Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylinositol-linked form of the neural cell adhesion molecule.

The neural cell adhesion molecule (N-CAM) of rodents comprises three distinct proteins of Mr 180,000, 140,000, and 120,000 (designated N-CAM-180, -140, and -120). They are expressed in different proportions by different tissues and cell types. but the individual contribution of each form to cell adhesion is presently unknown. Previous studies have shown that the two N-CAM species of higher relative molecular mass span the membrane whereas N-CAM-120 lacks a transmembrane domain and can be released from the cell surface by phosphatidylinositol-specific phospholipase C. In this report, we provided evidence that N-CAM-120 contained covalently bound phosphatidylinositol and studied N-CAM-120 from its biosynthesis to its membrane insertion and finally to its release from the cell surface. Evidence was presented showing that the lipid tail of N-CAM-120 contained ethanolamine as is the case for other lipid-linked molecules. The phospholipid anchor was attached to the protein during the first minutes after completion of the polypeptide chain. This process took place in the endoplasmic reticulum as judged from endoglycosidase H digestion experiments. Immediately after a 2-min pulse with [35S]methionine, we detected also a short-lived precursor that had not yet acquired the lipid tail. Pulse-chase studies established that N-CAM-120 was transported to the cell surface from which it was slowly released into the extracellular milieu. The molecules recovered in the incubation medium appeared to have lost all of their bound fatty acid but only around half of the ethanolamine. Upon fractionation of brain tissue, approximately 75% of N-CAM-120 was recovered with a membrane fraction and approximately 25% in a membrane-free supernatant. A small proportion (approximately 6%) was found to be resistant to extraction by non-ionic detergent. A major posttranslational modification of N-CAM is polysialylation. Our results showed that also N-CAM-120 was polysialylated in the young postnatal brain and released in this form from cultured cerebellar cells. The presence of N-CAM in a form that can be released from the cell surface and accumulates in the extracellular fluid suggests a novel mechanism by which N-CAM-mediated adhesion may be modulated.

Enzymatic basis for a lectin-resistant phenotype: increase in a fucosyltransferase in mouse melanoma cells.

In the search for the biochemical basis of the control of glycosylation of cell surface carbohydrates, revertant clones were isolated from previously characterized wheat germ agglutinin-resistant clones of B16 mouse melanoma cells by selection for resistance to Lotus tetragonolobus lectin or to ricin. Comparison of the wheat germ agglutinin-resistant clones with the parent and revertant clones indicated that this phenotype was correlated with an increased sensitivity to the Lotus lectin, a 60- to 70-fold increase in alpha 1 leads to 3 fucosyltransferase activity and a decreased sialic acid content of the N-glycosidic chains of glycoproteins. The results suggest a novel type of control mechanism for lectin resistance, an increase in a glycosyltransferase activity. The presence of alpha 1 leads to 3 bound fucose on N-acetylglucosamine residues would interfere with the addition of sialic acid by alpha 2 leads to 3 linkages to galactose residues in the carbohydrate units, and this change could explain the resistance to wheat germ agglutinin and the increased sensitivity to the Lotus lectin. A change in a regulatory gene for the fucosyltransferase as a possible primary cause for the changed phenotype is discussed.

Carbohydrate changes in glycoproteins of a poorly metastasizing wheat germ agglutinin-resistant melanoma clone.

Glycoproteins of a metastasizing line of B16 mouse melanoma and a poorly metastasizing wheat germ agglutinin-resistant clone were compared. Cell surface proteins and glycoproteins were isotopically labeled by lactoperoxidase-catalyzed iodination and by NaB3H4 reduction after oxidation by periodate or galactose oxidase and subsequently analyzed by gel electrophoresis and autoradiography. Differences were observed in the relative mobilities of several major cell surface components. Binding of 125I-labeled lectins to total cellular proteins on polyacrylamide gels following electrophoresis showed that the major wheat germ agglutinin-binding components of F1 cells were altered in Wa-4 cells. Similar differences were not observed in concanavalin A-binding components. Total cellular glycopeptides were analyzed after separation into structurally distinct classes. The acidic "complex" N-glycosidic glycopeptides from the resistant cells were of lower molecular weight than those from the parent cells. No differences were observed among the mannose-rich N-glycosidic glycopeptides or the alkali-labile O-glycosidic oligosaccharides. Structural studies involving methylation analysis revealed that in the altered glycopeptides of the resistant cells the amount of neuraminic acid residues was decreased to one-half, concomitant with an increase in the amount of fucose. The lost sialic acid was bound to C-3 of galactose, whereas the increased fucose was found on C-3 of 4-substituted N-acetylglucosamine. A possible basis for the glycosylation change and its relation to the biological behavior are discussed.

Structure of the O-glycosidically linked carbohydrate units of rat brain glycoproteins.

The O-glycosidically linked carbohydrate units of rat brain glycopeptides were released as reduced oligosaccharides with NaOH/NaBH4 treatment. Five oligosaccharides were isolated using gel filtration, ion-exchange chromatography and preparative thin-layer chromatography. Studies employing periodate oxidation, methylation analysis, chromium trixide oxidation and gas-liquid chromatography-mass spectrometry indicated the following structures: (I) alpha-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (II) beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol, (III) N-acetylneuraminyl-[beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol], (IV) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)-N-acetylgalactosaminitol and (V) N-acetylneuraminyl-(2 leads to 3)-beta-galactosyl-(1 leads to 3)[N-acetylneuraminyl-(2 leads to 6)]-N-acetylgalactosaminitol.

Disialosyl paragloboside. A novel ganglioside isolated from human kidney.

A ceramide hexasaccharide was purified from the gangliosides of human kidney. Its structure was studied by methylation analysis, neuraminidase treatment, weak acid hydrolysis and chromium trioxide oxidation. The structure is suggested to be a disialosyl derivative of paragloboside: formula: (see text) Data indicating the possible existence of a corresponding trisialosyl derivative are also presented.

No GIST-type c-kit gain of function mutations in neuroblastic tumours.

AIMS: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. METHODS: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). RESULTS: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. CONCLUSIONS: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated.

Interaction of meningococcal group B monoclonal antibody and its Fab fragment with alpha 2-8-linked sialic acid polymers: requirement of a long oligosaccharide segment for binding.

Mouse monoclonal IgG2a antibody (735D4) and other antibodies to the capsular polysaccharide of group B meningococci have been shown to require an unusually long segment of the alpha 2-8-linked N-acetylneuraminic acid polymer for binding. This property may be due to a conformational nature of the polysaccharide epitope recognized, or alternatively due to the requirement of bivalent binding of the antibody to the polysaccharide. In order to study the binding requirements, Fab fragments were prepared from the monoclonal antibody and their binding to alpha 2-8-linked sialic acid polymers of different lengths was studied. Both the intact antibody and its Fab fragment bound to sialic acid poly- and oligomers to similar extents, the critical chain length being about 10 sialyl units for both molecules. This excluded bivalency as the explanation for the requirement of a long oligosaccharide segment for binding. Although the binding was enhanced with increasing chain length, the first 10 monosaccharides were calculated to contribute to more than 90% of the total binding energy. This is in agreement with an oligosaccharide segment with defined conformational epitope binding to the antibody combining site. The antibody preparations also bound polysialic acid containing glycopeptides isolated from developing human and rat brain, suggesting, in quantitative binding assay, an average chain length of 10 or more sialic acid residues. The interaction of the antibody with both the bacterial and the tissue derived polysialic acids suggests that the conformational epitope critical for the interaction is formed by both classes of compounds.

Novel cell-binding activity specific for N-acetyl-D-glucosamine in an Escherichia coli strain.

Escherichia coli strains isolated from patients with different levels of urinary tract infection and from healthy persons were tested for their ability to haemagglutinate endo-beta-galactosidase-treated human erythrocytes. Among the 104 strains studied one revealed a strong agglutination reaction with the enzyme-treated erythrocytes. From the monosaccharides tested N-acetyl-D-glucosamine inhibited agglutination most effectively. Orosomucoid and asialo-orosomucoid had no effect on the haemagglutination whereas beta-galactosidase treated asialo-orosomucoid was inhibitory. These findings indicate that the E. coli strain studied contains a novel cell-binding activity with specificity for terminal N-acetyl-D-glucosamine residues.

Immunoblot analysis of bacterial polysaccharides: application to the type-specific polysaccharides of Streptococcus suis and Streptococcus agalactiae.

A method for the immunoblot analysis of the type-specific capsular polysaccharides of streptococci was developed. The capsular polysaccharides were extracted by sonication and subjected to polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulphate (SDS). After electrophoresis the polysaccharides were transferred to charged PVDF-N membranes and probed with the type specific antibodies. A characteristic ladder-like pattern of polysaccharide bands specific for the serotype (1, 2, 4, 7) was observed for capsular extracts of Streptococcus suis. Human immune sera against type-specific group B streptococcal polysaccharides reacted most strongly with the immunizing polysaccharide type (Ia, II, III). The previously observed crossreactions between the group B streptococcal type-specific capsular preparations were shown to be due to binding to the isomeric polysaccharide molecules. Thus, gel electrophoresis combined with immunoblot analysis seems to provide a novel method for the molecular and immunochemical characterization of bacterial polysaccharides and for the study of the specificity and properties of antibodies to capsular polysaccharides.

Hyperexcretion of free N-acetylneuraminic acid--a novel type of sialuria.

A 26-yr-old female with increased urinary excretion of free N-acetylneuraminic acid is described. Her early history was normal but she had difficulties at school and developed epilepsy at 14. She is mildly retarded, has slight changes in the spine, but no hepato- or splenomegaly. Cerebrospinal fluid leucocytes and IgG are elevated. Liver and skin biopsies disclosed no morphological abnormality. The daily excretion of free N-acetylneuraminic acid is ca. 0.5 g, intermediate to that found in other known types of sialuria. The clinical and morphological findings are also different suggesting a novel type of sialuria.

The influence of membrane mutations on metastasis.

In an effort to assess the effect of surface carbohydrates upon the metastasizing properties of tumor cells, lectin-resistant mouse melanoma cells were selected. Wheat-germ-agglutinin-resistant lines displayed mainly decreased metastasis properties as well as well-defined alterations in surface carbohydrates: in a glycopeptide with four side chains, two of them were missing their terminal sialic acid residues while two fucoses were newly attached to the oligosaccharide. The enzymatic defect could be pinpointed to an over-60-fold increase in fucosyltransferase, while the sialyltransferase did not decrease significantly. Revertants were again selected with lectins and their fucosyltransferase activities returned to normal values again. The metastasizing potential of the revertants was not yet assessed carefully but a return of some of the metastasizing potential was noted.

Determination (by methylation analysis) of the substitution pattern of 2-amino-2-deoxyhexitols obtained from O-glycosylic carbohydrate units of glycoproteins.

The derivatives obtained by permethylation of unsubstituted 2-amino-2-deoxy-hexitols and of these compounds monosubstituted at C-3, C-4, or C-6, and disubstituted at C-3 and C-6, have been analysed by g.l.c.-m.s. Each derivative can be identified on the basis of retention time and mass spectrum. In methylation analysis, methanolysis gave one derivative of each hexitol, whereas a mixture of products was formed when degradation was effected by acetolysis followed by hydrolysis. An application in the analysis of amino-sugar linkages in alkali-labile O-glycosylic oligosaccharides from rat-brain glycoproteins is described.

The Gal alpha 1-4 Gal-binding adhesin of Streptococcus suis, a gram-positive meningitis-associated bacterium.

Streptococcus suis causes septicaemia and meningitis in pigs and occasionally in humans. A major galactose-inhibitable adhesin recognizing the blood group P-related disaccharide Gal alpha 1-4 Gal beta 1--present in the GbO3 glycolipid was identified in S. suis. Two variant adhesins, inhibitable by galactose and N-acetylgalactosamine (type PN) or galactose only (type Po) both preferred the disaccharide in terminal position. The hydrogen bonding patterns were determined using deoxy and other derivatives of the receptor disaccharide, and were compared to that of E. coli PapG396 adhesin. The essential hydroxyls were the HO-4', HO-6', HO-2 and HO-3 hydroxyls; type Po adhesin also weakly interacted with HO-6 and HO-3'. The mechanism differed from that of E. coli which binds to a cluster of five hydroxyls, HO-6, HO-2', HO-3', HO-4' and HO-6'. The purified adhesin had a molecular weight of 18 kDa and an isoelectric point of 6.4. The agglutination of latex-bound purified adhesin was inhibited by the same inhibitors as agglutination with whole bacteria. The adhesin was detected by immunoblot analysis in all 23 S. suis strains examined representing different serotypes, was highly immunogenic and showed opsonizing activity. This represents the first example of the comparison of the saccharide receptor hydrogen bondings of two bacteria of different origin and shows that the same saccharide may be recognised by two different mechanisms. As a potential virulence factor present in different serotypes the adhesin represents a potential vaccine against S. suis infections.