Human Pro-Tumor Necrosis Factor: Molecular Determinants of Membrane Translocation, Sorting, and Maturation

Abstract

Human pro-tumor necrosis factor (pro-TNF) is a type II transmembrane protein with a highly conserved 76-residue leader sequence. We have analyzed the behavior, both in a microsomal translocational system and by transfection, of a series of mutants with deletions from the cytoplasmic, transmembrane, and linking domains. Cytoplasmic deletions included the Arg doublet at ؊49 and ؊48 and/or the Lys doublet at ؊58 and ؊57; additional mutants included deletion of residues ؊73 to ؊55 and ؊73 to ؊55, ؊49, and ؊48. The transmembrane and linking domain mutants included deletions in the ؊42 to ؊35 region, combined with the deletion of residues ؊32 to ؊1. Two hybrid mutants combined the cytoplasmic deletions with the deletion of residues ؊32 to ؊1. All of the cytoplasmic deletion mutants were properly translocated, as were the transmembrane deletion mutants with deletions up to residues ؊36, ؊35, ؊32 to ؊1, although the last one exhibited reduced efficiency; further incremental deletions, including deletions of residues ؊38 to ؊35 and ؊32 to ؊1, completely blocked translocation. Both hybrid mutants were effectively translocated; furthermore, transfection analysis revealed competent expression and maturation of both the cytoplasmic and hybrid mutants. Thus, proper expression and maturation of human pro-TNF can be accomplished with as few as ϳ12 of the 26 residues of the native transmembrane domain and with a net negative charge in the cytoplasmic domain flanking the transmembrane region.

Amino Acid Residue Penultimate to the Amino-terminal Gly Residue Strongly Affects Two Cotranslational Protein Modifications, N-Myristoylation andN-Acetylation

“…After blocking with nonfat milk, the membrane was probed with a specific goat anti-human TNF antibody as described previously (25). Immunoreactive proteins were specifically detected by incubation with horseradish peroxidaseconjugated anti-goat IgG antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).…”

“…In Vitro Transcription and Translation-Methods essentially identical to those described previously were employed (23). T3 polymerase was used to obtain transcripts of these cDNAs subcloned into pB vector.…”

“…Plasmid Construction-Plasmid pBluescript II SK(ϩ) lacking ApaI and HindIII sites was constructed as previously described (23) and designated pB. Plasmid pBpro-TNF, which contains the full-length human pro-TNF cDNA, and plasmid pB⌬pro-TNF, containing a cDNA coding for the mature domain of TNF, were constructed as described (21,23). Plasmid pBV2G-TNF was constructed by utilizing PCR.…”

“…Immunoprecipitation-Samples containing TNF mutants were immunoprecipitated with a specific goat anti-human TNF polyclonal antibody (R & D systems) as described (23).…”

A new coding mutation in the Tnf-α leader sequence in tuberculosis-sensitive I/St mice causes higher secretion levels of soluble TNF-α

“…However, translocation and release of soluble TNF-a has previously been shown, in the human form, to proceed even after deletion of all cytoplasmic charged residues including Arg8 and approximately 70% of the cytoplasmic domain. 42 Still, that this region is highly conserved indicates functionality. The Arg8His substitution may affect the processing selectivity.…”