Structural, functional and immunologic characterization of folded subdomains in the Ro52 protein targeted in Sjögren's syndrome.
Ro52, one of the major autoantigens in the rheumatic disease Sjögren's syndrome (SS), belongs to the tripartite motif (TRIM) or RING-B-box-coiled-coil (RBCC) protein family, thus comprising an N-terminal RING, followed by a B-box and a coiled-coil region. Several different proteomic functions have been suggested for Ro52, including DNA binding, protein interactions and Zn(2+)-binding. To analyze the presence and/or absence of these functions and, in particular, map those to different subregions, the modular composition of the Ro52 protein was experimentally characterized. Two structured parts of Ro52 were identified, corresponding to the RING-B-box and the coiled-coil regions, respectively. Secondary structure analysis by circular dichroism (CD) spectroscopy indicated that the two subregions are independently structured. The entire RING-B-box region displayed Zn(2+)-dependent stabilization against proteolysis in the presence of Zn2+, indicating functional Zn(2+)-binding sites in both the RING and the B-box. However, no stabilization with DNA was detected, irrespective of Zn(2+), thus suggesting that the RING-B-box region does not bind DNA. Oligomerization of the coiled-coil was investigated by analytical ultracentrifugation and in a mammalian two-hybrid system. Both methods show weak homodimer affinity, in parity with other coiled-coil domains involved in regulatory interactions. The C-terminal B30.2 region was rapidly degraded both during cellular expression and refolding, indicating a less stable structure. Immunologic analysis of the stable protein regions with sera from patients with Sjögren's syndrome shows that immunodominant epitopes to a large extent are localized in the structurally stable parts of Ro52. The results form a basis for further Ro52 functional studies on the proteome level.
Ottosson
Lars
L
Rheumatology Unit, Department of Medicine, CMM L8:04, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
Hennig
Janosch
J
Espinosa
Alexander
A
Brauner
Susanna
S
Wahren-Herlenius
Marie
M
Sunnerhagen
Maria
M
D001324:Autoantigens; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D016056:Immunodominant Epitopes; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000276:immunology* / Q000502:physiology; D012859:Sjogren's Syndrome / Q000276:immunology*; D013696:Temperature; D015032:Zinc; D000099207:SS-A Antigen
D001324:Autoantigens; D016056:Immunodominant Epitopes; D012261:Ribonucleoproteins; D015032:Zinc; D000099207:SS-A Antigen