Isolation and characterization of a novel glycosyl‐phosphatidylinositol‐anchored glycoconjugate expressed by developing neurons

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Abstract
In search of new markers for studying thymic and nervous system ontogeny, we raised rat monoclonal antibodies against glycosyl‐phosphatidylinositol‐anchored molecules among which larger groupings have been shown to be ectoenzymes and adhesion molecules. Two of these monoclonal antibodies (H193–4 and H194–563, IgG) were found to recognize glycosyl‐phosphatidylinositol‐anchored glycoconjugates of 28–33 kDa (P31) and 50–70 kDa in developing mouse brain and thymus respectively, when these tissues were analysed by immunoblot experiments. P31 antigen was found to be transiently expressed by neurons in neural primary cultures [Rougon, G., Alterman, L., Dennis, K., Guo, X. J. & Kinnon, K. (1991) Eur. J. Immunol. 21, 1397–1402]. We show in this report that, in developing mouse brain, a maximal expression occurred between embryonic day 17 and post‐natal day 5, a period that corresponds to the formation of neuronal networks. P31 antigen was immunopurified and found to possess the following properties: (a) it was soluble in alkaline solvents; (b) it bound to DEAE‐cellulose and was eluted by a salt gradient of 0–1 M NaCl; (c) it was sensitive to endoglycosidase F digestion; (d) it was insensitive to heparinase, hyaluronidase, chondroitinase ABC, endo‐β‐galactosidase and sialidase treatment; (e) it was labile to mild acid hydrolysis without loss of immunoractivity; (f) it contained phosphate; (g) it lost its immunoreactivity after treatment with phosphatidylinositol phospholipase C and treatment. These characteristics combine to suggest that P31 is an anionic glycoconjugate sharing similarities with Leishmania donovani lipophosphoglycan and with the heat‐stable antigen recognized by J11d antibody on murine hematopoïetic cells. This last hypothesis was further confirmed by the observation that oligonucleotide probes derived from the heat‐stable antigen‐encoding cDNA detect, in developing brain, a 1.8‐kb mRNA species similar in size to that reported for the heat‐stable antigen mRNA and following the same developmental expression as P31 antigen.