The induction of α1-acid glycoprotein by methylmercury

• 1.1. 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).
• 2.2. 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.
• 3.3. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of α₁-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.

     

Glycoforms of serum α1-acid glycoprotein as markers of inflammation and cancer

1-acid glycoprotein (AGP) is a serum acute phase glycoprotein which possesses five N-linked complex type heteroglycan side chains which may be present as bi-, tri- and tetraantennary structures. Depending upon the content of biantennary structure on AGP, up to four glycoforms of AGP are present in serum. These glycoforms can be easily estimated in body fluids by means of crossed affinity-immunoelectrophoresis (CAIE) with the lectin, Concanavalin A (Con A). Con A selectively binds biantennary structures; the more biantennary structures on AGP, the stronger the binding. In acute inflammation, a relative increase of AGP glycoforms with biantennary units is observed - a type I glycosylation change. In some chronic inflammatory states there is an relative decrease of AGP glycoforms with biantennary heteroglycans — a type II glycosylation change. Moreover, in certain other states such as pregnancy, estrogen administration or liver damage, type II glycosylation changes are also seen. A detailed analysis of the clinical applications of the assessment of AGP glycoforms in sera of patients with rheumatic diseases, AIDS and various types of cancers is presented. Accumulated data shows that AGP glycoforms may be very useful in the detection of intercurrent infections in the course of rheumatoid arthritis, systemic lupus erythematosus, or myeloblastic leukaemia, and in the detection of secondary infections in human immunodeficiency virus infected individuals. AGP glycoforms are also very useful in differentiation between various forms of trophoblastic disease and are helpful in monitoring the treatment of these patients. Finally, AGP glycoforms provide valuable information for differentiation between primary and secondary liver cancer.     

Intracellular precursor forms of transferrin, α1-acid glycoprotein,and α1-antitrypsin in human liver

Ion-exchange chromatography of dialyzed human plasma and of buffer extracts of acetone-dried powder from human liver was used to analyze 13 different plasma proteins which are synthesized in the liver. Specific intracellular forms which differ from the plasma forms were found for transferrin, α₁-acid glycoprotein, α₁-antitrypsin, and albumin. The intracellular forms were labeled earlier than the plasma forms, when liver slices were incubated with radioactive leucine, suggesting that they are precursor forms of the proteins in the bloodstream. The liver form of transferrin was found to have the same molecular weight and N-terminus as the plasma form, but it differed from the plasma form by the absence of sialic acid. For α₁-acid glycoprotein two different liver forms were observed, both of which had lower molecular weights than the plasma form. One of these liver forms was analyzed further. Its polypeptide chain was found to have a blocked N-terminus, as does the plasma form. However, in contrast to the plasma form, it did not contain sialic acid. Its content of N-acetyl glucosamine was about one-third and the content of neutral hexoses about two-thirds of that found in the plasma form. Circular dichroism spectra were similar for liver and plasma forms and indicated a predominant β structure with very little α-helix content for both.     

Osmotic water permeability through liposomes in the presence of α1-acid glycoprotein

¹-acid glycoprotein (orosomucoid) from human blood serum was isolated in pure form and then reconstituted into large multilamellar liposomes, consisting of a binary mixture of hen-egg phosphatidylcholine and cholesterol. These liposomes were found to be osmotically sensitive. The osmotic water permeability of proteoliposomes was determined by light-scattering measurements of the osmotic volume changes after mixing with hyperosmotic solutions of potassium salts and aminoglycoside antibiotics. The initial rate of water outflow was measured as a function of glycoprotein concentration in the mixture for the preparation of proteoliposomes. This can serve as an indication for membrane permeability to the solutes used in these experiments. It was shown that aminoglycoside antibiotics passed much faster across the membrane than potassium salts, in the presence of glycoprotein in the liposomes. A recognition pattern in the osmotic behavior of these proteoliposomes was assumed.     

Changes in expression and microheterogeneity of the genetic variants of human α1-acid glycoprotein in malignant mesothelioma

Human α₁-acid glycoprotein (AAG), an acute-phase plasma protein, is heterogeneous in the native state and polymorphic in the desialylated state. The AAG heterogeneity is mainly explained by a variable glycan chain composition in its five glycosylation sites. The AAG polymorphism is due to the presence of genetic variants. Three main variants are observed for AAG, ORM1 F1, ORM1 S and ORM2 A, which have a separate genetic origin. In this paper, we have used different isoelectric focusing (IEF) methods and chromatography on immobilized metal affinity adsorbent to study the relative occurrence of the genetic variants of AAG in relation to changes in microheterogeneity, in plasma and pleural effusions of patients with malignant mesothelioma (MM). The results were compared to those obtained with the variants in plasma of healthy individuals. Significant changes in variant distribution were observed in the MM samples, that corresponded to a rise in the proportion of the ORM1 variants and a fall in that of the ORM2 variant. However, the concentration in MM plasma increased for both variants. The AAG in MM plasma and effusion fluids was found to be more heterogeneous on IEF than AAG of healthy plasma. The evidence of stronger concentrations of both the high and low pI forms of AAG in the MM samples suggested two kinds of changes in charge heterogeneity. These two changes were shown to be attributed to different variants — i.e. the high pI forms to ORM1 F1 and S and the low pI forms to ORM2 A, after fractionation of AAG by chromatography on immobilized copper(II) ions. These results indicate specific changes in both the expression and glycosylation for each AAG variant, according to its separate genetic origin, in MM.     

Characterization of the mechanism of interaction between α1-acid glycoprotein and lipid membranes by vacuum-ultraviolet circular-dichroism spectroscopy

α₁-Acid glycoprotein (AGP) interacts with lipid membranes as a peripheral membrane protein so as to decrease the drug-binding capacity accompanying the β→α conformational change that is considered a protein-mediated uptake mechanism for releasing drugs into membranes or cells. This study characterized the mechanism of interaction between AGP and lipid membranes by measuring the vacuum-ultraviolet circular-dichroism (VUVCD) spectra of AGP down to 170 nm using synchrotron radiation in the presence of five types of liposomes whose constituent phospholipid molecules have different molecular characteristics in the head groups (e.g., different net charges). The VUVCD analysis showed that the α-helix and β-strand contents and the numbers of segments of AGP varied with the constituent phospholipid molecules of liposomes, while combining VUVCD data with a neural-network method predicted that these membrane-bound conformations comprised several common long helix and small strand segments. The amino-acid composition of each helical segment of the conformations indicated that amphiphilic and positively charged helices formed at the N- and C-terminal regions of AGP, respectively, were candidate sites for the membrane interaction. The addition of 1 M sodium chloride shortened the C-terminal helix while having no effect on the length of the N-terminal one. These results suggest that the N- and C-terminal helices can interact with the membrane via hydrophobic and electrostatic interactions, respectively, demonstrating that the liposome-dependent conformations of AGP analyzed using VUVCD spectroscopy provide useful information for characterizing the mechanism of interaction between AGP and lipid membranes.     

Two-step chromatographic purification of human plasma α1-acid glycoprotein Its application to the purification of rare phenotype samples of the protein and their study by chromatography on immobilized metal chelate affinity adsorbent

α₁-Acid glycoprotein (AAG) or orosomucoid was purified to homogeneity from human plasma by a separate two-step method using chromatography on immobilized Cibacron Blue F3G-A to cross-linked agarose and chromatography on hydroxyapatite. The conditions for the pre-purification of AAG by chromatography on immobilized Cibacron Blue F3G-A were first optimized using different buffer systems with different pH values. The overall yield of the combined techniques was 80% and ca. 12 mg of AAG were purified from an initial total amount of ca. 15 mg in a ca. 40 ml sample of human plasma. This method was applied to the purification of AAG samples corresponding to the three main phenotypes of the protein (F1^s*S/A, F1/A and S/A), from individual human plasma previously phenotyped for AAG. A study by isoelectric focusing with carrier ampholytes showed that the microheterogeneity of the purified F1^s*S/A, F1/A and S/A AAG samples was similar to that of AAG in the corresponding plasma, thus suggesting that no apparent desialylation of the glycoprotein occurred during the purification steps. This method was also applied to the purification of AAG samples corresponding to rare phenotypes of the protein (F1/A¹AD, S/A¹X₀ and F1/A¹C1) and the interactions of these variants with immobilized copper(II) ions were then studied at pH 7, by chromatography on an iminodiacetate Sepharose-Cu(II) gel. It was found that the different variants encoded by the first of the two genes coding for AAG in humans (i.e. the F1 and S variants) interacted non-specifically with the immobilized ligand, whereas those encoded by the second gene of AAG (i.e. the A, AD, X₀ and C1 variants) strongly bound to immobilized Cu(II) ions. These results suggested that chromatography on an immobilized affinity Cu(II) adsorbent could be helpful to distinguish between the respective products of the two highly polymorphic genes which code for human AAG.     

Severe rheumatoid arthritis prohibits the pregnancy-induced decrease in α3-fucosylation of α1-acid glycoprotein

Patients suffering from rheumatoid arthritis (RA) may experience a temporary reduction of disease symptoms during pregnancy. As indicated by the occurrence of RA-disease symptoms during pregnancy, three categories of patients were defined, namely, remission, relapse and unchanged. In all three categories changes in the plasma level and glycosylation of α1-acid glycoprotein (AGP) were determined longitudinally in comparison to those occurring in pregnancy of healthy women. In healthy pregnancy, we observed: (i) a peak in the plasma concentration at week 18 and a minimum at week 30; (ii) a continuous increase in the degree of branching of the glycans during the entire pregnancy period, and (iii) a decrease in the degree of α3-fucosylation of AGP-glycans with a minimum occurring at week 25. Comparable pregnancy-induced changes in glycosylation were found for two other acute-phase proteins α1-protease inhibitor (PI) and α1-antichymotrypsin (ACT). Increased oestrogen levels, known to occur during pregnancy, may be one of the factors that induce these changes, because the increased branching and decreased α3-fucosylation is in agreement with our earlier findings regarding an involvement of this hormone in the regulation of acute phase protein glycosylation in oestrogen-treated males as well as females. In all three clinical categories in RA, pregnancy also induced a continuous increase in the degree of branching of the glycans of AGP. However, similar changes in concentration and fucosylation were only found during remission of the disease symptoms. In the relapse and unchanged categories in RA, the degree of fucosylation and the plasma concentration of AGP remained constant throughout pregnancy. This indicates a relationship between changes in α3-fucosylation of AGP and RA disease activity.     

Improved purification of α-amylase isolated fromRhizomucor pusillus by affinity chromatography

A highly purified extracellular -amylase was isolated fromRhizomucor pusillus with minimum loss of enzymatic activity. The enzyme was purified from the mycelium-free liquid filtrate of the thermophilic moldRhizomucor pusillus. Maximum enzyme yields were attained after 5 days of growth on liquid starch-yeast extract at 45°C and pH 7.0. The crude enzyme preparation was first concentrated 80-fold by ultrafiltration. Purification was recently achieved with high-performance liquid chromatography and Waters Protein Pak 300 SW. Improved purification was then achieved with a dextrin-bound affinity column, with a 59-fold increase in specific activity from the crude enzyme preparation. This final enzyme preparation produced a single band on polyacrylamide gel electrophoresis. The molecular weight determined by SDS gel electrophoresis was 52,000 daltons.     

Glycosylation ofα 1 glycoprotein in septic shock: Changes in degree of branching and in expression of sialyl Lewisx groups

The occurrence of differences in acute-phase response, with respect to concentration and glycosylation of ₁-acid glycoprotein (AGP) was studied in the sera of patients surviving or not from septic shock. Crossed affino-immunoelectrophoresis was used with concanavalin A andAleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of sialyl Lewis^x (SLeX) groups on AGP. Septic shock apparently induced an acute-phase response as indicated by the increased serum levels and changed glycosylation of AGP. In the survivor group a transient increase in diantennary glycan content was accompanied by a gradually increasing fucosylation and SLeX expression, comparable to those observed in the early phase of an acute-inflammatory response. Remarkably, in the non-survivor group a modest increase in diantennary glycan content was accompanied by a strong elevation of the fucosylation of AGP and the expression of SLeX groups on AGP, typical for the late phase of an acute-phase response. Our results suggest that these changes in glycosylation of AGP can have a prognostic value for the outcome of septic shock.Abbreviations AAL Aleuria aurantia lectin - AGP ₁-acid glycoprotein - CAIE crossed affinoimmunoelectrophoresis - Con A Concanavalin A - HSPC human serum protein calibrator - IL-1 interleukin 1 - IL-6 interleukin 6 - LIF leukaemia inhibitory factor - LPS lipopolysaccharide - SLeX sialyl Lewis^x - TNF tumour necrosis factor     

Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein

Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in blood system which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we studied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperine caused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching of proteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The binding constants obtained from fluorescence emission were found to be K_piperine?=?5.7 ± .2 × 10⁵ M^?1 and K_piperine = 9.3± .25 × 10⁴ M^?1 which correspond to the free energy of ?7.8 and ?6.71 kcal M^?1at 25 °C for HSA and AGP, respectively. Further, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA due to partial destabilization of HSA–piperine complexes. Consequently, inference drawn from the site-specific markers (phenylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), which was further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and free energy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilic interactions when piperine binds to HSA. Additionally, the MD studies have showed that HSA–piperine complex reaches equilibration state at around 3 ns, which prove that the HSA–piperine complex is stable in nature.     

Studies on the carbohydrate moiety of α1-acid glycoprotein (orosomucoid) by using alkaline hydrolysis and deamination by nitrous acid

Alkaline hydrolysis followed by deamination with nitrous acid was applied for the first time to a glycoprotein, human plasma alpha(1)-acid glycoprotein (orosomucoid). This procedure, which specifically cleaves the glycosaminidic bonds, yielded well-defined oligosaccharides. The trisaccharides, which were obtained from the native protein, consisted of a sialic acid derivative, galactose and 2,5-anhydromannose. The linkage between galactose and 2,5-anhydromannose is most probably a (1-->4)-glycosidic bond. A hitherto unknown linkage between N-acetylneuraminic acid and galactose was also established, namely a (2-->2)-linkage. The three linkages between sialic acid and galactose described in this paper appear to be about equally resistant to mild acid hydrolysis. The disaccharide that was derived from the desialized glycoprotein consisted of galactose and 2,5-anhydromannose. Evidence was obtained for the presence of a new terminal sialyl-->N-acetylglucosamine disaccharide accounting for approximately 1mol/mol of protein. The presence of this disaccharide may explain the relatively severe requirements for the complete acid hydrolysis of the sialyl residues. The present study indicates that alkaline hydrolysis followed by nitrous acid deamination in conjunction with gas-liquid chromatography will afford relatively rapid determination of the partial structure of the complex carbohydrate moiety of glycoproteins.     

Metal-binding sites of concanavalin A and their role in the binding of α-methyl d-glucopyranoside

Binding of a transition metal ion to specific sites in concanavalin A induces the formation of specific Ca(2+) ion-binding sites. Sites for binding alpha-methyl d-glucopyranoside exist only when a transition metal ion and Ca(2+) ion are bound.     

Facile analysis of ribonucleotides in d,l-α-difluoromethylornithine-treated mouse lymphoid cells by high-performance anion-exchange column chromatography

Using high-performance liquid chromatography with the strong anion-exchange resin column CDR-10 for analysis of ribonucleotides, the effects of d,l-α-difluoromethylornithine (DFMO) on ribonucleotides of mouse leukemic lymphoid cells SC-1 have been studied. More than 16 nucleoside mono-, di and triphosphates, and unknown peaks were clearly separated and measured without increase in baseline rise using the gradient systems of phosphate buffer. The ATP level in DFMO-treated SC-1 cells was reduced, but was reversed by exogenous putrescine. These facts may suggest that polyamine depletion by DFMO during a short time (6 h) caused mitochondrial damage in mammalian cells.     

Different susceptibilities of complex-, hybrid- and high-mannose-type α1- inhibitor and α1-acid glycoprotein to endo-β-N-acetylglucosaminidase F and peptide:N-glycosidase F

Endo--N-acetylglucosaminidase F (endo F, EC 3.2.1.96) and peptide:N-glycosidase F (PNGase F, EC 3.2.2.18) fromFlavobacterium meningosepticum were used for the deglycosylation of ₁-proteinase inhibitor and ₁-acid glycoprotein carrying oligosaccharide side chains of the complex-, high-mannose- and hybrid-type. High-mannose-and hybrid-type glycoproteins were obtained by the incubation of rat hepatocyte primary cultures with 1-deoxymannojirimycin or swainsonine, respectively. It was found that endo F cleaves hybrid- and high-mannose-type ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 as well as at pH 8.5 in the presence or absence of 1% octyl--d-glucopyranoside. Complex-type ₁-proteinase inhibitor or ₁-acid glycoprotein were not cleaved by endo F even in the presence of octyl--d-glucopyranoside.PNGase F was found to cleave complex-, hybrid- and high-mannose-type oligosaccharide side chains of ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 and pH 8.5 in the presence of 0.75% octyl--d-glucopyranoside. The deglycosylation of both protein substrates was very poor without detergents.Abbreviations Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - PNGase F peptide:N-glycosidase F (EC 3.2.2.18) Dedicated to Prof. Dr. Wolfgang Gerok on the occasion of his 60th birthday     

The major serum protein of fetal and newborn pigs: Biochemical properties and identification as a fetal form of α1-acid glycoprotein

• 1.I. The major protein component of fetal pig serum, has been immunologically identified as α₁-acid glycoprotein (orosomucoid).
• 2.2. Amino acid composition and total carbohydrate content (around 38% by weight) were similar in the adult and fetal forms of α₁-acid glycoprotein. These forms differ, however, in the proportion of individual monosaccharides.
• 3.3. Fucose, represented the 1.5% (by weight) in the fetal protein, and the 2.5% in its adult counterpart. The latter was more susceptible to ncuraminidase and also possesses a higher mannose/galactose ratio than the fetal form.
• 4.4. Insolubilized Concanavalin A (Con A) retained 80%, of the adult protein, whereas the fetal form was mostly Con A-non reactive. The proportion of this -non reactive fraction, as revealed by crossed immuno-affino-electrophoresis experiments, was age-dependent and varied from 62% at fetal age of 50–60 days to 80% at birth.

     

Chaperone-like activity of the acute-phase component human serum α1-acid glycoprotein: Inhibition of thermal- and chemical-induced aggregation of various proteins

In vitro chaperone-like activity of the acute-phase component and plasma drug transporter human α₁-acid glycoprotein (AAG) has been shown for the first time. AAG suppressed thermal aggregation of a variety of unrelated enzymatic (e.g., aldolase, catalase, enolase, carbonic anhydrase) and non-enzymatic proteins (β-lactoglobulin, ovotransferrin) and it also prevented dithiothreitol induced aggregation of insulin. The anti-aggregation ability of AAG was abolished/reduced upon drug binding suggesting that protein–protein interactions established between the lipocalin β-barrel fold of AAG and hydrophobic surfaces of the stressed proteins are involved in the chaperone-like activity. The results shed some light on the possible biological function of this enigmatic protein and suggest that besides haptoglobin, clusterin, fibrinogen and α₂-macroglobulin AAG can be considered as a novel member of the extracellular molecular chaperones found in human body fluids.     

Effects of nicotinamide on hepatocyte viability and secretion of albumin and α1-acid glycoprotein by adult rat hepatocytes in primoculture. Comparison with dexamethasone and recombinant human interleukin-6

The effects of nicotinamide on hepatocyte viability and secretion of albumin and α₁-acid glycoprotein were studied in the absence or presence of dexamethasone and/or recombinant human interleukin-6 either after cell attachment (2 h) or after 24, 48, and 72 h of culture. The evolution of hepatocyte survival during the culture was appreciated by measurement of total DNA content. The secretion of albumin and α₁-acid glycoprotein was measured after a 4-h period following cell attachment or after 24, 48 and 72 h of culture. The important decrease of DNA content, mRNA levels and secretion of albumin and α₁-acid glycoprotein in control cultures after 2–3 days was not prevented by the addition of nicotinamide. In contrast, dexamethasone alone or with recombinant human interleukin-6 improved DNA content and albumin secretion with no additional effect of nicotinamide. The secretion of α₁-acid glycoprotein was largely induced by dexamethasone alone or dexamethasone and recombinant human interleukin-6. The increase of α₁-acid glycoprotein secretion was not modified by the addition of nicotinamide and averaged respectively 27- and 60-fold for dexamethasone alone and dexamethasone and recombinant human interleukin-6 after 48 h. These observations suggested that nicotinamide, at least in the conditions tested here, is unable to prevent alterations of hepatocyte viability and gene expression of cultured hepatocytes.     

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.