Characterization of a partial cDNA clone for the NILE glycoprotein and identification of the encoded polypeptide domain

Abstract

A partial cDNA clone [2.4 kilobase (kb)] for the nerve growth factor-inducible large external (NILE) glycoprotein was selected from a lambda gt 11 expression library constructed using mRNA from PC 12 cells. A 0.2 kb subclone (pNILE-1B) was used for Northern blot analysis of NILE message present in 2 NILE-positive neuronal cell lines and 2 NILE-negative glial cell lines. pNILE-1B hybridizes with components of 6.8 and 2.0 kb in the 2 neuronal cell lines but fails to show hybridization with any components in the 2 glial cell lines. Only the 6.8 kb species would be large enough to code for the NILE polypeptide. A rabbit antiserum was prepared against the NILE-beta-galactosidase fusion protein produced by the NILE clone. This antiserum (anti-NILE-beta-gal) immunoprecipitates NILE glycoprotein from neuronal cell lines, further confirming the authenticity of the NILE cDNA clone. The epitope recognized by anti-NILE-beta-gal is contained in an 85 kDa tryptic fragment from the phosphorylated carboxy terminus of NILE. The 160 kDa tryptic fragment containing the amino terminus is not recognized by anti-NILE-beta-gal. Both immunoprecipitation and immunofluorescence experiments indicate that the anti-NILE-beta-gal epitope is not exposed on the cell surface but is accessible only after cells are treated with detergent. The cytoplasmic nature of the determinant is also indicated by its absence on a truncated, soluble form of NILE released from cells (possibly by a proteolytic mechanism) into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)