Crossed Affinoimmunoelectrophoresis Research Articles

Abstract A27: Comprehensive analysis of glycans on α1-acid glycoprotein from various cancer patients by MALDI-TOF-MS for assigning a prognostic predictor

Abstract α1-Acid glycoprotein (AGP) is well-known as an acute-phase protein, a major plasma glycoprotein with highly glycosylated, branched N-linked glycans and one of the markers for inflammation. In our previous study (Cancer 101:2825–2836, 2004), with the aid of crossed affinoimmunoelectrophoresis (CAIE), glycoforms of plasma AGP from various cancer patients could be easily determined based on the degrees of branching and extent of fucosylation characterized by reactivities with Con A lectin and Aleuria aurantia lectin, respectively. Results clearly indicated that patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated tri- and tetraantennary glycans for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms with no such changes were expected to have a good prognosis irrespective of their clinical stages. In this study, we purified AGP from plasma samples of the same patients and then analysed detailed structures of their glycans involving a new tumor marker using a high throughput glycosylation kit (BlotGlyco, Sumitomo Bakelite, Tokyo, Japan) combined with MALDI-TOF-MS analysis. More than two hundreds of glycan structures were assigned and relative abundance of each glycan was calculated. Follow-up studies of the relative abundance of glycans indicated that the abundance of fucosylated tri- and tetraantennary glycans in patients with a poor prognosis was quite high, and most of which were estimated to form sialyl-LeX structures (NeuAcα2,3Galβ1,4[Fucα1,3]GlcNAcβ) with the non-reducing end sialyl residues. Whereas, in those with a good prognosis, such an abundance was found to be as low as in healthy controls. These results indicated that no distinct difference was observed from our previous CAIE data and that more precise glycan structures instead of glycoforms could be easily assigned by the present method, providing an aberrant marker for predicting the fate of patients after surgery. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A27.

α1‐Acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis

Serum alpha1-acid glycoprotein (AGP), an acute-phase protein secreted by the liver, carries alpha(1,3)-fucosylated structures on its 5 highly branched, N-linked sugar chains. Serum AGP levels in patients with various types of malignancies (n=214 patients) were measured using an enzyme-linked immunosorbent assay with anti-AGP antibody. To investigate glycoforms that differed in their degree of branching and extent of fucosylation, serum AGP samples were analyzed by crossed affinoimmunoelectrophoresis (CAIE) with concanavalin A, and Aleuria aurantia lectin (AAL), and anti-AGP antibody. A significant difference (P <0.001) in serum AGP levels was observed in preoperative patients compared with levels in the healthy control group, but the levels in individual patients did not reflect their clinical status. Conversely, it was found not only that the patterns of AGP glycoforms differed widely in the patient group compared with the healthy control group, but they also changed depending on each patient's clinical status. Furthermore, AGP glycoforms seemed to be appropriate markers of disease progression and prognosis according to follow-up studies of 45 patients during prolonged preoperative and postoperative periods. Patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated triantennary and tetraantennary sugar chains for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms without such changes were expected to have a good prognosis.

Highly enhanced fucosylation of serum glycoproteins in patients with hepatocellular carcinoma.

We recently reported that the measurement of Lens culinaris agglutinin-reactive species of alpha-fetoprotein (AFP), alpha-1-antitrypsin (AAT) and transferrin (TF) is useful for the diagnosis of hepatocellular carcinoma (HCC) and that the molecular basis for this reactivity is fucosylation at the innermost N-acetylglucosamine residue of a biantennary sugar chain. However, the precise relationship of the fucosylation of AFP, AAT and TF in patients with HCC and liver cirrhosis is not fully understood. The aim of this study is to delineate the relationship of the fucosylation between these three glycoproteins in HCC. Three hundred and thirty-four patients with HCC were referred to our university hospital from 1987 to 1997. An increase in serum AFP (> 20 ng/mL) was observed in 233 (69.8%) patients with HCC. From these 233 patients with AFP-producing HCC, 60 serum samples were randomly selected and used in the present study. As a reference, samples from 60 patients with liver cirrhosis, in which 30 had increased AFP, were used. Lens culinaris agglutinin (LCA)-reactive species were determined by crossed immunoaffinoelectrophoresis (CIAE). The contents of the fucosylated biantennary chain of purified AAT and TF samples were determined as pyridylamino derivatives of each oligosaccharide with high-performance liquid chromatography (HPLC). There was a highly significant correlation between LCA-reactive species by CIAE and pyridyl-amino-fucosylated biantennary sugar chain by HPLC in both AAT and TF. Lens culinaris agglutinin-reactive species of AFP, AAT and TF in HCC were significantly higher than those in liver cirrhosis. A highly statistically significant positive correlation of fucosylated glycans was observed between AAT and TF in both HCC and liver cirrhosis, but not between AFP and AAT or between AFP and TF. Accordingly, the present results indicate that highly enhanced fucosylation of serum glycoproteins was found in HCC compared with liver cirrhosis and that the combination of measurements of fucosylated AFP with AAT or TF were useful for the diagnosis of HCC.

P-22 Nifedipine steady-state pharmacokinetics in prophylactic treatment of mood disorders

Total serum α1-acid glycoprotein (AGP) concentration and concanavalin A-dependent microheterogeneity were studied in 31 healthy elderly subjects (18 men, 13 women, 71 to 76 yr old).Crossed affino-immunoelectrophoresis (CAIE) revealed three microheterogeneity variants of AGP: non-reactive, weakly reactive and strongly reactive with ConA. Two patterns were found in both elderly men and women, i.e. a normal pattern and one with an increase in the non-reactive form.Mean serum AGP levels in the elderly subjects with slighly higher than in a reference group of younger subjects. The Con A non-reactive form of AGP was increased in 42% of the elderly population.An increase in the non-reactive form of AGP in CAIE should be considered as general expression of chronic inflammation which is of no clinical relevance.

Comparative study of the asparagine-linked oligosaccharide structures of normal and acute-phase rat plasma thiostatin

Rat plasma thiostatin is a 68 kDa glycoprotein with kinin donor and cysteine proteinase inhibitor properties. Thiostatin is an acute-phase plasma protein (APPP) with dramatically elevated plasma levels in response to inflammatory stimuli. APPPs have been shown to possess altered glycan structures in inflammation. This study compares the carbohydrate structure of normal thiostatin with that expressed during the acute-phase response. Thiostatin from both normal and acute-phase plasma was purified by carboxymethyl-papain Sepharose 4B affinity chromatography. Sugar composition analysis by gas chromatography and the Warren method yielded similar mean values for both proteins on a mole sugar per mole protein basis (normal/acute phase): fucose, 2.4/1.7; mannose, 7.5/8.0; galactose, 11.2/10.6; and sialic acid, 14.2/13.0. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot identified a homogeneous 68–70 kDa molecular species for normal and acute-phase thiostatin. Inter-sugar linkage analysis was carried out for permethylated oligosaccharides released by hydrazinolysis. Gas chromatography yielded the following partially methylated alditol acetates relative to 1.0 mole of 1,3,6-tri-O-linked mannose (mean normal/mean acute phase): galactose: 1,3-di-O-, 1.44/1.01; 1,6-di-O-, 1.02/0.68; mannose: 1,2-di-O-, 1.64/1.42; 1,2,4-tri-O-, 0.24/0.13; 1,3,6-tri-O-, 1.0/1.0; 2-deoxy-2-N-methylacetamidoglucose: 1,4-di-O-, 1.42/1.12. These analytical studies indicated that corresponding carbohydrate structures are present in normal and acute-phase thiostatin. Crossed affinoimmunoelectrophoresis (CAIE) further confirmed the structural similarity between the glycan moieties.

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 α1-acid glycoprotein (AGP) is subject to marked changes in branching of its glycans, its degree of fucosylation and the expression of sialyl-Lewisx)(SLex) 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 SLex 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.

Glycan microheterogeneity of α1-antitrypsin in serum and meconium from normal and cystic fibrosis patients by crossed immunoaffinoelectrophoresis with different lectins (Con A, LCA, WGA)

In order to test whether abnormalities of glycosylation occur in cystic fibrosis (CF), the glycan microheterogeneity of α 1-antitrypsin ( α 1-AT) was studied in serum and meconium from normal individuals and patients with cystic fibrosis, by crossed immunoaffinoelectrophoresis (CIAE) using free Concanavalin A (Con A), Lens culinaris lectin (LCA) and wheat germ agglutinin (WGA). Thre3 main results emerged from this study: (1) modification of glycosylation in serum α 1-AT from patients with cystic fibrosis were only significant with free Con A and WGA; this probably results from a reduced synthesis of the bi-antennary side-chains or by their increased catabolism. (2) Differences in isoforms found in α 1-AT from normal individuals and patients with CF using free Con A, LCA, were more pronounced in the meconium than in the serum; this may provide a useful test in diagnosis of cystic fibrosis. (3) There was parallelism between the behaviour of α 1-AT in serum and meconium from patients with CF using LCA, Con A; this may be explained by different types of levels of disfunction affecting a glycosylation mechanism.

Concanavalin A crossed affinoimmunoelectrophoretic analysis of the major pig serum proteins during fetal development.

The interaction between concanavalin A (Con A) and alpha-fetoprotein (AFP), transferrin (TF), fetuin, alpha 1-antitrypsin (AT) and alpha 1-acid glycoprotein (AGP) has been analyzed by crossed affinoimmunoelectrophoresis (CAIE) in fetal extracts or sera, from 26-day-old porcine fetuses to birth, and in adult pigs. Most of the TF and AFP (100 and 85-90%, respectively) reacted with Con A during the entire developmental period. AGP showed both two reactive and one nonreactive Con A isoforms, whose proportions change greatly during development. In younger fetuses 100% of the protein was Con A-nonreactive. This variant represented 64% in 50-day-old fetuses, 80% in newborn pigs and 20% in adult sera. Fetuin and AT showed a maximum of three Con A-reactive microforms and one Con A-nonreactive microform, which was always a minor form. These microforms were detected mainly in young fetuses. Although the proportion of Con A-reactive variants of fetuin and AT changes during fetal development, the predominant microform was always that with intermediate affinity against Con A. The same microform was also predominant in adult AT, whereas the more reactive microform in respect to Con A predominates in adult fetuin.

Changes in α 1-acid glycoprotein serum concentrations and glycoforms in the developing human fetus

α 1-Acid glycoprotein concentrations and reactivity to concanavalin A were measured in maternal and fetal serum and amniotic fluid obtained from 24 women undergoing diagnostic cordocentesis at 20 to 33 wk gestation and in 30 additional fetal sera (19 to 34 weeks gestation). Maternal α 1-acid glycoprotein serum levels were five to ten times higher than fetal and amniotic levels. Fetal α 1-acid glycoprotein levels were found to increase with advancing gestational age. Using crossed immunoaffino electrophoresis with concanavalin A, α 1-acid glycoprotein patterns were identical in maternal serum and amniotic fluid but totally different in fetal serum. The fetal concanavalin A pattern changed progressively during fetal life towards that of the newborn. These data confirm earlier assumptions of fetal synthesis of α 1-acid glycoprotein and provide normal reference values for α 1-acid glycoprotein in fetal serum. In addition, the specific fetal concanavalin A pattern indicates that the α 1-acid glycoprotein glycosylation process during fetal life differs from that in post-natal life.

Removal of the artifactual peak associated with concanavalin a crossed affinoimmunoelectrophoresis of human serum transferrin

During the first dimension of crossed affinoimmuno-electrophoresis (CAIE) with the lectin concanavalin A (Con A), an immobile glycoprotein—Con A affinoprecipitate may form near the application well and, subsequently, produce an artifactual peak in the second-dimension gel. In this study, we examined the effects of sample glycoprotein concentration and gel Con A concentration on the magnitude of the transferrin artifactual peak present in the analysis of human serum. In addition, we examined the potential for reducing or eliminating the artifact by including a competitive inhibitor of glycoprotein-lectin interaction, α-methylmannoside (αMM), in the application well. We demonstrate that the artifact can be eliminated through an appropriate choice of glycoprotein, Con A, and αMM concentrations. This approach should be applicable for diagnosing and eliminating the artifact in the Con A CAIE analyses of other glycoproteins.

Microheterogeneity of α1-acid glycoprotein in healthy elderly subjects: Patterns obtained by crossed affino-immunoelectrophoresis

Total serum α1-acid glycoprotein (AGP) concentration and concanavalin A-dependent microheterogeneity were studied in 31 healthy elderly subjects (18 men, 13 women, 71 to 76 yr old). Crossed affino-immunoelectrophoresis (CAIE) revealed three microheterogeneity variants of AGP: non-reactive, weakly reactive and strongly reactive with ConA. Two patterns were found in both elderly men and women, i.e. a normal pattern and one with an increase in the non-reactive form. Mean serum AGP levels in the elderly subjects with slighly higher than in a reference group of younger subjects. The Con A non-reactive form of AGP was increased in 42% of the elderly population. An increase in the non-reactive form of AGP in CAIE should be considered as general expression of chronic inflammation which is of no clinical relevance.

Role of sialic acid residues in crossed immuno-affinoelectrophoresis of α 1-proteinase inhibitor

We have investigated the importance of the degree of sialylation when an acute phase glycoprotein, α 1-proteinase inhibitor (α 1-Pi), was analysed both by affinity chromatography on concanavalin A (Con A)-Sepharose and by crossed immuno-affinoelectrophoresis (CIAE) using Con A in the first dimension. Human α 1-Pi was isolated by immunosorption chromatography and then more or less desialylated. On Con A-Sepharose chromatography no significant difference was observed in the percentage of the two fractions (retained or not retained) whatever the degree of desialylation. In contrast by CIAE this degree was largely involved in the separation of the different isoforms obtained in the first dimension.

A Lectin-Based Monoclonal Enzyme Immunoassay to Distinguish Fucosylated and Non-Fucosylated α-Fetoprotein Molecular Variants

Purified alpha-fetoprotein (AFP), fucosylated AFP mixtures, and 40 sera from patients with AFP-producing hepatocellular carcinoma were analysed by monoclonal enzyme immunoassay (EIA) to distinguish fucosylated and nonfucosylated AFP molecular variants. FUC-AFP-25 was discriminated from FUC-AFP-75 by the EIA using monoclonal antibody 18H4 in the range of total AFP concentrations from 100 to 800 ng/mL. In addition, sera from 40 patients with hepatocellular carcinoma, with AFP concentrations from 100 to 1270 ng/mL and with fucosylated AFP from 0 to 100% by conventional cross immuno-affinoelectrophoresis, were also analysed by the present EIA. A statistically significant correlation was obtained between the data from the present EIA and from the conventional crossed immuno-affinoelectrophoresis in the range of fucosylated AFP more than 20% and the serum concentration of AFP more than 100 ng/mL. These results indicate that the present EIA is useful for clinical detection of hepatocellular carcinoma during the follow-up of patients with chronic liver diseases.

Con A affinity of rat α1-acid glycoprotein (rAGP): changes during inflammation, dexamethasone or phenobarbital treatment as detected by crossed affino immunoelectrophoresis (CAIE) are not only a reflection of biantennary glycan content

Using crossed affino immunoelectrophoresis (CAIE), the secretion of the Con A most reactive form (CAIE-3) of rat α1-acid glycoprotein (rAGP) has been shown to be increased in sera of Wistar and Sprague Dawley rats during inflammation and treatment with dexamethasone or phenobarbital. Primary hepatocyte cultures prepared from experimentally treated Wistar rats reflect these in vivo findings, since rAGP as present in corresponding secretion media shows similar changes in Con A reactivity. In this study, the relation of this increase towards the amount of biantennary glycans was investigated for both differently treated rat strains. For this purpose, metabolically labelled rAGP, secreted by isolated hepatocytes under the various conditions, was separated on Con A-Sepharose into four fractions. For each fraction of rAGP its behaviour in CAIE was established, revealing a positive correlation for Con A reactivity between the two methods. However, the enormous increase in Con A reactivity of rAGP in CAIE during inflammation and other conditions (increase in CAIE-3), could not be shown using Con A-Sepharose chromatography. Glycopeptides of each fraction were prepared and the amount of biantennary glycans was assessed. Contrary to expectations, an increase, of the total amount of biantennary glycans of rAGP, secreted during conditions associated with an increase in CAIE-3 was not found. The independency of the results with regard to rat strain and procedures used underlined the generality of these findings. Consequently, not only the biantennary glycan content is responsible for the separation of rAGP in CAIE. The importance of other differences in glycosylation, e.g. sialylation, for the increase of rAGP CAIE-3 during various experimental conditions is discussed.

Isolation of line 10 hepatoma-cell membrane by the water extraction method and immunochemical analysis.

Water extraction method was applied to isolate the cell membrane from line 10 hepatoma cells and normal liver cells in strain 2 guinea pig. The materials isolated by this method were further analyzed by different immunochemical techniques including SDS-PAGE, crossed immunoelectrophoresis and crossed affino immunoelectrophoresis to demonstrate the major components and their antigenicities. Five major glycoproteins of apparent molecular weights of 44, 46, 62, 64, and 68 kDa were prominent in line 10 tumor cell materials, whereas one band of molecular weight of 82 kDa was prominent in the materials from normal liver cells. Also four minor components from line 10 tumor cells were found to be glycoprotein in nature.

Modifications of Concanavalin A patterns of α1-acid glycoprotein and α2-HS glycoprotein in alcoholic liver disease

In order to test whether abnormalities in hepatocytes affect the glycoprotein carbohydrate moiety, crossed immunoaffinoelectrophoresis (CIAE) with Concanavalin A (Con A) was used to study serum alpha 1-acid glycoprotein (α1-AGP) and alpha 2-HS glycoprotein (α2-HS) obtained from alcoholic patients with biopsy-proven liver disease. Cirrhotic patients, placed in groups C1, C2 or C3, according to Pugh's classification, were compared to healthy donors (N) and to steatosic non-cirrhotic patients (S). Con A CIAE patterns revealed in group N three subpopulations for α2-HS and four for α1-AGP. Two main results emerged from this study: (1) in the alcoholic groups, the proportions of Con A-unreactive subpopulations of both glycoproteins increased. Moreover, group N could be separated from group S and group S from all the cirrhotic groups. (2) There was a good correlation between the relative amounts in Con A-unreactive subpopulations of α1-AGP and α2-HS. The increases observed in Con A-unreactive subpopulations are probably a general phenomenom related to alterations in glycosylation processing during liver cell damage.

Studies on Lectin Affinities of Chicken Alpha-Fetoprotein During Ontogeny

Alpha-fetoprotein in the sera of chicken embryos (ch-AFP) of the 7th, 10th, 13th, 16th, and 19th day of incubation and of one-day-old chickens were examined in crossed-immuno-affino-electrophoresis (CIAE) using different lectins [concanavalin A (Con A), lentil, wheat germ lectin, and soybean lectin] as ligands. Differences in lectin affinities, suggesting different degrees or forms of glycosylation of ch-AFP during ontogeny, were demonstrated by this method. Sera of chicken embryos of the same stages were also subjected to Con A affinity chromatography and the elution patterns were evaluated qualitatively in CIAE and quantitatively in rocket Immunoelectro-phoresis. These experiments revealed a major Con A-nonreactive and a minor Con A-reactive fraction at the 7th day of incubation. In contrast, a minor Con A nonreactive fraction and a major Con A-reactive fraction were observed at later stages of embryonic life. Extracts from embryos of the 8th and 15th day of incubation did not exhibit heterogeneity when subjected to CIAE. The biological significance of these observations, as well as the biological role of AFP in general, remains to be elucidated. Examination of ch-AFP glycosylation patterns might be included into the spectrum of anlayses when certain early teratological processes in the chicken embryo are to be monitored. In addition, these molecular changes have to be considered when in vitro or in vivo experiments on the biological, especially immunological, role of ch-AFP are envisaged.

Distinct molecular species of human alpha-fetoprotein due to differential affinities to lectins.

Resolution of human alpha-fetoprotein (AFP) into four distinct molecular species was demonstrated by a combination of two affinity chromatographies with crossed-immuno-affino-electrophoresis (CIAE) using concanavalin A (Con A) and Lens culinaris hemagglutinin (LcH)-A and LcH-B as affinity media. Of the four AFPs, AFP1 had no affinity for Con A, LcH-A, or LcH-B; AFP2 showed a high affinity for Con A, a low affinity for LcH-A, and an intermediate affinity for LcH-B (or a low affinity, depending on the lot of LcH-B preparations used); AFP3 revealed strong affinities for all of the three lectins; and AFP4, a trace component of hepatoma AFP in the present study, showed no interaction with Con A, but a definite interaction with LcH-A or LcH-B. These results were based on the determination of dissociation constants (Kd) of AFP-lectin complex by CIAE on isolated preparations of the three major hepatoma AFPs. These AFPs had identical electrophoretic mobilities of 0.86-0.87 (relative to human albumin) in the absence of lectins. The calculated mobilities of AFP2 and AFP3 were both reduced to 0.50-0.58 by saturation with lectins, but these two AFPs were clearly separated by 1 mg/ml LcH-A or LcH-B because of their large differences in Kd.

Placental affinity for pregnancy‐associated murine protein‐1 examined by crossed affino immuno‐electrophoresis

AbstractUsing crossed affino immunoelectrophoresis and tissue extracts added to the first‐dimension gels the affinity between pregnancy‐associated murine protein‐1 (PAMP‐1) and various tissues was examined. Substances binding to PAMP‐1 in the liver and in the placenta were found to affect the electrophoretic migration velocity of PAMP‐1 when added to first‐dimension gels.