Forssman Hapten Research Articles

Extracellular carbohydrate-signal triggering camp-dependent protein kinase-dependent neuronal actin-reorganization

Cell surface glycoconjugates are thought to mediate cell–cell recognition and to play roles in neuronal development and functions. We demonstrated here that exposure of neuronal cells to nanomolar levels of glyco-chains with an N-acetylgalactosamine (GalNAc) residue at the non-reducing termini (GalNAc-S) such as GalNAcβ4(Neu5Acα3)Galβ4GlcCer (GM2) ganglioside, its oligosaccharide portion, GalNAcβ4Galβ4GlcCer (Gg 3) Cer, GalNAcα3GalNAcβ3Galα4Galβ4GlcCer (Gb 5) Cer (Forssman hapten) and α1–4 linked oligomers of GalNAc, induced a rapid and transient activation of cAMP-dependent protein kinase (PKA) in subplasmalemma. The treatment was accompanied by peripheral actin polymerization and filopodia formation in NG108–15 cells and primary cultured hippocampal neurons, but not in glial cells. A cAMP-dependent protein kinase (PKA) selective inhibitor and an adenylate cyclase inhibitor blocked both PKA activation and the subsequent filopodia formation. A small GTPase cdc42 was a potential downstream target of GalNAc-S-activated PKA. These results suggest that extracellular GalNAc-S serve as potential regulators of the filopodia formation in neuronal cells by triggering the activation of PKA followed by cdc42 up-regulation via a cell surface receptor-like component. Filopodia formation induced by GalNAc-S may have a physiological relevance because long-term exposure to GalNAc-S enhanced F-actin-rich dendrite generation of primary cultured hippocampal neurons, and PKA-dependent dendritic outgrowth and branch formation of primary cultured cerebellar Purkinje neurons, in which actin isoforms were localized to motile structures in dendrites. These findings provide evidence for a novel GalNAc/PKA-signaling cascade in regulating some neuronal maturation.

Purification and Characterization of Blood Group A-degrading Isoforms of α-N-Acetylgalactosaminidase from Ruminococcus torques Strain IX-70

To cleave blood group A immunodeterminants from erythrocytes (Hoskins, L. C., Larson, G., and Naff, G. B. (1995) Transfusion 35, 813-821), we purified and characterized alpha-N-acetylgalactosaminidase (EC 3.2.1.49) activity from culture supernatants of the human fecal bacterium Ruminococcus torques strain IX-70. Three isoforms separated during hydrophobic interaction chromatography. Hydroxyapatite chromatography further resolved the most hydrophilic, isoform I, into isoforms IA and IB. The most hydrophobic, isoform III, differed from IA and IB by a more acidic pH optimum, greater heat resistance, greater sensitivity to alkylating agents, and anomalous retardation during gel filtration chromatography. Isoform IB differed from IA and III in N-terminal amino acid sequence and in sensitivity to EDTA inhibition. Each cleaved nonreducing alpha(1-->3)-N-acetylgalactosamine residues from human blood group A and AB mucin glycoproteins, Forssman hapten, and blood group A lacto series glycolipids. The apparent molecular mass of denatured isoform subunits of IA, IB, and III-PII (158, 173, and 201 kDa, respectively) bore no integer relationship to the apparent molecular mass of the native isoforms (265, 417, and 530 kDa), but the latter bore a ratio of 1.96:3.09:3.93 to the weight-average apparent molecular mass of native IA (135 kDa), suggesting that the isoforms are multimers of a 135-kDa sequence. Isoforms IA and III-PII had an identical N-terminal amino acid sequence which showed homologies to the N-terminal sequence of sialidases produced by Bacteroides fragilis SBT3182, another commensal enteric bacterium.

Serological study using α- N-acetylgalactosaminidase from Acremonium sp.

α- N-Acetylgalactosaminidase, produced by Acremonium sp. destroyed blood group A active substance. This enzyme converted human erythrocytes from group A to O(H). The enzyme was ascertained to liberate N-acetylgalactosamine from group A erythrocytes membranes accompanying a decrease in hemagglutination inhibition activity of A erythrocyte-anti-A serum by the erythrocyte membranes. Glycoproteins and glycolipids extracted from A erythrocytes reduced their blood group A activity after the enzyme treatment. The enzyme also worked on Forssman hapten glycolipid.

Anti-peripheral nerve myelin antibodies in Guillain-Barre syndrome bind a neutral glycolipid of peripheral myelin and cross-react with Forssman antigen.

During acute-phase illness, serum of patients with Guillain-Barre syndrome (GBS) contain complement-fixing antibodies (Ab) to peripheral nerve myelin (PNM). We investigated PNM lipids as putative antigens for these Ab since GBS serum retained significant reactivity to PNM treated with protease. Ab binding to specific lipids was studied with a C1 fixation and transfer (C1FT) assay using fractions of PNM lipid reincorporated into liposomes as antigen targets or to lipids on HPTLC plates with peroxidase-labeled goat Ab to human IgM. Reactivity was detected to a neutral glycolipid (NGL) of human PNM with a similar number of carbohydrates residues to that of Forssman hapten (Forss). Anti-NGL Ab titers in GBS patients (50-220 U/ml) were significantly elevated over disease and normal controls (0-5 and 0-6 U/ml). We studied possible antigenic cross-reactivity of these Ab with Forss by first quantitating Ab activity with C1FT assay and liposomes containing Forss. All 12 GBS sera tested showed titers (54-272 U/ml) significantly elevated over 11 disease controls (0-22 U/ml) and 25 normal controls (0-11 U/ml). GBS serum Ab reacted with Forss isolated from dog nerve or sheep erythrocytes on HPTLC plates. Further, absorption of 80-100% of anti-NGL Ab activity and 17-97% of anti-PNM Ab activity from eight GBS patient serums was accomplished with liposomes containing Forss but not with control liposomes. In seven GBS patients anti-NGL Ab activity represented only a portion of anti-PNM Ab activity. These results suggest that a glycolipid with antigenic cross-reactivity to Forssman hapten may be responsible for some of the anti-PNM Ab activity in GBS.

Carbon-13 nuclear-magnetic-resonance spectroscopy of Forssman hapten.

The 13C n.m.r. spectrum of Forssman hapten was obtained at 25.16 MHz in [3H] chloroform/[2H] methanol (1:1, v/v), using purified glycosphinogolipid from canine intestinal mucosa (glycolipid I). All amide, olefin, anomeric, intersaccharide glycosidic ether, amide linkage, methyl and many methylene resonances were resolved and assigned. Analysis of the anomeric region reveals the following pentaglycosylceramide structure as originally proposed [Siddiqui & Hakomori (1971) J. Biol. Chem. 246, 5766-5769]: GalNAc (alpha 1 leads to 3) GalNAc (beta 1 leads to 3) Gal (alpha 1 leads to 4) Gal (beta 1 leads to 1) ceramide. Analysis of the amide, olefin and methylene regions reveals no alpha-hydroxy fatty acyl group and less than or equal to 6 mol% unsaturated fatty acyl groups are present. Chemical-shift assignments are reported for the anomeric and glycosidic ether carbon atoms of intersaccharide-linked alpha-galactose and N-acetyl-alpha-galactosamine residues. Two rules are proposed for the assignment of the anomeric form of 1 leads to 3 and 1 leads to 4 linkages of galactose and N-acetylgalactosamine residues present in the glycone of glyco-conjugates. The present study emphasizes the importance of the anomeric "window" (80-120 p.p.m.) in studies of glycone structure.

In vitro effects of glycosphingolipids on human tumor cell proliferation.

AbstractA drug-resistant subpopulation of cells (HTFU) was previously isolated from the human colonic carcinoma cell line HT29. The cell lines varied significantly in in vitro growth patterns with HTFU cells showing contact inhibition while HT29 cells demonstrated uncontrolled growth (10). The malignant cell lines were grown in the presence of various concentrations of blood group A fucolipid, Lewisb, Forssman hapten, and ceramide tri-hexoside for 2 weeks. Although HTFU cells demonstrated an equal or enhanced sensitivity to the lipids relative to HT29 cells, the cell lines responded in similar fashion to each lipid. Treatment with Forssman and ceramide trihexoside induced a temporary inhibition of cell proliferation, however, continued exposure to these lipids became stimulatory. Exposure to Lewisb and blood group A fucolipids caused a permanent retardation of cell proliferation.

Radioimmunoassay of glycosphingolipids: Application for the detection of forssman glycolipid in tissue extracts and cell membranes

Development of a radioimmunoassay for detecting glycosphingolipids has been difficult, primarily because of transfer of radiolabeled glycolipid antigen to the unlabeled antigen pool. This difficulty has been overcome by the use of a radiolabeled glycolipidpolymer. Thus, an assay system has been developed for measuring picomolar quantities of Forssman hapten glycolipid. This assay is based on competition for rabbit anti-Forssman antibodies between Forssman glycolipid and a radiolabeled Forssman polyacrylic hydrazide polymer. Antigen-antibody complexes are removed quickly and efficiently by binding to formalin-fixed Staphylococcus aureus and subsequent centrifugation. One nanogram of Forssman glycolipid can be readily detected both in plasma membrane preparations and in purified glycolipid fractions. The isoantigenic expression of Forssman glycolipid in human gastrointestinal tissues has been reported previously (Hakomori et al., 1977). Using the radioimmunoassay, the Forssman status of several additional cases has been determined quantitatively.

Proton nuclear magnetic resonance analysis of anomeric structure of glycosphingolipids: The globo-series (one to five sugars)

Proton nuclear magnetic resonance spectroscopy has been reevaluated concerning the assignment of anomeric structure of glycosphingolipids. Solubility problems due to a varying number of sugars are avoided by permethylation, allowing a wide range of glycolipids to be compared. High resolution spectra were recorded in chloroform solution for the following substances with known structure, most of them representing a successive building up of members of the globo-series: ceramide, Galβ1 → 1Cer, a mixture of Glcα1 → 1Cer and Glcβ1 → 1Cer, lactosylceramide, globotriaosylceramide, globotetraosylceramide (globoside), and GalNAcα1 → 3globotetraosylceramide (Forssman hapten). Resonances originating in anomeric protons were identified and possible interference from other signals was defined. A complex set of resonances from H-1 of hexosamines was probably due to two separate conformers of the acetamido group caused by N-methylation. The complexity disappeared upon reduction with LiAlH 4. The chemical shifts and coupling constants were characteristic for the configuration of the glycosidic bond, the type of monomer, and in part for its location in the chain. At present, spectra may be recorded from 200-μg samples. It is concluded that the good quality and resolution obtained make this technique an alternative method to the presently used enzymatic degradation for establishing anomeric structure of glycosphingolipids.

Studies on aminoglycolipid contents and their synthesis in hamster cell lines: Polyoma-transformed bhk cell lines with low β- and high aw-acetylgalactosaminyltransferase activities

Hamster cell lines, BHK and their polyoma transformant, contained a very small amount of globoside and Forssman hapten. This observation was also confirmed with hemolysis inhibition test using the antisera. On the other hand, an appreciable incorporation of radioactivity into these glycolipids was found in both the cell lines after the cells were labeled with [ 14C]glucosamine. When glycolipid β- and α- N-acetylgalactosaminyl transferases were assayed with enzyme preparations, BHK-Py cells showed little activity in the β-transferase (globoside synthesis) in contrast to much elevated activity of the α-transferase (Forssman hapten synthesis) as compared with those in the parent BHK cells, suggesting that the little activity of the β-transferase results in low content of both the aminoglycolipids. The properties of the α-transferase of both the cell lines were examined. The enzyme of BHK-Py cells showed similar kinetic and other properties to those of BHK cells. Neither inhibitor in BHK cells, nor activator in BHK-Py cells was noted for the α-transferase.

Solubilization and partial characterization of UDP- N-acetylgalactosamine:globoside α- N-acetylgalactosaminyltransferase from dog spleen microsomes

UDP- N-acetylgalactosamine:globoside α- N-acetylgalactosaminyltransferase (EC 2.4.1.-) synthesizing Forssman hapten was solubilized from dog spleen microsomes by a combination of Triton X-100 treatment and sonication. The solubilized enzyme was partially purified by calcium phosphate gel, ammonium sulfate fractionation and then DEAE-cellulose column chromatography. The enzymatic activity of the purified preparation was stimulated by exogenously added phosphatidylserine, as found in the particulate enzyme. When the properties of the purified enzyme were examined in the presence of exogenous phosphatidylserine, the enzyme had an absolute requirement for Mn 2+; this was not substituted by Ca 2+ or Mg 2+. Apparent K m values for UDP- N-acetylgalactosamine and globoside were 1 · 10 −5 and 5 · 10 −4 M, respectively. It had a pH optimum of 6.55 regardless of the presence or absence of exogenous lipids. Since the partially purified enzyme was completely free of uridine diphosphatase which was found in the particulate preparation, the effect of UDP on the transferase activity could be studied. Thus, UDP inhibited 85% of the activity at a concentration of 1.5 mM. p-Chloromercuribenzoate inhibited over 90% of the activity at 2 mM, indicating the transferase to be SH-enzyme.

The identification of α-galactosidase B from human liver as an α-acetylgalactosaminidase

Previously reported differences in the substrate specificities of α-galactosidases A and B have been further investigated. A highly purified fraction of α-galactosidase B from normal human liver was found to possess α-N-acetylgalactosaminidase activity as well. The Michaelis constants with o-nitrophenyl-α-N-acetylgalactosaminide (o-NP-α-GalNAc) and 4-methylumbelliferyl-α-D-galactopyranoside (4-MU-α-Gal) were 1.3 and 6.8 mM, respectively. The hydrolysis of 4-MU-α-Gal was competitively inhibited by o-NP-α-GalNAc with a Kj of 2.7 mM. In addition, the B form uniquely catalyzed the hydrolysis of Forssman hapten, a glycolipid containing a terminal α-N-acetylgalactosaminyl residue, with a Michaelis constant of 0.59 mM.

Water-soluble glycosphingolipids of dog gastric mucosa. Characterization of a branched ceramide heptasaccharide.

Two glycosphingolipids, the carbohydrate portion of which consisted of galactose, glucose and N-acetylgalactosamine, have been isolated from the aqueous phase of buffered tetrahydrofuran extract of dog gastric mucosa. The structures of these glycolipids were identified by partial acid hydrolysis, sequential degradation with specific glycosidases and methylation analysis. The structure of glycolipid I (GalNAcalpha1 leads to 3GalNAcbeta1 leads to 3Galalpha1 leads to 4Galbeta1 leads to 4Glc leads to ceramide) was found to be identical to that of Forssman hapten. The branched structure of glycolipid II, as determined by the combination of enzymatic degradation and comparison of the permethylation products of the enzyme-degraded compounds, is proposed to be: See Source.

Effects of uridine nucleotides and nucleotide pyrophosphatase on glycolipid α- and β- N-acetylgalactosaminyltransferase activities in guinea pig microsomes

Membrane-bound α- and β- N-acetylgalactosaminyltransferases (EC 2.4.1.0) which catalyze formation of non-reducing terminal linkages of Forssman hapten and globoside, respectively, could be differentiated with respect to the different effects of UDP on the two enzyme activities. UDP markedly inhibited the α-transferase activity, in contrast to its stimulatory action on the β-transferase. These effects of UDP were similar to those of UDPglucose, which was demonstrated to be a competitive inhibitor ( K i, 3.3·10 −5 M for UDP- N-acetylgalactosamine) for the α-transferase reaction. Other uridine derivatives tested suppressed both the transferase activities, being more inhibitory for the α-transferase than for the β-transferase. Under the synthetic conditions of these aminoglycolipids, UDP- N-acetylgalactosamine as a donor was simultaneously degraded into N-acetylgalactosamine-1-phosphate and finally into N-acetylgalactosamine by UDP- N-acetylgalactosamine pyrophosphatase, which is part of the membrane system. UDP-glucose was confirmed as being able to prevent the enzymatic hydrolysis of UDP- N-acetylgalactosamine. UDPglucose, therefore, acts to suppress both the α- N-acetylgalactosaminyltransferase (but not the β-transferase) and the pyrophosphatase activities. The inhibitory effect of UDPglucose on the α-transferase activity was most probably due to its direct action on the transferase rather than its function in protecting UDP- N-acetylgalactosamine donor from pyrophosphatase action.

Alterations of glycolipid glycosyltrasnferase levels in lymphoma and lymphoid tissues of lymphoma-bearing hamsters.

Thymus from lymphoma-bearing hamsters in advanced stages of tumor growth showed enhanced activities of four glycolipid-glycosyltransferases which lead to the step-wise synthesis of lactosylceramide to Forssman hapten. The alpha-galactosyltransferase activity of the thymus was decreased until day 6 of inoculation but increased thereafter. The activities of all the transferases tested were significantly lowered in lymphoma tissue. The level of UDP-galactose pyrophosphatase did not correlate with the transferase activities.

Liposome spin immunoassay: A new sensitive method for detecting lipid substances in aqueous media

A new sensitive immunoassay procedure is described for quantitative detection of glycolipids and other lipids in aqueous media. As with other immunoassays, specific antiserum is first reacted with the free lipid hapten. The amount of antibody activity remaining is measured by assaying the release, in the presence of complement, of spin label marker from liposomes containing the same lipid hapten. Using this method, 2.6 pmol of aqueous Forssman hapten was detected, and the sensitivity could be increased further.

FORSSMAN HAPTEN N‐ACETYL‐α‐D‐GALACTOSAMINIDASE IN RAT BRAIN AND KIDNEY1

Abstract—Forssman hapten (N‐acetyl‐α‐galactosaminosyl‐N‐acetyl‐β‐galactosaminosyl‐α‐galactosyl‐β‐galactosyl‐glucosylceramide), prepared from sheep erythrocytes was specifically labelled with tritium at the terminal N‐acetyl‐α‐galactosamine moiety by the galactose oxidase‐sodium [3H]borohydride method. Activities to cleave the terminal N‐acetyl‐α‐galactosamine from Forssman hapten were detected in the high‐speed supernatant of the frozen‐thawed and sonicated crude mitochondrial fraction from adult rat brain and kidney. The optimal pH of the reaction was approximately 4·4. The reaction was linear for at least 1 h for the kidney enzyme and up to 3 h for the brain enzyme. Taurocholate was required for the activity. The optimal concentration was 1·5‐2 mg/ml. Several other detergents and bile salts tested could not replace taurocholate. The apparent Km of the brain and kidney enzymes were 1·0×10−4M and 3·5×10−4m, respectively. During development, Forssman hapten‐cleaving activities of both brain and kidney gradually declined in specific activity as the animal matured. These changes were similar to those of nonspecific p‐nitrophenyl N‐acetyl‐α‐galactosaminidase. Several rat organs examined all showed detectable activities to cleave Forssman hapten.

Structure of forssman hapten glycosphingolipid from sheep erythrocytes

The structure of Forssman glycosphingolipid hapten of sheep erythrocytes was worked out as follows. Earlier composition data showing it to be a pentahexosyl ceramid containing 2 mole of N-acetylgalatosamine, 2 mole of galactose and 1 mole of glucose per mole of hapten were confirmed. Positions of linkages between monosaccharides were determined by methylation-gas chromatographic-mass spectrometric analysis. Results of this study were confirmed by periodate oxidation which also established the sequence of monosaccharide residues. Double-labeling of Forssman hapten with 3H and 14C followed by selective enzymatic hydrolysis determined anomeric configurations of the acetal linkages. These results were verified by chromium trioxide oxidation of peracetylated hapten. The propose structure was supported by sequential enzymatic hydrolysis and assay of the products by inhibition of complement-medited hemolysis. It agrees with the carbohydrate sequences reported by Siddiqui and Hakomori [ J. biol. Chem. 246, 5766 (1971)] for horse kidney Forssman hapten and by Sung et al. [ J. biol. Chem. 248, 6528 (1973)] for canine kidney hapten. Gas chromatographic analysis revealed that sphinganine and lignoceric, nervonic and behenic acids were components. A limited interchangeability of fatty acid moieties is suggested. The structure proposed for Forssman hapten from sheep erythrocytes is N-acetylgalactosaminosyl-( α1 → 3)-N-acetylgalactosaminosyl-( β1 →3)-galactosyl-( α1 → 4)-galactosyl -( gb1 → 4)-glucosyl-( β1 → 1)-O-(acyl-N-sphinganine).

Biosynthesis of globoside and Forssman-related glycosphingolipid in mouse adrenal Y-1 tumor cells

Summary The activities of four glycolipid glycosyltransferases involved in the biosynthesis in vitro of globoside and Forssman hapten were measured in normal mouse adrenal tissues, Y-1 mouse adrenal tumor cells, and tumors derived therefrom. These enzyme activities, found in Golgi-rich membranes isolated on a sucrose density gradient, were higher in the tumor cell cultures than in normal mouse adrenal tissue. The ratio between long chain oligoglycosylceramides and short chain glycosphingolipids was higher in the case of Y-l-K cells treated with dibutyryl cAMP than in concanavalin A, colchicine treated, or untreated control cells in culture.

Forssman-Containing Liposomes: Complement-Dependent Damage due to Interaction with a Monoclonal IgM

Abstract Since 1968 liposomes have been employed as a model for studying the molecular basis of immune lysis (1). The liposomes consist of closed, concentric shells of lipid bilayers alternating with aqueous interspaces (2). The aqueous portions within the liposomes generally contain marker molecules (e.g., glucose) and the membrane damage is measured by release of the marker compound (2, 3). By using liposomes with various amphipathic lipids as antigens, a wide variety of specificities have been described (1, 4). Forssman substance was the first antigen used in these studies and its properties in liposomes have now been thoroughly investigated (5). The initial work demonstrated that liposomes containing Forssman hapten could bind anti-Forssman antibodies (hemolysin) and this led to membrane damage and glucose release due to complement fixation (6, 7). A subsequent report showed that this was a highly specific phenomenon, and anti-Forssman antibodies did not cross-react with globoside I, which is a closely related glycolipid (8).

Biosynthesis of globoside and forssman hapten from trihexosylceramide and properties of β- N-acetyl-galactosaminyltransferase of guinea pig kidney

The enzyme(s) that catalyze the transfer of N-acetylgalactosamine from UDP- N-acetylgalactosamine to exogenous human kidney galactosyl-galactosyl-glucosylceramide was detected in young guinea pig tissues. The enzymatic products from trihexosylceramide were identified as globoside and Forssman hapten by thin-layechromatography, chemical and enzymatic degradations and by immunological methods. The β- N-acetylgalactosaminyltransferase activity for globoside synthesis was found in the microsomal as well as mitochondrial fractions. The optimum pH was 6.9. K m values for trihexosylceramide and for UDP- N-acetylgalactosamine were shown to be 1.4. 10 −4M and 2.3. 10 −5M, respectively. As to the specificity for the glycolipid acceptor of this enzyme preparation, maximum incorporation of the amino sugar was found with trihexosylceramide, but an appreciable activity was found with human kidney globoside and equine erythrocyte hematoside when they were used as acceptors.