A lectin immunosensor technique for determination of alpha(1)-acid glycoprotein fucosylation

The fucosylation of alpha(1)-acid glycoprotein (AGP), an acute-phase protein, is known to change in association with inflammatory diseases. Thus, fucosylation of AGP could be a potential diagnostic or prognostic marker. The change in fucosylation has previously been investigated using crossed affinoimmunoelectrophoresis, high-pH anion-exchange chromatography, and lectin ELISA. This study describes a surface plasmon resonance-based affinity biosensor assay for quantification of the fucosylation of AGP. Diluted EDTA plasma or serum was injected directly in a BIACORE 2000 biosensor. AGP was captured on the sensor surface using immobilized antibodies and a fucose-binding lectin from Aleuria aurentia was then used for the detection of fucosylation. The feature of the biosensor makes it possible to determine both the amount of bound AGP and the amount of bound lectin. Using a calibration curve it was possible to obtain a fucosylation ratio that was independent of AGP concentration. The assay was validated against a lectin ELISA and used to follow inflammation in patients with severe burns.     

Lymphocyte receptors for trophoblastic beta 1-glycoprotein

The authors have devised a method for rosette-formation to determine TBG receptors on the surface of T lymphocytes. It has been established that among woman peripheral blood lymphocytes, 7.1% of the cells carry TBG receptors, with this percentage increasing maximally up to 50.2% (the first trimester). Among T gamma lymphocytes, 1.2% carry TBG receptors. The amount of such lymphocytes rises 10-fold by the end of pregnancy.     

Immunochemical study of the heterogeneity of trophoblastic beta 1-glycoprotein

It has been found that in mild acid (pH 6.6) and mild-alkaline media (pH 7.7) both pregnancy proteins form complete precipitates. In more alkaline buffer solutions the form of alpha 2-glycoprotein (alpha 2-GP) precipitate is preserved, while trophoblastic beta 1-glycoprotein (TBG) shows three immunochemically identical components with different electrophoretic mobility. The form with beta-globulins mobility predominates, and minor fragments are presented by alpha- and gamma-components. All TBG forms are clearly seen at pH 8.6. In more alkaline medium (pH 10.0) the clarity of the precipitates drastically decreases. It is shown that heparin introduction into the gel of first dimension electrophoresis increases anode electrophoretic mobility of both proteins at polysaccharide concentration of at least 0.1 mg/ml. Large amounts of heparin cause the increase in TBG alpha-component precipitate area and the decrease in the form with beta-globulins mobility. At the same time alpha 2-GP precipitate area and form remain unchanged.     

Detection of trophoblastic beta 1-glycoprotein in tumor tissue and blood in ovarian neoplasms

Using immunochemical analysis methods (the reaction of precipitation in agar, immunoenzymatic method, immunofluorescence), trophoblastic beta 1-glycoprotein (TBG) concentration in tumour tissue and in the blood serum of patients with ovarian cancer was studied. By rabbits immunization with glycoprotein fraction of ovarian adenocarcinoma, dissolved in 0.6 M sulfosalicylic acid, the authors obtained antibodies to TBG. Immunoenzymatic method showed, that TBG level is raised during ovarian cancer (more than 3 micrograms/l): in 18% of tumour extracts, in 12.5% of blood sera samples and in 41.6% of cases in ascites fluid. Utilizing indirect immunofluorescence method morphological structures of trophoblastic type were identified in paraffin sections of ovarian adenocarcinoma. The authors suppose, that such structures may be responsible for TBG biosynthesis in ovarian tumours.     

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.     

Comparison of the action of the sera of pregnant women and of trophoblastic beta 1-glycoprotein in a number of immunological tests

The effects of blood sera of pregnant women in the I, II and III trimesters (SPWI, SPWII, SPWIII) were compared with those of purified preparations of trophoblastic beta 1-glycoproteid (TBG) on phytohemagglutinin (PHA) induced proliferative response of lymphocytes of the intact donors and on the production of a factor inhibiting the migration of leucocytes (FIM-L). SPWI and purified TBG preparations in doses corresponding to its level in SPWI were shown to produce similar effects. With the increase of TBG concentration, however, the inhibiting effect of its preparations on the proliferation of lymphocytes increased much more intensively than that of SPWII and SPWIII containing the same dose of this protein. The stimulating effect of TBG on the production of FIM-L increased with the dose as well while SPWII and SPWIII did not exert any effect on the migration of leucocytes. A suggestion is put forward that SPWII and SPWIII contain factors modifying the effect of TBG.     

Effect of specific placental proteins--trophoblastic beta 1-glycoprotein chorionic alpha 1-microglobulin--on the proliferation of lymphocytes and malignant fibroblasts in vitro

A study was made of the action of trophoblastic beta 1-glycoprotein (TBG) and chorionic alpha 1-microglobulin (CAG1) on proliferation of malignant fibroblasts (transplanted L line) and on phytohemagglutinin-stimulated lymphocytes. TBG depressed proliferation of the stimulated lymphocytes and transformed fibroblasts (according to 3H-thymidine incorporation). A dose-response dependence was ascertained. CAG1 did not affect cell division. The inhibitory effect of TBG was seen to be reversed or decreased provided the lymphocyte culture was supplemented with CAG1. The decreased inhibitory effect of TBG in the presence of CAG1 was also noted in the L cell culture. It is likely that in vivo protection of intensely proliferating fetal tissues or tumor from the inhibitors is effected just in this way since placental proteins are synthesized both by embryonic and tumorous cells.     

Comparative study of the immunosuppressive properties of trophoblastic beta 1-glycoprotein and placental alpha 2-microglobulin in vitro

It has been shown that trophoblastic beta 1-glycoprotein (TBG) and placental alpha 2-microglobulin (PAMG-2) in concentrations 60-120 micrograms/ml suppresses both the inductive and proliferative phase of unidirectional mixed lymphocyte reaction in mice, as well as proliferative responses to phytohemagglutinin or pokeweed mitogen. TBG protein was more effective. The proteins were not toxic for lymphocytes.     

Effects of trophoblastic beta 1-glycoprotein (TBG) on the functional activity of different cell lines

The effect of TBG on the functional activity of different cell lines, spontaneous and Con A induced proliferation of PBL was studied. If concentration of TBG is higher than 50 mu kg/ml it suppresses the proliferation in many used cell lines, except choriocarcinoma and cancer of uterus. The reliable increasing of spontaneous proliferation of PBL, Jurkat and K-562 cells may be observed if concentration is more lower (0.5-15 mu kg/ml). However proliferation of other cell lines corresponds to control level, and Con A induced proliferation of PBL is inhibited. The effect was more marked at 48, as compared to 24 hours of cell incubation with TBG.     

The role of branching glycan of human alpha1-acid glycoprotein in enantioselective binding to basic drugs as studied by capillary electrophoresis

The role of the branching glycan structure of human alpha1-acid glycoprotein (AGP) in the interaction with basic drugs was investigated in terms of enantioselectivity in binding ability. AGP was separated by concanavalin A lectin affinity chromatography into two subfractions, the unretained AGP (UR-AGP) which has no biantennary glycan chain and the retained AGP (R-AGP) which possesses biantennary oligosaccharide chain(s). The unbound concentrations of propranolol (PRO) enantiomers and verapamil (VER) enantiomers in UR-AGP solution and R-AGP solution were determined by high-performance frontal analysis combined with capillary electrophoresis. It was found that (S)-PRO is bound to UR-AGP and R-AGP more strongly than (R)-PRO, whereas the reverse applies to VER enantiomers, and that such enantioselectivity is common to these proteins. This suggests that the branching type of glycan chains of AGP does not play significant role in the chiral recognition in binding these basic drugs.     

Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique

A method based on a surface plasmon resonance technique for detection of changes in concentration and glycosylation of proteins in cell culture supernatant is described. The method was used to analyze alpha(1)-acid glycoprotein (AGP) produced by a human hepatoma cell line (HepG2). Cell culture supernatant was injected to a BIACORE 2000 instrument and AGP was captured on the sensor chip by immobilized antibodies. The captured glycoprotein was then analyzed for content of carbohydrate epitopes using three different lectins, Aleuria aurantia lectin (AAL), Sambucus nigra agglutinin (SNA), and Triticum vulgaris agglutinin (wheat germ agglutinin, WGA). The method was used to analyze changes in concentration and glycosylation of AGP produced by HepG2 cells grown with or without three different cytokines, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and transforming growth factor beta-1 (TGF beta(1)). Using the described method it was shown that when HepG2 cells were grown in the presence of IL-6 both AGP concentration and fucosylation increased. When HepG2 cells instead were grown in the presence of TGF beta(1) AGP fucosylation increased whereas AGP concentration decreased.     

Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex

Arabinogalactan-protein (AGP, "beta-lectin") was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect beta-linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected beta-glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Binding of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended throughout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.     

Separation of alpha-acid glycoprotein glycoforms using affinity-based reversed micellar extraction and separation

A new method for the preparation of the glycoforms of bovine alpha(1)-acid glycoprotein (AGP) is described relying on affinity-reversed micellar extraction and separation (ARMES). This method has proven effective in separating structurally similar glycoproteins and separating glycoproteins from nonglycosylated proteins from natural sources. In this method, individual glycoforms complex with the lectin, concanavalin A (ConA) are extracted into an organic-phase reversed micellar solution formed by Aerosol OT (AOT). The purity of three AGP glycoforms isolated was assessed by hydroxyapatite high-performance liquid chromatography (HPLC), gel-permeation chromatography and SDS-PAGE. The glycan structure of the pure glycoforms was analyzed. Oligosaccharide mapping using capillary electrophoresis (CE) and PAGE showed the glycans obtained from each glycoform to be distinctly different. ARMES can be used for the semi-preparative scale resolution of the glycoforms of bovine AGP or other therapeutic glycoproteins.     

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     

Enhanced expression of α1-acid glycoprotein and fucosylation in hepatitis B patients provides an insight into pathogenesis

Altered glycosylation and concentration of α1-acid glycoprotein has been known to be related to the pathogenesis of the hepatic diseases. The present study investigated enhanced fucosylation of AGP in the sera of chronic hepatitis B (HBV-CH) and hepatitis B cirrhosis (HBV-LC) patients by high performance anion exchange chromatography and by ELISA using fucose binding Aleuria aurantia lectin. The concentration of AGP determined by ELISA using monoclonal anti-human AGP (mAb-AGP) showed high level of AGP in HBV-CH and HBV-LC patients. This was further judged by association constant (K _A) measured by surface plasmon resonance analysis. There was no apparent linkage variation of sialic acid among different patient groups when tested with two sialic acid binding lectins viz., Maackia amurensis agglutinin (MAA, NeuAc α2-3-) and Sambucus nigra agglutinin (SNA, NeuAc α2-6-) respectively. There was no change of oligosaccharide branching in HBV-CH in comparison to controls whereas a slight change was observed in HBV-LC using ConA. The above results suggest that the changes in concentration of AGP and fucosylation have a prognostic value of hepatitis diseases and it could be possible to use AGP as diagnostic marker besides clinical examination and routine laboratory investigation.     

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

Detection of human asialo-α1-acid glycoprotein using a heterosandwich immunoassay in conjunction with the light addressable potentiometric sensor

Highly specific detection of human ₁-acid glycoprotein (AGP) and asialo-₁-acid glycoprotein (asialo-AGP) was made possible by use of a sandwich immunoassay. The glycoproteins were sandwiched between biotinylated and fluoresceinated polyclonal rabbit anti-human AGP antibodies. Additionally, asialo-AGP could be distinctly detected, apart from AGP, via the formation of a heterosandwich immunoassay using biotinylated polyclonal rabbit antihuman AGP and the lectin, fluoresceinated ricin toxin. Streptavidin was added to the formed immunocomplexes and the immunocomplexes captured on a biotinylated nitrocellulose membrane. The signal generator, urease conjugate of an anti-fluorescein antibody, was then bound to the complex on membrane. The rate of pH change under microvolume conditions (0.6 µl) was monitored using a silicon chip-based, light addressable potentiometer sensor. Results indicated that AGP and asialo-AGP can be detected to the 2 pg level when two antibodies are used to form the immunocomplex. Asialo-AGP can be detected down to 250 pg when the heterosandwich immunoassay is used; this assay exhibited no response up to 10 ng for native AGP or asialofetuin. Both immunoassays can be used to quantify the level of AGP and asialo-AGP in solution. Although the assay presented is very specific for AGP, asialo-AGP and terminal galactose, it is readily adaptable for the detection of any glycoprotein and terminal carbohydrate (or branched structure) by use of a protein-specific antibody and various lectins.     

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.     

Altered glycosylation and expression of plasma alpha-1-acid glycoprotein and haptoglobin in rheumatoid arthritis

Altered glycosylation patterns in plasma proteins are found to be associated with the pathogenesis of various malignancies and autoimmune disorders. Our previous studies demonstrated the occurrence of some differentially glycosylated plasma proteins in rheumatoid arthritis (RA) patients. The current study was conducted to evaluate the alterations in expression and glycosylation of major acute phase proteins from wheat germ agglutinin enriched RA patients' plasma. Immunoblotting studies revealed a significant enhancement in the plasma levels of alpha-1 acid glycoprotein (AGP) and haptoglobin (Hp) in RA patients with respect to healthy controls. Monosaccharide analysis by high performance anion exchange-chromatography with pulse amperometric detection showed significant variations in the relative percentage of galactose, glucosamine and mannose in AGP and of mannose in Hp in RA patients. Altered patterns of mannosylation in AGP and Hp were also established by enzyme linked immunosorbent assay and Western blotting using Concanavalin-A lectin. These results could give information for understanding the disease pathogenesis and may provide an insight into the development and progression of the disease.     

Preparative affinity electrophoresis of different glycoforms of serum glycoproteins: Application for the study of inflammation-induced expression of sialyl-Lewisx groups onα 1-acid glycoprotein (orosomucoid)

During acute inflammation, human ₁-acid glycoprotein (AGP) is subject to marked changes in branching of its glycans, its degree of fucosylation and the expression of sialyl-Lewis^x)(SLe^x) groups. To be able to study these changes in glycosylation in more detail, a procedure was developed to isolate the different glycoforms of AGP in milligram amounts by preparative affinity electrophoresis (AE) with a free lectin as fractionating agent. The method was applied to isolate differently fucosylated forms of AGP with the fucose-specific lectinAleuria aurantia (AAL). AGP was separated into one non-reactive (AO) and four reactive (A1-A4) fractions. It was found that, in particular, the highly fucosylated fractions A3 and A4 contained the inflammation-induced SLe^x groups of AGP. Analysis by crossed affinoimmunoelectrophoresis (CAIE) with concanavalin A (Con A) of these different glycoforms of AGP showed that the presence of tri-and/or tetraantennary glycans, instead of diantennary glycans, was associated with a higher degree of fucosylation. Identical results were obtained by subjecting Con A-fractionated forms of AGP to CAIE with AAL as the affinocomponent. It is expected that this method of preparative AE can generally be applied to other glycoproteins, which can be separated in different glycoforms by CAIE using lectins.Part of this work was published in abstract form,Glycoconjugate J 1993;10; 317.