title: 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.affiliation: Department of Biochemistry and the Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Shetzline
authors.forename: Camille
authors.initials: C
authors.lastname: Martinand-Mari
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Reichenbach
authors.forename: Zivjena
authors.initials: Z
authors.lastname: Buletic
authors.forename: Bernard
authors.initials: B
authors.lastname: Lebleu
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Pfleiderer
authors.forename: Ramamurthy
authors.initials: R
authors.lastname: Charubala
authors.forename: Kenny
authors.initials: K
authors.lastname: De Meirleir
authors.forename: Pascale
authors.initials: P
authors.lastname: De Becker
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Peterson
authors.forename: C V T
authors.initials: CV
authors.lastname: Herst
authors.forename: Patrick
authors.initials: P
authors.lastname: Englebienne
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Suhadolnik
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002457:Cell Extracts / Q000032:analysis; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D015673:Fatigue Syndrome, Chronic / Q000201:enzymology*; D006801:Humans; D020014:K562 Cells; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D008970:Molecular Weight; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000345:Affinity Labels; D001386:Azides; D002457:Cell Extracts; D004220:Disulfides; D012333:RNA, Messenger; C012760:8-azidoadenosine; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D000241:Adenosine