Vase mini-exon usage by NCAM is not restricted to tumours of neuroectodermal origin.

The neural cell adhesion molecule (NCAM) plays an important role in normal development. Many variants of NCAM are generated through post-transcriptional and post-translational modifications. These variants are tissue-specific and their expression is developmentally regulated. NCAM is also re-expressed in a number of human tumours, including neuroblastoma, rhabdomyosarcoma, Wilms' tumour and Ewing's sarcoma. We have characterized the NCAM variants associated with rhabdomyosarcoma. Polysialylated NCAMs are present in this tumour and, after neuraminidase treatment, they resolve into 2 bands of 140 and 120 kDa. These data were corroborated by Northern-blot analysis where mRNA species of 6.7 and 5.5 kb are detected. These mRNA code for the 140- and 120-kDa NCAM proteins respectively. PCR analysis shows that the previously described VASE mini-exon is also present in NCAM found in rhabdomyosarcoma. The VASE mini-exon, spliced at exon 7-8 junctions, has previously been detected in neural and heart NCAM, as well as in NCAMs found in human small-cell lung carcinoma (SCLC). DNA sequencing confirmed that the VASE mini-exon in rhabdomyosarcoma is identical to that found in neuroblastoma and SCLC.

A discordance between the human muscle NCAM sequence and those seen in other NCAM cDNA clones.

A cDNA clone, designated NC7, has been isolated from human foetal kidney that partially codes for the 140 kDa isoform of human NCAM. This clone contains a 6 bp insert that is not present in the human muscle cDNA clone lambda 4.4. This same sequence has also been found in both a cDNA clone obtained from a human Small Cell Lung Carcinoma (SCLC) line and in human genomic DNA. Furthermore, an equivalent sequence to the 6 bp region identified in the above samples is present in mouse, rat and chicken NCAM. The 6 bp insertion does not lie at a predicted intron/exon boundary as extrapolated by homology studies with the chicken and, therefore, the mechanism by which the sequence is deleted from the human muscle clone lambda 4.4 remains unclear.

Splicing of the VASE exon of neural cell adhesion molecule (NCAM) in human small-cell lung carcinoma (SCLC).

Expression of the neural cell adhesion molecule (NCAM) on small-cell lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.

A comparison of the lectin-binding properties of glycoconjugates from a range of Leishmania species.

Constituent glycoconjugates of promastigotes of 14 different Leishmania strains from 6 different species were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently stained with 14 125I-labelled lectins of different specificities. Autoradiography of the gels revealed lectin-specific glycoproteins and other glycoconjugates of known molecular weight. Similarities and differences in antigens and glycoproteins present in the strains are described. The possibility of identification or characterisation of Leishmania species from their electrophoretic behaviour and lectin-binding patterns is unlikely, but these results should be an aid to purification of glycoconjugates from particular strains by lectin-affinity chromatography.

Monoclonal antibody UJ13A recognizes the neural cell adhesion molecule (NCAM).

Monoclonal antibody (MAb) UJ13A, raised against 16-week-old human foetal brain, recognizes an antigen present on the majority of tissues of neuro-ectodermal origin. The binding profile of this antibody is similar to the known distribution of the neural cell adhesion molecule (NCAM). Although detailed immunohistological and immuno-cytochemical studies with the MAb have been published previously, we demonstrate here, through investigations of selected tissues and cell lines, that the binding profile of an anti-NCAM antiserum is similar to UJ13A. Additional indirect evidence suggesting that UJ13A recognizes NCAM comes from Northern blot analysis of cell lines either binding or not binding UJ13A as determined by indirect immunofluorescence. Only those cell lines known to bind UJ13A express high levels of NCAM mRNA. Western blot analysis of extracts of human brain show that UJ13A recognizes proteins of 180 and 140 kDa in addition to a very weak band of 120 kDa. This data corroborates the suggestion that UJ13A recognizes NCAM as these 3 isoforms of the protein are identified in human brain. Final confirmation of the specificity of UJ13A comes from the study of 3T3 fibroblasts transfected with a cDNA coding for the 125 kDa isoform of human muscle NCAM. UJ13A selectively recognizes these transfectants and binds to a 125 kDa protein isolated from the cells by Western blot analysis. Thus we conclude from these immunological, biochemical and molecular studies that the antigen recognized by the UJ13A MAb is the neural cell adhesion molecule.

Monoclonal antibody 3F8 recognises the neural cell adhesion molecule (NCAM) in addition to the ganglioside GD2.

The monoclonal antibody 3F8 has been described as binding to the ganglioside GD2. This antibody, of the IgG3 isotype, has been used in immunotherapy, radioimmunolocalisation and targeted radiation therapy. 3F8 was originally observed to have a binding profile similar to two monoclonal antibodies, UJ13A and 5.1.H11, characterised as binding to the neural cell adhesion molecule (NCAM). This observation has also been confirmed using a hetero-antiserum prepared against purified NCAM. The cross-reactivity of 3F8 with NCAM has been confirmed by cross-blocking studies with an anti-NCAM antiserum, and by direct immunoprecipitation and gel electrophoresis. In addition, we show that 3F8 binds to human NCAM from 3T3 fibroblasts transfected with NCAM cDNA constructs. It is possible that the common epitope shared by GD2 ganglioside and NCAM involves sialic acid residues common to both the ganglioside and the glycoprotein.

Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma.

We describe reagents from 2 workshops which had been identified as recognizing the same or very similar antigens based on their tissue reactivity. Examination of their tissue specificity led us to the conclusion that this was similar to the expression of the neural cell adhesion molecule (NCAM). We also describe the use of a transfection-based assay to show that these reagents do recognize NCAM. 3T3 cells were transfected with a full-length clone of human NCAM. Indirect immunofluorescence studies showed binding of all related antibodies to the transfectants, but not to the control 3T3 cells. In addition, biochemical analysis using certain antibodies in the cluster confirm that they detect NCAM in the transfectants. Our study shows the benefits of using workshops to compare monoclonal antibodies and a molecular approach to define the antigens recognized by such reagents.