A new coding mutation in the Tnf-alpha leader sequence in tuberculosis-sensitive I/St mice causes higher secretion levels of soluble TNF-alpha.
I/St and A/Sn mice are polar extremes in terms of several parameters defining sensitivity to Mycobacterium tuberculosis. TNF-alpha, mainly produced by activated macrophages, can mediate both physiological and pathophysiological processes. Adequate TNF-alpha levels are essential for a forceful protective response to M. tuberculosis. We have functionally characterized a nonsynonymous substitution, Arg8 His, in the highly conserved cytoplasmic domain of the pro-TNF-alpha leader peptide from extremely M. tuberculosis-sensitive I/St mice. This was compared to the common pro-TNF-alpha variant found in A/Sn mice. Using cDNA constructs, both variants were constitutively expressed in HEK293A cells. A significantly higher secretion level of Arg8 His TNF-alpha was shown using flow cytometry and ELISA analysis (P=0.0063), while intracellular levels were similar for both protein variants. An even TNF-alpha distribution throughout the cells was seen using confocal microscopy. This suggests that the Arg8 His substitution affects pro-TNF-alpha processing. The I/St mouse may serve as a model to further explore the function of the well-conserved cytoplasmic region of TNF-alpha. However, other identified substitutions in the I/St promoter, introns and 3'UTR of Tnf-alpha, as well as the cellular environment in vivo may affect the balance between soluble and intracellular Arg8 His TNF-alpha before and during M. tuberculosis infection.
Kähler
A K
AK
Department of Molecular Medicine, Karolinska Institutet, CMM, Karolinska Hospital, Stockholm, Sweden.
Persson
A-S
AS
Sánchez
F
F
Källström
H
H
Apt
A S
AS
Schurr
E
E
Lavebratt
C
C
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000235:genetics; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry; D006639:Histidine / Q000235:genetics; D006801:Humans; D051379:Mice; D008817:Mice, Mutant Strains; D009154:Mutation; D011110:Polymorphism, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D012995:Solubility; D014376:Tuberculosis / Q000235:genetics* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*
D021382:Protein Sorting Signals; D014409:Tumor Necrosis Factor-alpha; D006639:Histidine; D001120:Arginine