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.     

Inflammation-induced expression of sialyl Lewisx is not restricted to α1-acid glycoprotein but also occurs to a lesser extent on α1-antichymotrypsin and haptoglobin

Acute and chronic inflammation-induced expression of sialyl Lewisx has already been shown to occur on α1-acid glycoprotein. We now demonstrate that this phenomenon is not restricted to α1-acid glycoprotein but also occurs on two other acute-phase proteins. ie on α-antichymotrypsin and on haptoglobin. The level of expression of sialyl Lewisx on these proteins was lower than on α1-acid glycoprotein, in all likelihood because α1-acid glycoprotein is the only acute-phase protein containing tetraantennary glycans. No expression of sialyl Lewisx was detectable on α1-protease inhibitor, a protein with a high diantennary glycan content. Non-sialylated Lewisx was not detectable on these major acute-phase proteins in any of the conditions studied. This indicates that the majority of the α3-linked fucose residues are present as sialyl Lewisx on α1-acid glycoprotein, α1-antichymotrypsin and haptoglobin. The absolute contribution to the total phenotype in plasma of protein containing this determinant in a multivalent form was highest for α1-acid glycoprotein. This leads us to propose that α1-acid glycoprotein is, among the acute-phase proteins studied, the one with the highest potential for interference with the extravasation of leukocytes by binding to the selectins.     

Glycosylation of site-specific glycans of α1-acid glycoprotein and alterations in acute and chronic inflammation

Backgroundα₁-Acid glycoprotein (AGP), an acute phase reactant, is extensively glycosylated at five Asn-linked glycosylation sites. In a number of pathophysiological states, including inflammation, rheumatoid arthritis, and cancer, alterations of Asn-linked glycans (N-glycans) have been reported. We investigated alteration of N-glycans at each of glycosylation sites of AGP in the sera of patients with acute and chronic inflammation.MethodsAGP purified from sera was digested with Glu-C and the liberated glycopeptides were isolated by reverse phase HPLC. N-glycans released with peptide N-glycosidase F and followed by neuraminidase treatment were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.ResultsSite-specific differences in branching structures were observed among N-glycosylation sites 1, 3, 4 and 5. Within the sera of patients with acute inflammation, increases in bi-antennary and decreases in tri- and tetra-antennary structures were observed, as well as increases in α1,3-fucosylation, at most glycosylation sites. In the sera of patients with chronic inflammation, increased rates of tri-antennary α1,3-fucosylation at sites 3 and 4 and tetra-antennary α1,3-fucosylation at sites 3, 4 and 5 were detected. Although there were no significant differences between acute and chronic sera in site directed branching structures, significant differences of α1,3-fucosylation were detected in tri-antennary at sites 2, 4 and 5 and in tetra-antennary at sites 3 and 4.ConclusionLittle variation in the N-glycan composition of the glycosylation sites of AGP was observed among healthy individuals, while the sera of patients with acute inflammation demonstrated increased numbers of bi-antennary and α1,3-fucosylated N-glycan structures at each glycosylation site.     

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.     

Glycosylation of α1-acid glycoprotein in inflammatory disease: analysis by high-pH anion-exchange chromatography and concanavalin A crossed affinity immunoelectrophoresis

High-pH anion-exchange chromatography with pulsed amperometric detection is a highly sensitive technique that can be used for detecting changes in sialylation and fucosylation, as well as different branching patterns of N-linked oligosaccharides in glycoproteins. We examined the N-glycans of α1-acid glycoprotein obtained from twelve patients with various inflammatory conditions with this technique, as well as traditional concanavalin A crossed affinity immunoelectrophoresis. We found the chromatographic profiles of N-glycans in all patients with rheumatoid arthritis to be very similar, but significantly different from normal controls. N-glycans from patients with ulcerative colitis also showed specific alterations in their chromatographic profiles. However, some heterogeneity was found between these patients, perhaps reflecting changes in glycosylation secondary to certain states of the disease, or to medical treatment. We conclude that this technique is useful for detailed mapping of glycosylation changes in α1-acid glycoprotein in clinical samples, and that it may be used to further increase our knowledge about glycosylation changes in response to inflammatory disease. Abbreviations: AC, acute cholangitis; AGP, α1-acid glycoprotein; CAIE, crossed affinity immunoelectrophoresis; Con A, concanavalin A; HPAEC-PAD, high-pH anion-exchange chromatography with pulsed amperometric detection; IEC, ion exchange chromatography; RA, rheumatoid arthritis; SLex, sialyl Lex; UC, ulcerative colitis This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

Inhibitory effect of SJSZ glycoprotein (38?kDa) on expression of heat shock protein 27 and 70 in chromium (VI)-treated hepatocytes

Chromium (VI) is as an extremely toxic chemical substance, and is also an internationally recognized human carcinogen. The principal objective of this study was to determine whether or not Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents hepatocarcinogenesis in chromium-treated BNL CL.2 cells and ICR mice. Firstly, it was evaluated that SJSZ glycoprotein has strong antioxidant character and scavenges radicals. In an effort to assess the chemopreventive effects of SJSZ glycoprotein on hepatocarcinogenesis, ICR mice were intraperitoneally injected with chromium (10?mg/kg, BW) for 8?weeks. After sacrifice, we evaluated indicators of liver tissue damage [the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS)], antioxidative enzymes [activities of superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (GPx)], and initiating hepatocarcinogenic indicator [heat shock protein (HSP) 27 and 70] and protein kinase C (PKC), p38 MAPK and PCNA via biochemical methods and immunoblot analysis. The results obtained from this study demonstrated that the SJSZ glycoprotein (50?μg/ml) inhibited the production of intracellular ROS in BNL CL.2 cells. In addition, the SJSZ glycoprotein (10?mg/kg, BW) attenuated the levels of LDH, ALT, and TBARS, whereas it increased antioxidative enzymes in mouse serum. SJSZ glycoprotein attenuated the activity of HSP27, HSP70, PKC, MAPKs, and PCNA in BNL CL.2 cells and liver tissue. Taken together, our results indicate that SJSZ glycoprotein might be have a potent preventive effect against hepatocarcinogenesis induced by oxidative stress.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone α-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common α-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was α-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and α-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the buryrate-mediated induction of α-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6–24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-α. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2′,5′-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of α-subunit mRNA. There was a good correlation between α-subunit accumulation and corresponding levels of α-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Conformation and cooperative order-disorder transition in aqueous solutions of β-1,3-d-glucan with different degree of branching varied by the Smith degradation

β-1,3-d -glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded β-1,3-d -glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide–hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.     

Involvement of CTP:phosphocholine cytidylyltransferase-β2 in axonal phosphatidylcholine synthesis and branching of neurons

In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTβ2. In mice, CTβ2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTβ2 plays an important role in the nervous system. We, therefore, investigated the importance of CTβ2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTβ2, but not CTα, mRNA and protein. The brains of mice lacking CTβ2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTβ2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTβ2 deficiency in distal axons reduced the incorporation of [(3)H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTβ2 is the major CT isoform involved in PC synthesis in axons. Axons of CTβ2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTβ2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.     

Changes in expression of two endogenous β-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of -galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.     

Changes in adhesive and migratory characteristics of hepatocellular carcinoma (HCC) cells induced by expression of α3β1 integrin

The invasive and metastatic potentials of hepatocellular carcinoma are positively correlated with the expression level of α3β1 integrin, a high-affinity adhesion receptor for laminin isoforms including laminin-5. In this study, we investigated changes in the adhesive and invasive behaviors of human HCC HepG2 cells after transfection with cDNA for α3 integrin in order to elucidate the direct involvement of this integrin in these cellular processes. We introduced cDNA for splice variants of α3 integrin (α3A and α3B) into the cells, and selected two transfectant clones (HepG2-3A and HepG2-3B), which express the α3A and α3B integrins, respectively. Both transfectant cells adhered almost equally to laminin-5-coated plates in an α3 integrin-dependent manner, indicating that transfected α3Aβ1 and α3Bβ1 integrins were functionally active in these cells. The migratory and invasive potentials of the transfectant cells were assessed by scratch wound assay and in vitro chemoinvasion assay. The results demonstrated that the migration of HepG2-3A and HepG2-3B cells but not of mock transfectant (HepG2-M) cells was stimulated on the plates coated with laminin-5. Furthermore, HepG2-3A and HepG2-3B cells were found to be more invasive into laminin-5-containing matrices than were HepG2-M cells. These results strongly suggest that enhanced expression of α3β1 integrin on HCC cells is directly involved in their malignant phenotypes such as invasion and metastasis.     

Cardiac and skeletal muscle expression of mutant β-myosin heavy chains, degree of functional impairment and phenotypic heterogeneity in hypertrophic cardiomyopathy

Several mutations in distinct genes, all coding for sarcomeric proteins, have been reported in unrelated kindreds with familial hypertrophic cardiomyopathy (FHC). We have identified nine individuals from three families harboring two distinct mutations in one copy of the β-myosin heavy chain (β-MHC) gene. In this study, the expression of the mutant β-myosin protein isoform, isolated from slow-twitch fibers of skeletal muscle, was demonstrated by Northern and Western blot analysis; this myosin showed a decreased in vitro motility activity and produced a lower actin-activated ATPase activity. Isometric tension, measured in single slow-twitch fibers isolated from the affected individuals, also showed a significant decrease. The degree of impairment of β-myosin function, as well as the loss in isometric tension development, were strictly dependent on the amount of the isoform transcribed from the mutated allele. Interestingly, a strong correlation was also demonstrated between mutant β-myosin content and clinical features of FHC. On the other hand, we were unable to detect any correlation between mutant β-myosin expression and degree of cardiac hypertrophy, thereby strengthening the hypothesis that hypertrophy, one of the hallmarks of FHC, might not necessarily be related to the clinical evolution of this disease. These findings lend support to the notion that additional factors rather than the mutated gene may play a pathogenetic role in cardiac wall thickening, whereas the prognosis appears to be strongly related to the amount of mutant protein.     

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.