AG265 Sigma-AldrichNeural Cell Adhesion Molecule, chicken

A unique structural feature of the neural cell adhesion molecule N-CAM is the presence of homopolymers of alpha (2----8)-linked sialic acid units. We have used two specific probes for the detection of poly(sialic acid) in normal human kidney and Wilms tumor: a monoclonal antibody against meningococci group B capsular polysaccharide (homopolymers of alpha (2----8)-linked sialic acid units), which shows no crossreactivity with polynucleotides and denaturated DNA, and bacteriophage-induced endosialidases specifically hydrolyzing alpha (2----8)-linked poly(sialic acid) units. Additionally, for the detection of N-CAM, antibodies recognizing the polypeptide portion of the molecule and biotinylated antisense RNA transcribed from a cDNA clone for N-CAM were applied. Poly(sialic acid) was regionally detectable in human embryonic kidney but undetectable in normal adult kidney, as already reported for rat kidney. The malignant Wilms tumor, which is characterized by the presence of structural components resembling those found in embryonic kidney, reexpressed poly(sialic acid) units and showed positive immunostaining for the polypeptide portion of N-CAM. Immunoblot analysis of Wilms tumor as well as human embryonic kidney and brain with the monoclonal anti-poly(sialic acid) antibody revealed in each case the same high molecular mass broad band. In situ hybridization demonstrated the presence of mRNA for N-CAM in Wilms tumor. We conclude that poly(sialic acid), most probably present on N-CAM, is an oncodevelopmental antigen in human kidney.

The neural cell adhesion molecule, N-CAM, is a cell surface glycoprotein found on embryonic and adult neurons and on a variety of ectodermal and mesodermal tissues in very early embryos. During development, it shows local variations in prevalence at the cell surface as well as conversion from an embryonic form (E form) with high sialic acid content to an adult form (A form) with lesser amounts of this sugar. This E leads to A conversion occurs on different schedules in different brain regions, and it has been hypothesized that both the conversion and the prevalence changes are related to early regulation of pattern formation and connectivity. In order to identify precisely the consequences of these mechanisms of local cell surface modulation of N-CAM, an assay was developed to measure the rate of aggregation either of vesicles reconstituted from lipid and purified N-CAM or of native brain membrane vesicles. In both preparations, aggregation was greater than 95% inhibitable by specific anti-(N-CAM) Fab' fragments. The rates of aggregation of reconstituted N-CAM vesicles and native brain vesicles were found to be inversely related to the sialic acid content of their N-CAM molecules, with full desialylation resulting in about a 4-fold increase in rate over E-form N-CAM. Intermediate rates were obtained both with A-form N-CAM (which contains only one-third of the sialic acid content of E-form N-CAM) and with partially desialylated E-form N-CAM. The rate of coaggregation of reconstituted vesicles containing E-form N-CAM with reconstituted vesicles containing A-form N-CAM was also intermediate, implying that desialylation did not change the nature of (N-CAM)-(N-CAM) binding but only its rate. Even larger alterations in vesicle aggregation rate were seen when the amount of N-CAM per vesicle was altered. A 2-fold increase in the N-CAM-to-lipid ratio of reconstituted vesicles resulted in a greater than 30-fold increase in their rate of aggregation. Moreover, desialylation did not cause a further increase in the rate of aggregation of these already rapidly aggregating vesicles. These results in a model system demonstrate the large range of binding rates that are obtainable by various forms of local surface modulation of N-CAM. They are consistent with the proposal that similar alterations affecting (N-CAM)-mediated cell adhesion in vivo may be major factors in pattern formation during development of the nervous system.