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.     

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.     

Effect of transgenic expression of human alpha 1-acid glycoprotein (AGP) on the glycosylation of human and mouse AGP in various transgenic mouse sera.

The occurrence and the glycosylation of human alpha 1-acid glycoprotein (AGP) was studied in two classes of transgenic mice expressing either the A, B and B' genes (ABB'-mice) or only the A gene of human AGP (A-mice). The glycosylation of the human AGP molecules in the transgenic mouse sera was compared with the glycosylation of mouse AGP in the same animal and with human AGP in normal human serum by studying their heterogeneity in binding to concanavalin A (Con A), using crossed affino immunoelectrophoresis (CAIE) with Con A as the affinocomponent in the first dimension gel. Three to four different glycosylated fractions of human as well as mouse AGP were revealed by this method in all the transgenic mouse sera. A close relationship was apparent between the heterogeneities in Con A binding of human and mouse AGP in the same transgenic mouse. The magnitude of this so-called Con A reactivity was, however, strongly dependent on the transgenic mouse studied. Especially within the group of ABB'-mice dramatic changes in Con A reactivity were found when the human AGP genes were expressed. This indicates in the first place that the oligosaccharide chains of the human AGP molecules expressed also mouse-specific features. Secondly, and more importantly, these findings indicate that the expression of the human AGP genes affected the glycosylation process of the transgenic mouse liver. This organ is the source of the AGP forms occurring in serum. We do not know whether this effect has been caused by the introduction or the expression of the human gene(s) or by the presence of human AGP in the Golgi system or in serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute phase protein alpha 1-acid glycoprotein interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity.

alpha(1)-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators. This conclusion is based on the following observations: (a) alpha(1)-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system. Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of alpha(1)-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or alpha(1)-acid glycoprotein. Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of alpha(1)-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparent K(D) values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively). The on rate for binding of PAI-1 to alpha(1)-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by alpha(1)-acid glycoprotein. On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the alpha(1)-acid glycoprotein, as indicated by 4-fold lower k(off) values. Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of alpha(1)-acid glycoprotein. These observations suggest that the complex of PAI-1 with alpha(1)-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions.     

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.     

Effect of the threonine analog beta-hydroxynorvaline on the glycosylation and secretion of alpha 1-acid glycoprotein by rat hepatocytes

The threonine analog beta-hydroxynorvaline (Hnv) is an inhibitor of asparagine-linked glycosylation. In the presence of the analog hepatocytes synthesized immunoreactive alpha 1-acid glycoprotein with 0-6 oligosaccharide chains. Pulse-chase experiments were conducted to compare the rates of secretion of alpha 1-acid glycoprotein from untreated, tunicamycin-treated, and Hnv-treated cells. Partially glycosylated (1-5 oligosaccharide chains) and unglycosylated (tunicamycin-inhibited) molecules exited the cells more slowly than native alpha 1-acid glycoprotein. In addition, secretion of fully glycosylated (6 oligosaccharide chains) alpha 1-acid glycoprotein was retarded in Hnv-treated cells when compared to controls. The slowest rate of secretion was exhibited by the unglycosylated form from Hnv-treated cells. These results suggest that Hnv-induced changes either in the extent of glycosylation or in the peptide sequence of alpha 1-acid glycoprotein can interfere with its transport through the cell. The major intracellular forms of alpha 1-acid glycoprotein from control and Hnv-treated cells were endoglycosidase H-sensitive and contained Man9-8 GlcNAc2 oligosaccharide structures. The oligosaccharide chains on the secreted molecules from control and Hnv-treated cells were entirely of the endoglycosidase H-resistant, complex type.     

Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted.     

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.     

Induction of alpha 1-acid glycoprotein by recombinant human interleukin-1 in rat hepatoma cells

The induction of alpha 1-acid glycoprotein mRNA by recombinant murine interleukin-1, recombinant human interleukin-1 alpha, and recombinant human interleukin-1 beta has been studied in the rat hepatoma cell line Fao. Whereas the stimulatory capacities of recombinant human interleukin-1 alpha and recombinant murine interleukin-1 were almost identical, the concentrations of recombinant human interleukin-1 beta needed for half-maximal induction of alpha 1-acid glycoprotein mRNA were lower by three orders of magnitude. A 60-fold increase in alpha 1-acid glycoprotein mRNA levels was observed 18 h after the addition of recombinant interleukin-1 beta. In parallel albumin mRNA levels decreased to about 30%. The alpha 1-acid glycoprotein mRNA induction was strictly dependent on the presence of dexamethasone. For a full stimulation dexamethasone concentrations of greater than 10(-7) M were needed, whereas concentrations of less than 10(-12) M were ineffective. The increase in alpha 1-acid glycoprotein mRNA after recombinant human interleukin-1 beta was followed by a 36-fold stimulation in alpha 1-acid glycoprotein synthesis and secretion. When protein synthesis was blocked by either cycloheximide, puromycin, or emetine, the induction of alpha 1-acid glycoprotein mRNA by recombinant human interleukin-1 beta was impaired suggesting the involvement of a short-lived protein in the induction of alpha 1-acid glycoprotein mRNA.     

Variations in the rate of secretion of different glycosylated forms of rat alpha 1-acid glycoprotein.

Various studies have shown that oligosaccharides play an important role in the intracellular transport and secretion of glycoproteins. We show here a difference in the rate of secretion of two mature glycoforms of a single protein, alpha 1-acid glycoprotein. This indicates the existence of kinetically different pathways for these two forms for transport from the medial Golgi to the extracellular medium.     

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.