Structural and functional features of the 37-kDa 2-5A-dependent RNase L in chronic fatigue syndrome.

Abstract

A 2',5'-oligoadenylate (2-5A)-dependent 37-kDa form of RNase L has been reported in extracts of peripheral blood mononuclear cells (PBMC) from individuals with chronic fatigue syndrome (CFS). In the current study, analytic gel permeation FPLC, azido photoaffinity labeling, two-dimensional (2-D) gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been used to examine the biochemical relationship between the 80-kDa RNase L in healthy control PBMC and the 37-kDa RNase L in PBMC from individuals with CFS. Like the 80-kDa RNase L, the 37-kDa RNase L is present as a catalytically inactive heterodimer complex with the RNase L inhibitor (RLI). Formation of a 37-kDa RNase L-RLI complex indicates that the 37-kDa RNase L is structurally similar to the 80-kDa RNase L at the N-terminus, which contains the 2-5A binding domain. The enzymatically active monomer form of 37-kDa RNase L resolved by 2-D gel electrophoresis has a pI of 6.1. RT-PCR and Southern blot analyses demonstrated that the 37-kDa RNase L is not formed by alternative splicing. In-gel tryptic digestion of the 37-kDa RNase L that was excised from 2-D gels and subsequent MALDI-MS analysis identified three peptide masses that are identical to three predicted peptide masses in the 80-kDa RNase L. The electrophoretic mobility of 2-5A azido photolabeled/immunoprecipitated 37-kDa RNase L was the same under reducing and nonreducing conditions. The results presented show that the 37-kDa form of RNase L in PBMC shares structural and functional features with the native 80-kDa RNase L, in particular in the 2-5A binding and catalytic domains.

• Authors: Susan E Shetzline, Camille Martinand-Mari, Nancy L Reichenbach, Zivjena Buletic, Bernard Lebleu, Wolfgang Pfleiderer, Ramamurthy Charubala, Kenny De Meirleir, Pascale De Becker, Daniel L Peterson, C V T Herst, Patrick Englebienne, Robert J Suhadolnik
• Journal: Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research (J Interferon Cytokine Res) Vol. 22 Issue 4 Pg. 443-56 (Apr 2002) ISSN: 1079-9907 [Print] United States
• PMID: 12034027 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Affinity Labels
• Azides
• Cell Extracts
• Disulfides
• RNA, Messenger
• 8-azidoadenosine
• Endoribonucleases
• 2-5A-dependent ribonuclease
• Adenosine

Topics

• Adenosine (analogs & derivatives, chemistry)
• Affinity Labels (chemistry)
• Azides (chemistry)
• Cell Extracts (analysis)
• Cells, Cultured
• Chromatography, Gel
• Disulfides (metabolism)
• Electrophoresis, Gel, Two-Dimensional
• Endoribonucleases (chemistry, genetics, physiology)
• Fatigue Syndrome, Chronic (enzymology)
• Humans
• K562 Cells
• Leukocytes, Mononuclear (enzymology)
• Molecular Weight
• RNA, Messenger (biosynthesis)
• Reverse Transcriptase Polymerase Chain Reaction
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization