Isolation and characterization of a novel glycosyl-phosphatidylinositol-anchored glycoconjugate expressed by developing neurons.
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-beta-galactosidase and sialidase treatment; (e) it was labile to mild acid hydrolysis without loss of immunoreactivity; (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.
CNRS 179, Biologie de la Différenciation Cellulaire, Marseille, France.
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J
Nedelec
M
M
Pierres
H
H
Moreau
J
J
Barbet
P
P
Naquet
C
C
Faivre-Sarrailh
G
G
Rougon
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002808:Chondroitin Lyases / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002855:Chromatography, Thin Layer; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000378:metabolism; D005434:Flow Cytometry; D006017:Glycolipids / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols; D019763:Heparin Lyase; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009439:Neuraminidase / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D012313:RNA; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D009439:Neuraminidase; D001616:beta-Galactosidase; D006821:Hyaluronoglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002808:Chondroitin Lyases; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase