title: Cleavage of recombinant human corticotropin-releasing factor (CRF)-binding protein produces a 27-kilodalton fragment capable of binding CRF.
abstract: CRF is both a peripheral and a central mediator of inflammation, the activity of which is modified by the presence of a 37-kDa binding protein (CRF-BP). The objective of this study was to measure and characterize this protein in the synovial fluid of rheumatoid arthritis patients and to observe the effects of this inflammatory condition on its structure and properties. Measured by immunoradiometric assays, the mean CRF-BP concentration in synovial fluid from 27 arthritic patients was 0.51 nmol/L (SD = 0.24 nmol/L); that for CRF was 6.31 pmol/L. The mean plasma concentration of CRF-BP in 24 control subjects was 1.38 nmol/L (SD = 0.35 nmol/L) and that for 10 arthritic patients was 2.89 nmol/L (SD = 0.84 nmol/L). Synovial fluids were found by immunoblotting to contain intact CRF-BP and a 10-kDa C-terminal CRF-BP fragment; synovial fluid from healthy controls was not examined. We previously reported that after purification of recombinant CRF-BP, spontaneous cleavage frequently occurs, resulting in a 27-kDa N-terminal and a 10-kDa C-terminal fragment. Because concentrations of native CRF-BP in synovial fluid were insufficient to study the effects of cleavage on ligand binding, they were determined using recombinant human CRF-BP. Tryptophan excitation fluorescence spectra of intact and cleaved recombinant CRF-BP revealed that cleavage was accompanied by conformational change in the N-terminal fragment, leading to exposure of the sole tryptophan residue to polar molecules (emission peak shift from 310 to 250 nm). Using gel filtration chromatography to separate the N- and C-terminal fragments, it was found that the N-terminal fragment of the recombinant protein bound human CRF, although dimerization was somewhat impaired. The C-terminal fragment did not bind CRF. Scatchard analysis confirmed that the affinity of both intact and cleaved CRF-BP for CRF was 1 x 10(10) L/mol. We conclude that synovial fluid contains intact CRF-BP in molar excess to CRF and fragmented CRF-BP. The significance of cleavage and the role of cleavage products have yet to be determined, although they may represent the generation of a novel bioactivity.
authors.affiliation: School of Animal and Microbial Sciences, University of Reading, Whiteknights, United Kingdom. r.j.woods@reading.ac.uk
authors.forename: R J
authors.initials: RJ
authors.lastname: Woods
authors.forename: C F
authors.initials: CF
authors.lastname: Kemp
authors.forename: J
authors.initials: J
authors.lastname: David
authors.forename: I G
authors.initials: IG
authors.lastname: Sumner
authors.forename: P J
authors.initials: PJ
authors.lastname: Lowry
mesh_terms: D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism*; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000378:metabolism; D013582:Synovial Fluid / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; D011994:Recombinant Proteins; C067133:corticotropin releasing factor-binding protein; D003346:Corticotropin-Releasing Hormone