Deglycosylated Form Research Articles

Antibody to C1 Domain of Factor VIII Alters Interaction of Factor Xase Complex with Factor X.

The role of the C1 domain in function of factor VIII has not been clearly defined. In contrast, functional interactions have been identified for the three A domains and the C2 domain. We hypothesized that the C1 domain of factor VIII participates in both phospholipid binding and interaction with factor X and/or factor IXa. We evaluated inhibition of the factor Xase complex by LE2E9, a human inhibitor IgG4k mAb against C1. We utilized altered catalytic activity of the factor Xase complex in a defined assay to report the inhibition by LE2E9. Inhibition by LE2E9 was also evaluated when soluble phosphatidylserine replaced vesicles to support the factor Xase complex and when Gla-domainless factor X was the substrate. The deglycosylated form of LE2E9 was also evaluated to better define the mechanism through which LE2E9 exerts its effect. We found that LE2E9 bound to factor VIIIa with an apparent KD of 0.5 nM. The apparent affinity of factor VIIIa for sonicated phospholipid vesicles of phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine 4:20:76 increased 3-fold in the presence of LE2E9. The apparent affinity of factor VIIIa for factor IXa was not significantly changed. The KM of the factor VIIIa-factor IXa complex was 20 ± 2 nM with LE2E9 vs. 40 ± 2 nM without. LE2E9 decreased the Vmax by 77 ± 6% indicating that the affinity of factor X for the factor Xase complex is increased while the rate of cleavage is decreased. When Gla-domainless factor X was used as the substrate for the factor Xase complex, LE2E9 did not inhibit activity indicating that inhibition occurs via an interaction that involves the factor X Gla domain. When the factor VIIIa-factor IX complex was supported by dihexanoyl phosphatidylserine rather than phospholipid vesicles the inhibition of Vmax was 47% indicating that the inhibitory effect does not require a phospholipid bilayer. Deglycosylated LE2E9 did not significantly change the KM but decreased the Vmax by 22% while both antibodies bound to factor VIII with the same affinity. These results suggest that LE2E9 inhibition relates largely to interaction of a carbohydrate moiety with factor VIII or factor X rather than binding the core C1 epitope. We conclude that LE2E9 decreases the KM, and the Vmax for the factor VIIIa-factor IXa complex, but only when the factor X Gla domain is present. These results suggest that in the factor Xase complex the C1 domain of factor VIII is intimately associated with the Gla domain of factor X and that interaction between these domains enhances the kcat for the factor VIIIa-factor IXa complex.

Soluble, Oligomeric, and Ligand-binding Extracellular Domain of the Human α7 Acetylcholine Receptor Expressed in Yeast: REPLACEMENT OF THE HYDROPHOBIC CYSTEINE LOOP BY THE HYDROPHILIC LOOP OF THE ACh-BINDING PROTEIN ENHANCES PROTEIN SOLUBILITY

The N-terminal extracellular domain (ECD; amino acids 1-208) of the neuronal nicotinic acetylcholine receptor (AChR) alpha7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This molecule, alpha7-ECD, although able to bind the specific ligand alpha-bungarotoxin, existed mainly in the form of microaggregates. Substitution of Cys-116 in the alpha7-ECD with serine led to a decrease in microaggregate size. A second mutant form, alpha7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydrophobic Cys-loop (Cys(128)-Cys(142)) was replaced by the corresponding hydrophilic Cys-loop from the snail glial cell acetylcholine-binding protein. This second mutant protein was water-soluble, expressed at a moderate level (0.5 +/- 0.1 mg/liter), and had a size corresponding approximately to a pentamer as judged by gel filtration and electron microscopy studies. It also bound (125)I-alpha-bungarotoxin with relatively high affinity (K(d) = 57 nm), the binding being inhibited by unlabeled alpha-bungarotoxin, d-tubocurarine, or nicotine (K(i) = 0.8 x 10(-7) m, K(i) = 1 x 10(-5) m, and K(i) = 0.9 x 10(-2) m, respectively). All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity without affecting their ligand binding properties. These results show that alpha7-ECD(C116S,Cys-loop) was expressed in P. pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for structural studies on the ligand-binding domain of a neurotransmitter receptor.

Identification of Distinct N-terminal Truncated Forms of Prion Protein in Different Creutzfeldt-Jakob Disease Subtypes

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules.

Effect of glycosylation on the structure of Erythrina corallodendron lectin.

The three-dimensional structure of the recombinant form of Erythrina corallodendron lectin, complexed with lactose, has been elucidated by X-ray crystallography at 2.55 A resolution. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites (Asn17). These changes take place in such a way that hydrogen bonds with the neighboring protein molecules in rECorL compensate those made by the glycan with the protein in ECorL. Contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well-defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.

Import and export of nuclear proteins: focus on the nucleocytoplasmic movements of two different species of mammalian estrogen receptor.

There is a wealth of information regarding the import and export of nuclear proteins in general. Nevertheless, the available data that deals with the nucleocytoplasmic movement of steroid hormone receptors remains highly limited. Some research findings reported during the past five years have succeeded in identifying proteins related to the movement of estrogen receptor alpha from the cytoplasm to the nucleus. What is striking in these findings is the facilitatory role of estradiol in the transport process. A similar conclusion has been drawn from the studies on the plasma membrane-to nucleus movement of the alternative form of estrogen receptor, the non-activated estrogen receptor (naER). The internalization of naER from the plasma membrane takes place only in the presence of estradiol. While the gene regulatory functions of ER alpha appear to get terminated following its ubiquitinization within the nucleus, the naER, through its deglycosylated form, the nuclear estrogen receptor II (nER II) continues to remain functional even beyond its existence within the nucleus. Recent studies have indicated the possibility that the estrogen receptor that regulates the nucleo cytoplasmic transport of m RNP is the nERII. This appears to be the result of the interaction between nERII and three proteins belonging to a group of small nuclear ribonucleo proteins (snRNP). The interaction of nERII with two of this protein appears to activate the inherent Mg2+ ATPase activity of the complex, which leads to the exit of the RNP through the nuclear pore complex.

Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I.

Recently, the role of N-linked glycans in the process of ERAD (endoplasmic reticulum-associated degradation) of proteins has been widely recognized. In the present study, we attempted to delineate further the sequence of events leading from a fully glycosylated soluble protein to its deglycosylated form. Degradation intermediates of a truncated form of ribophorin I, namely RI(332), which contains a single N-linked oligosaccharide and is a substrate for the ERAD/ubiquitin-proteasome pathway, were characterized in HeLa cells under conditions blocking proteasomal degradation. The action of a deoxymannojirimycin- and kifunensine-sensitive alpha1,2-mannosidase was shown here to be required for both further glycan processing and progression of RI(332) in the ERAD pathway. In a first step, the Man(8) isomer B, generated by ER mannosidase I, appears to be the major oligomannoside structure associated with RI(332) intermediates. Some other trimmed N-glycan species, in particular Glc(1)Man(7)GlcNAc(2), were also found on the protein, indicating that several mannosidases might be implicated in the initial trimming of the oligomannoside. Secondly, another intermediate of degradation of RI(332) accumulated after proteasome inhibition. We demonstrated that this completely deglycosylated form arose from the action of an N-glycanase closely linked to the ER membrane. Indeed, the deglycosylated form of the protein remained membrane-associated, while being accessible from the cytoplasm to ubiquitinating enzymes and to added protease. Our results indicate that deglycosylation of a soluble ERAD substrate glycoprotein occurs in at least two distinct steps and is coupled with the retro-translocation of the protein preceding its proteasomal degradation.

Effect of pH, temperature and alcohols on the stability of glycosylated and deglycosylated stem bromelain.

The biological significance of the carbohydrate moiety of a glycoprotein has been a matter of much speculation. In the present work, we have chosen stem bromelain from Ananas comosus as a model to investigate the role of glycosylation of proteins. Stem bromelain is a thiol protease which contains a single hetero-oligosaccharide unit per molecule. Here, the deglycosylated form of the enzyme was obtained by periodate oxidation. The differences in the glycosylated and deglycosylated forms of the glycoprotein have been studied at various temperatures and pH values, using probes such as loss of enzyme activity and by the changes in fluorescence and circular dichroism spectra. Deglycosylated bromelain showed decreased enzyme activity and perturbed fluorescence and circular dichroism spectra. In addition to this, a comparative study of their activities in different organic solvents showed a marked decrease in case of deglycosylated form of the enzyme. It is thus concluded that glycosylation contributes towards the functional stability of glycoenzymes.

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.

The Reaction of Antibodies with the Native and Deglycosylated Thyrotropin Receptor obtained from Transfected Insect Cells

The role of glycan moieties in thyrotropin receptor molecule in binding of antibodies is a subject of intense debate. To approach the function of sugars in recognition by antibody of the extracellular part of the receptor (ETSHR) we studied the reaction of the HPLC purified ETSHR from insect cells in the reaction with autoantibodies and antibodies of animal origin. None of the autoantibodies from Graves' patients sera bound to ETSHR. In contrast, each of the animal antibodies: three monoclonal, five polyclonal antireceptor and two polyclonal anti peptide corresponding to the amino acid sequence present in the receptor, became bound to the native receptor from insect cells as well as to its deglycosylated form. The shape of the dilution curves of particular antibodies in the reaction with either form of the receptor was almost the same. The coefficients of correlation was about 0.9. It seems that the correct receptor glycosylation is not crucial for binding of animal origin antibodies.

Guanidine hydrochloride denaturation of glycosylated and deglycosylated stem bromelain.

Glycosylation is one of the major naturally occurring covalent modifications of proteins. We have used stem bromelain, a thiol protease with a single, N-glycosylated polypeptide chain as a model to investigate the role of glycosylation of proteins. Periodate oxidation was used to obtain the deglycosylated form of the enzyme. Denaturation studies in the presence of guanidine hydrochloride (Gn*HCl) were performed using fluorescence and circular dichroism spectroscopy. The glycosylated stem bromelain was found to be stabilized by 1.9 kcal/mol as compared to the deglycosylated one. At a given concentration of denaturant, the fraction of denatured protein was higher in the case of deglycosylated stem bromelain. In short, deglycosylated bromelain showed more susceptibility towards guanidine hydrochloride denaturation, indicating the contribution of the carbohydrate part of the glycoprotein to the stability of the enzyme.

Identification of the amino terminal subunit of the glycoprotein of Borna disease virus

The only surface membrane glycoprotein of Borna disease virus (BDV) is synthesized as a polypeptide with a molecular mass of 57 kDa and N-glycosylated to a precursor glycoprotein (GP) of about 94 kDa. It is processed by the cellular protease furin into the C-terminal membrane-anchored subunit GP-C, also known as gp43, and a presumptive N-terminal subunit GP-N, that is highly glycosylated and has a molecular mass of about 51 kDa. However, up to now the latter remained undetected in BDV-infected material. We describe a novel approach to identify glycan masked linear antigenic epitopes. In the present study, GP-N was identified in BDV-infected cells by a combination of lectin precipitation, enzymatic deglycosylation on blot and immunochemistry using an N-terminal specific antiserum. The GP-N has an apparent molecular mass of 45–50 kDa in its glycosylated form and 27 kDa in its deglycosylated form. N-glycan analysis revealed that the precursor GP contains only mannose-rich N-glycans, whereas GP-N and GP-C contain mannose-rich and complex-type N-glycans.

The systemin receptor SR160 from Lycopersicon peruvianum is a member of the LRR receptor kinase family.

The isolation to homogeneity of the 160-kDa systemin cell-surface receptor (SR160) from plasma membranes of suspension cultured cells of Lycopersicon peruvianum is reported. The purification procedure resulted in recovery of 13 microg of pure receptor protein, representing an 8,200-fold purification. Gel blot analyses using SR160-specific antibodies confirmed that a cross-reacting protein in the membranes of suspension-cultured cells comigrates with both the native and a deglycosylated form of the radiolabeled receptor. Internal amino acid sequences of the purified protein facilitated the isolation of a cDNA clone encoding the 160-kDa receptor. The identity of the encoded protein as SR160 was further confirmed by a comparison of its sequence with a mass spectral fingerprint of the SR160 protein. The deduced amino acid sequence of SR160 revealed that it is a member of the leucine-rich repeat (LRR) receptor kinase family, closely related to the brassinolide receptor kinase, BRI1.

The fate of newly synthesized V-ATPase accessory subunit Ac45 in the secretory pathway.

The vacuolar H+-ATPase (V-ATPase) is a multimeric enzyme complex that acidifies organelles of the vacuolar system in eukaryotic cells. Proteins that interact with the V-ATPase may play an important role in controlling the intracellular localization and activity of the proton pump. The neuroendocrine-enriched V-ATPase accessory subunit Ac45 may represent such a protein as it has been shown to interact with the membrane sector of the V-ATPase in only a subset of organelles. Here, we examined the fate of newly synthesized Ac45 in the secretory pathway of a neuroendocrine cell. A major portion of intact approximately 46-kDa Ac45 was found to be N-linked glycosylated to approximately 62 kDa and a minor fraction to approximately 64 kDa. Trimming of the N-linked glycans gave rise to glycosylated Ac45-forms of approximately 61 and approximately 63 kDa that are cleaved to a C-terminal fragment of 42-44 kDa (the deglycosylated form is approximately 23 kDa), and a previously not detected approximately 22-kDa N-terminal cleavage fragment (the deglycosylated form is approximately 20 kDa). Degradation of the N-terminal fragment is rapid, does not occur in lysosomes and is inhibited by brefeldin A. Both the N- and C-terminal fragment pass the medial Golgi, as they become partially endoglycosidase H resistant. The Ac45 cleavage event is a relatively slow process (half-life of intact Ac45 is 4-6 h) and takes place in the early secretory pathway, as it is not affected by brefeldin A and monensin. Tunicamycin inhibited N-linked glycosylation of Ac45 and interfered with the cleavage process, suggesting that Ac45 needs proper folding for the cleavage to occur. Together, our results indicate that Ac45 folding and cleavage occur slowly and early in the secretory pathway, and that the cleavage event may be linked to V-ATPase activation.

Characterization of a Kunitz Trypsin Inhibitor with One Disulfide Bridge Purified from Swartzia pickellii

Swartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. The primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130.

G1-dependent prion protein expression in human glioblastoma cell line T98G.

Human glioblastoma cell line T98G produced a cellular form of prion protein (PrP(C)), and we confirmed expression of PrP mRNA by RT-PCR. Immunoblot analysis of whole cell lysate revealed one major (35 kDa) and two faint bands (31, 25 kDa) that reacted with monoclonal anti-human PrP antibody 3F4. Cells treated with tunicamycin produced only a 25 kDa band, representing a deglycosylated form of PrP. Similarly, peptide: N-glycosidase F treatment of whole cell lysate altered the Asn-linked form to the deglycosylated form. When T98G cells were cultured for a longer period, the amount of PrP(C) per cell increased on Day 4 to 16 in a time-dependent manner. When the cells were cultured at high cell-density, the cells on Day 4 produced the same amount of PrP(C) as those on Day 16 of the usual culture. Moreover, in a serum-free medium, cells cultured at a low cell-density produced the same amount of PrP(C) as those cultured at the high cell-density. These results demonstrate that PrP(C) production in T98G cells was dependent on the phase of the cell cycle, probably the G1 phase.

Performance evaluation of capillary surface treatments for peptide mapping by capillary zone electrophoresis

The performance of different capillary coatings and their ability to reduce peptide/wall interactions in capillary zone electrophoresis (CZE) was evaluated through the separations of standard peptides with PM ranging from 720 to 3500 and pl ranging from 3.0 to 11.5. The study was performed in acidic buffer with untreated fused silica capillary, poly(vinylalcohol) (PVA) modified capillary (generated in our laboratory by immobilization with thermal treatment of a PVA solution) and polybrene coated capillary. PVA and polybrene modified capillaries were shown to produce higher reproducibilities for migration times and superior efficiencies in comparison to the uncoated capillary. Peak efficiencies of acidic peptides were 300000 to 650000 (N/m) with polybrene capillary coating while PVA coating produced high efficiencies (N/m>330000) for the most basic peptides. An efficient peptide mapping of the β-lactoglobulin was obtained in less than 20 min using the polybrene coating. Attempts to separate the peptide digest from a deglycosylated form of the rgp 160, a very complex and heterogeneous HIV envelope glycoprotein, were not fully satisfactory but the results demonstrate the potential of capillary surface treatment in producing peptide maps.

Crystallization and functional analysis of a soluble deglycosylated form of the human costimulatory molecule B7-1.

The interactions of B7-1 with CD28 and CTLA-4 modulate the course of human immune responses, making B7-1 an important target for developing structure-based therapeutics. B7-1 is, however, one of the most heavily glycosylated proteins found at the leukocyte cell surface, complicating the structural analysis of this molecule. Methods for the production, crystallization and selenomethionine labelling of a soluble deglycosylated form of this molecule are described. The protein readily forms both tetragonal plate and bipyramidal crystals belonging to space groups I4(1)22, with unit-cell parameters a = b = 56.9, c = 298.7 A, and P4(1)22 (or P4(3)22), with unit-cell parameters a = b = 89.0, c = 261.9 A, respectively. The I4(1)22 and primitive crystal forms diffract to 2.7 and 3.5 A, respectively. Surface plasmon resonance-based assays indicate that the ligand-binding properties of sB7-1 are unaffected by deglycosylation. Since none of the methods relied on any special structural properties of sB7-1, it is proposed that this novel combination of procedures could in principle be adapted to the systematic analysis of many other glycoproteins of structural or functional interest.

Characterization of Binding Properties of Urinary Trypsin Inhibitor to Cell-associated Binding Sites on Human Chondrosarcoma Cell Line HCS-2/8

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness. It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP(40)), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP(45)). Here we characterize binding properties of UTI-BPs.UTI complexes in the cells. In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP(40) and UTI-BP(45) bind (125)I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP(40). Additional experiments, using various reagents to block binding of (125)I-UTI and NG-UTI to the UTI-BP(40) and UTI-BP(45) confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP(45). Analysis of binding of (125)I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP(40) (which can bind NG-UTI), and the high affinity sites are the UTI-BP(45). In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.

Carbohydrate-Carbohydrate Binding of Ganglioside to Integrin α5 Modulates α5β1Function

Gangliosides GT1b and GD3, components of keratinocyte membranes, inhibit keratinocyte adhesion to fibronectin. Although ganglioside sialylation is known to be important, the mechanism of inhibition is unknown. Using purified insect recombinant alpha(5) and beta(1) proteins and alpha(5)beta(1) integrin from lysed keratinocyte-derived SCC12 cells, we have shown that GT1b and GD3 inhibit the binding of alpha(5)beta(1) to fibronectin. Co-immunoprecipitation of GT1b and alpha(5)beta(1) from SCC12 cells and direct binding of GT1b and GD3 to affinity-purified alpha(5)beta(1) from SCC12 cells and insect recombinant alpha(5)beta(1), particularly the alpha(5) subunit, further suggest interaction between ganglioside and alpha(5)beta(1). The carbohydrate moieties of integrin appear to be critical since gangliosides are unable to bind deglycosylated forms of alpha(5)beta(1) from SCC12 and insect cells or poorly glycosylated recombinant alpha(5)beta(1) from Escherichia coli cells. The GT1b-alpha(5)beta(1) interaction is inhibited by concanavalin A, suggesting that GT1b binds to mannose structures in alpha(5)beta(1). The preferential binding of GT1b to high mannose rather than reduced mannose ovalbumin further implicates the binding of GT1b to mannose structures. These data provide evidence that highly sialylated gangliosides regulate alpha(5)beta(1)-mediated adhesion of epithelial cells to fibronectin through carbohydrate-carbohydrate interactions between GT1b and the alpha(5) subunit of alpha(5)beta(1) integrin.

Chemical construction of immunotoxins.

Immunotoxins are chimeric proteins consisting of an antibody linked to a toxin. The antibodies most frequently used for the preparation of immunotoxins are murine monoclonal antibodies belonging to IgG isotype. The most used toxins for the chemical construction of immunotoxins are Ricin toxin A chain in its deglycosylated form and recombinant Pseudomonas endotoxin with the cell-binding domain deleted. The linkage of the antibody to the toxin can be accomplished by chemical methods using reagents that crosslink antibody to toxin. The usual crosslinkers attach disulfide groups into the antibody molecule to form a disulfide bond between the antibody and the toxin. Disulfide bonds are susceptible to reduction in the cytoplasm of the targeted cells thereby releasing the toxin so that it can exert its cytotoxic activity only into the cells (e.g., tumor cells) binding the antibody moiety. This article describes various methods to obtain antibodies and toxins and several procedures for their crosslinking as well as "in vitro" and "in vivo" testing of the immunotoxins efficacy.