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Preparation of affinity-purified, biotinylated tetanus toxin, and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

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Abstract

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 1∶1 to 20∶1 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these “primed” cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.

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Abbreviations

PBS:

phosphate-buffered saline

STS:

sucrose-Tris-serum solution

NGF:

nerve growth factor

C:

collagen

PL:

polylysine

BBG:

bovine brain ganglioside mixture

GM1 :

gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide

GD1a :

[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide

GT1a :

[N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide

GD1b :

galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide

GT1b :

[N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide

NANA:

N-acetylneuraminic acid

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Authors and Affiliations

  1. Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, 20892, Bethesda, Maryland

    Ko Fujita,  Gordon Guroff & Philip Lazarovici

  2. Department of Neurobiology, The Weizmann Institute of Science, 26100, Rehovot, Israel

    Ephraim Yavin

  3. Laboratory of Cell Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892, Bethesda, Maryland

    Gerthrud Goping & Richard Orenberg

  4. National Institute of Child Health and Human Development, National Institutes of Health, Building 6, Room 130, 20892, Bethesda, MD

    Gordon Guroff

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Fujita, K., Guroff, G., Yavin, E. et al. Preparation of affinity-purified, biotinylated tetanus toxin, and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells. Neurochem Res 15, 373–383 (1990). https://doi.org/10.1007/BF00969922

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