G. Durand

Alterations in relative proportions of microheterogenous forms of human α1-acid glycoprotein in liver disease

To determine whether liver damage correlates with typical changes in alpha 1-acid glycoprotein (alpha 1-AGP) carbohydrate branching, we selected patients with liver disease determined by histological liver findings. The severity of their illness was assessed by a clinical classification depending on the presence singly or together of four clinical complications (jaundice, ascites, hepatic encephalopathy and weight loss). An alpha 1-AGP crossed immunoelectrophoresis with Concanavalin A, an easy-to-perform method, revealed 3 or 4 subpopulations, the areas of which were calculated. A ratio R was determined as the most anodic peak area relative to the other ones. In our experimental conditions a ratio R value exceeding 1 correlated with the presence of one or more clinical complications. These results, evidencing fluctuations in the proportion of the carbohydrate variants of alpha 1-AGP, lead us to propose such a ratio as an index for grading liver damage. The sensitivity of this test was 86% and its specificity was 83%.

Alterations in the carbohydrate moiety of alpha-1-acid glycoprotein purified from human cirrhotic ascitic fluid.

The carbohydrate analysis of alpha 1-AGPc purified from cirrhotic ascitic fluid was performed by immunoaffinity chromatography. It showed a large increase in the fucosyl molar ratio and sugar content (47%). The molar ratio of the oligosaccharides which were released by hydrazinolysis and fractionated by high-performance liquid chromatography confirms the marked increase in fucosyl residues in each fraction. A shift towards fractions with a high degree of branching was also observed. Moreover, the studies of sugar molar ratios and methylation of the tetrasialylated fraction indicated the simultaneous presence of sialyl and fucosyl residues on one of the outer branches.

Microheterogeneity of the carbohydrate moiety of human alpha 1-acid glycoprotein in two benign liver diseases: Alcoholic cirrhosis and acute hepatitis

To determine whether alterations of the carbohydrate moiety of human alpha 1-acid glycoprotein constitute a marker of hepatic damage we studied purified alpha 1-acid glycoprotein from healthy individuals and two groups of patients with benign liver diseases: alcoholic cirrhosis and acute viral hepatitis. The results indicate: (1) increased concanavalin A-non reactive forms in cirrhosis and hepatitis, (2) a markedly increased proportion of fucosyl residues in all cirrhotic and some hepatitis patients. Although hyperfucosylation is generally considered to be a tumor marker, the observation here in the two benign liver diseases indicates that an increased fucosyl content should be considered as a more general expression of pathological glycoconjugate metabolism.

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

Abstract 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 Pughs 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.

STAT5B-mediated Growth Hormone Signaling Is Organized by Highly Dynamic Microtubules in Hepatic Cells

In the last decade, the notion that microtubules are critical to the spatial organization of signal transduction and contribute to the transmission of signals to downstream targets has been proposed. Because the STAT5B transduction and transcription factor is the major STAT protein activated by growth hormone stimulation in hepatocytes and is a crossroads between many signaling pathways, we studied the involvement of microtubules in STAT5B-mediated growth hormone signaling pathway in the highly differentiated and polarized WIF-B hepatic cell line. We showed that depolymerization of the microtubule network impaired STAT5B translocation to the nucleus upon growth hormone treatment. A significant amount of STAT5B binds to microtubules, while STAT5A and STAT3 are exclusively compartmentalized in the cytosol. Moreover, taxol-induced stabilization of microtubules released STAT5B from its binding, and we show that STAT5B binds specifically to the highly dynamic microtubules and is absent of the stable microtubule subpopulation. The specific involvement of dynamic microtubule subpopulation in growth hormone signaling pathway was confirmed by the inhibition of growth hormone-induced STAT5B nuclear translocation after stabilization of microtubules or specific disruption of highly dynamic microtubules. Upon growth hormone treatment, MT-bound STAT5B was rapidly released from microtubules by a dynein-dependent transport to the nucleus. Altogether, our findings indicate that the labile microtubule subpopulation specifically and dynamically organizes STAT5B-mediated growth hormone signaling in hepatic cells.

400-MHz 1H-NMR spectroscopy of fucosylated tetrasialyl oligosaccharides isolated from normal and cirrhotic α1-acid glycoprotein

The comparative study of fucosylated tetrasialyl‐oligosaccharides isolated from normal and cirrhotic α1‐AGP was performed using permethylation and 400‐MHz 1H‐NMR spectroscopy. These results clearly show the tetraantennary structure of these two oligosaccharides with hyperfucosylation for the tetrasialylated fraction from cirrhotic α1‐AGP. In the latter oligosaccharide the simultaneous presence on two antennae (7 and 7′) of the sialosyl Lewis X determinant NeuAc‐(α2–3) Gal(β1–4) [Fuc(αl–3)l GlcNAc has been observed. Moreover the 5 and 5′ antennae were α2–6 sialylated but without fucose.

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 alpha 1-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.

One-step purification of rat plasma α1-acid glycoprotein by antibody affinity chromatography: Application to normal and inflamed rat sera

Abstract Most purification procedures used previously to isolate α 1 -acid glycoprotein (AGP) from plasma can lead to some alterations in its carbohydrate moiety. An immunoaffinity chromatographic method is proposed for purifying in one step rat plasma AGP without any detectable modification of its glycan moiety. Crossed immunoaffinoelectrophoresis with concanavalin A before and after purification showed identical patterns, suggesting no glycan selection during the purification. In the same way no desialylation occurred during the purification step. This immunoaffinity chromatographic procedure provided evidence of a decreased level of fucosyl residues in turpentine oil rat plasma AGP compared with normal rat plasma AGP.

Electroimmunodiffusion de Laurell. Intérêt et limite des diverses techniques à propos de l'étude d'un éluat de chromatographie d'immunoaffinité

Abstract Monodimensional and bidimensional electroimmunodiffusion techniques were applied to the study of immunochromatography eluates. They were obtained with human sera as samples, and rabbit anti human sera α 1 AGP antibodies coupled to CNBr Sepharose 4B packed in a chromatography column. By monodimensional electroimmunodiffusion α 1 AGP could be quantitated. By tandem bidimensional immunoelectrophoresis the eluate purity could be shown, as well as its identity to α 1 AGP. Besides, this last technique was used to the critical study of elution conditions, and to put forward the influence of some parameters (such as pH ionic strength) on α 1 AGP denaturation and its transformation into a protein which is still antigenic but has lost a part of its electrophoretic mobility at pH 8.6. This phenomenon could explain the α 1 AGP loss observed after column elution. The precautions in the interpretation, the interest and the sensitiveness of the electroimmunodiffusion techniques are exposed.