Isolation of NILE glycoprotein-related cDNA probes.
The nerve growth factor (NGF)-induced large external (NILE) glycoprotein is an NGF-inducible surface component of PC12 cells that is also widely distributed in the nervous system. It has recently been shown to be indistinguishable from the high-molecular-weight species of the brain antigens L1 and neuron-glia cell adhesion molecule (Ng-CAM) and may have a function in regulating cell adhesion in the developing nervous system. We have used polyclonal anti-NILE antisera to screen a lambda gt11 cDNA library made from NGF-treated PC12 cells. Four molecular probes have been isolated that encode parts of the apoprotein, related proteins, or both. These probes are 1,500, 800, 330, and 300 base pairs long, respectively, and in Northern blots they recognize a family of messages having lengths of approximately 5.9, 3.4, 2.4, and 1.9 kilobases. The two smaller messages are modestly but reproducibly up-regulated by NGF in PC12 cells, as is the NILE glycoprotein; however, only the two larger species would appear to be large enough to encode it. These messages are prominent in brain but not in nonneural tissues, in accordance with the observed levels of the protein. The recombinant phages produce fusion proteins that share specific epitopes with the NILE glycoprotein but not with other proteins. In these experiments, filters coated with recombinant fusion protein were prepared. Antibodies bound to and eluted from these filters specifically immunoprecipitate NILE glycoprotein, but not other proteins, from PC12 cell extracts. Other activities present in the original sera are not specifically retained by recombinant fusion proteins, and Escherichia coli lysates made with nonrecombinant lambda gt11 do not absorb the anti-NILE activity.(ABSTRACT TRUNCATED AT 250 WORDS)
D000310:Adrenal Gland Neoplasms / Q000032:analysis; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000235:genetics*; D006168:Guinea Pigs; D039842:Neural Cell Adhesion Molecule L1; D010673:Pheochromocytoma / Q000032:analysis; D011061:Poly A / Q000032:analysis; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D014018:Tissue Distribution
D006023:Glycoproteins; D039842:Neural Cell Adhesion Molecule L1; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA