Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in Chronic Fatigue Syndrome (CFS)

Previous studies from this laboratory have demonstrated a

statistically significant dysregulation in several key

components of the 2′,5′-oligoadenylate (2-5A) synthetase/RNase

L and PKR antiviral pathways in chronic fatigue syndrome (CFS)

(Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994;

Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies

have been developed to further examine the upregulated RNase L

activity in CFS. First, photoaffinity labeling of extracts of

peripheral blood mononuclear cells (PBMC) with the azido 2-5A

photoaffinity probe, [32P]pApAp(8-azidoA), followed by

immunoprecipitation with a polyclonal antibody against

recombinant, human 80-kDa RNase L and analysis under

denaturing conditions. A subset of individuals with CFS was

identified with only one 2-5A binding protein at 37 kDa,

whereas in extracts of PBMC from a second subset of CFS PBMC

and from healthy controls, photolabeled/immunoreactive 2-5A

binding proteins were detected at 80, 42, and 37 kDa. Second,

analytic gel permeation HPLC was completed under native

conditions. Extracts of healthy control PBMC revealed 2-5A

binding and 2-5A-dependent RNase L enzyme activity at 80 and

42 kDa as determined by hydrolysis of poly(U)-3′-[32P]pCp. A

subset of CFS PBMC contained 2-5A binding proteins with

2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa.

However, a second subset of CFS PBMC contained 2-5A binding

and 2-5A-dependent RNase L enzyme activity only at 30 kDa.

Evidence is provided indicating that the RNase L enzyme

dysfunction in CFS is more complex than previously reported.

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