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.     

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.     

Partial characterization of alpha 1-acid glycoprotein isolated from mouse serum

AGP was purified from mouse serum by perchloric acid treatment and CM-Sepharose chromatography. Induction of inflammation with turpentine resulted in a 10-fold increase in the serum level of mouse AGP, indicating mouse AGP is an acute phase reactant. Biochemical characterization of mouse AGP indicated similarity with human and rat 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.     

Structure of the human alpha 1-acid glycoprotein gene: sequence homology with other human acute phase protein genes.

We have determined the sequence coding for human alpha 1-acid glycoprotein from two independently isolated cDNA clones and a genomic clone. The aminoacid sequences deduced from the three clones, deriving from three different individuals, are identical. Southern blot analysis on human DNA indicates that there are at least two genes coding for alpha 1-AGP. We propose that alpha 1-AGP found in plasma is a mixture of the products of these two different genes. This is the simpler explanation for the heterogeneity in the aminoacid composition in purified alpha 1-AGP observed by Schmid et al. (1). DNA sequence comparison with cDNA clones coding for human alpha 1-antitrypsin and haptoglobin shows a conserved sequence within the 5' untranslated region which may play a role in the acute phase response.     

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.     

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.     

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 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.     

Structure and expression of mouse alpha 1-acid glycoprotein gene-3 (AGP-3).

The genome of Mus domesticus has multiple genes of the alpha 1-acid glycoprotein (AGP). Two cDNA clones were identified corresponding to AGP-1 and AGP-2. Moreover, two alleles of AGP-1 exist in inbred mice. The genomic DNA of the AGP-2 gene has been cloned and studied. Here we report the genomic organization of three M. domesticus AGP genes, the sequence analysis of the AGP-3 genomic DNA, and the expression of the AGP-3 gene. The major structural differences between AGP-2 and AGP-3 genes are located in introns 1 and 5. The low level of AGP-3 mRNA can be detected by the polymerase chain reaction (PCR). The molecular basis of the low level expression of AGP-3 and the possible classification of AGP-3 as a pseudogene are discussed.