state of the two disulfide bonds, established by differential reduction and alkylation with specific reagents, was shown to be of an intermediate type. Using carboxymethylation with bromoacetate at pH 7.0 for 8 days, the three histidines (97, 100, and 171) and methionine 111 could be shown to be in intermediate states. All lysines were treated with trinitrobenzenesulfonate and thus were assumed to be free. Of the 40 carboxylic groups, which were amidated with glycine methyl ester, 32 including the 14 sialyl residues were found to be free, six in an intermediate and the remaining two in a buried state. The present study describes the states of almost half of the amino acid residues of alpha1-acid glycoprotein, a knowledge important for the construction of a preliminary three-dimensional model of this conjugated protein.     

The interaction of collagen with alpha1-acid glycoprotein.

The influence of alpha1-acid glycoprotein on the formation of fibrous long spacing fibers of collagen has been investigated. It was observed that addition of the glycoprotein to dialyzed collagen solutions caused a significant decrease in the intensity of the circular dichroic spectrum of collagen. This phenomenon, which displays an optimum with respect to glycoprotein, is consistent with previous observations of fibrous long spacing fiber formation. Changes in viscosity of collagen initially dissolved in acetic acid were monitored during dialysis. It was found that a significant increase in viscosity must occur during dialysis of collagen before fibrous long spacing formation could take place. This increase in viscosity can be related directly to removal of acetic acid from the collagen solution. Removal of all sialyl residues from the alpha1-acid glycoprotein with neuraminidase prevents fibrous long spacing formation while removal of up to 35% of the sialyl residues has no effect on the interaction of glycoprotein with collagen. Amino acid composition and radioactivity studies suggest that 45-55% of the insoluble fibrous long spacing fibers is glycoprotein. In contrast to native collagen fibers, reduced fibrous long spacing fibers do not contain histidinohydroxymerodesmosine or hydroxylysinonorleucine. Instead, they contain significant quantities of allysine aldol and epsilon-hydroxynorleucine.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

A three-step purification of human alpha 1-acid glycoprotein

alpha 1-Acid glycoprotein (AGP) was purified to homogeneity by a 3-step procedure using pseudo-ligand affinity chromatography on immobilized Cibacron blue F3GA, Procion red HE3B, and preparative column isoelectric focusing. The overall yield of the combined techniques was 88%. Analysis of the purified AGP by lectin affinity chromatography on immobilized Con A and immunoaffino-electrophoresis indicated that the most acidic form did not interact with the lectin, while the two more basic fractions possessed different affinities for Con A. In addition, 3 different populations of AGP were clearly separated by Con A affinity chromatography.     

The induction of alpha 1-acid glycoprotein by methylmercury

The incorporation of [14C]leucine into protein was measured in liver preparations and blood of rats following the s.c. administration of methylmercury hydroxide (24 mg/kg body wt) or turpentine (5.0 ml/kg body wt). The translatability of the RNA obtained from polysomes in an mRNA-dependent reticulocyte lysate was elevated significantly in the preparations derived from the treated rats compared to control rats. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of alpha 1-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Interaction of propafenone enantiomers with human alpha 1-acid glycoprotein

The interaction of propafenone enantiomers with human alpha 1-acid glycoprotein was studied using high-performance liquid chromatography. Each of the two optical antipodes interacted with one class of high-affinity binding sites characterized by Ka(R) = (6.18 +/- 0.93) x 10(5) M-1, n(R) = 1.34 +/- 0.09 for the (R)-isomer and Ka(S) = (8.93 +/- 1.82) x 10(5) M-1, n(S) = 0.99 +/- 0.08 for the (S)-isomer. Nonspecific binding to secondary low-affinity high-capacity binding site(s) was only slightly greater in the case of the (S)-enantiomer (n'k'(S) = (1.06 +/- 0.09) x 10(4) M-1) compared to the (R)-enantiomer (n'k'(R) = (6.87 +/- 0.72) x 10(3) M-1). It was concluded that both enantiomers interact with common single class of high-affinity binding sites on AAG (along with nonspecific binding) exhibiting only slight stereoselectivity for propafenone.