Defining a novel 75-kDa phosphoprotein associated with SS-A/Ro and identification of distinct human autoantibodies
Dunrui Wang, … , Weiguo Zhu, Edward K.L. Chan
Dunrui Wang, … , Weiguo Zhu, Edward K.L. Chan
Published November 1, 1999
Citation Information: J Clin Invest. 1999;104(9):1265-1275. https://doi.org/10.1172/JCI8003.
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Defining a novel 75-kDa phosphoprotein associated with SS-A/Ro and identification of distinct human autoantibodies

Abstract

Mothers of children with neonatal lupus erythematosus (NLE) and heart block, as well as patients with Sjögren’s syndrome (SS) and systemic lupus erythematosus, have serum autoantibodies that recognize SS-A/Ro autoantigens including the 60-kDa ribonucleoprotein. By yeast 2-hybrid screening, we identified a novel 75-kDa protein (pp75) that interacts with the carboxyl 70% of 60-kDa SS-A/Ro. The specificity of interaction was confirmed using mammalian 2-hybrid and chemical crosslinking studies. Immunoprecipitation with radiolabeled HeLa cell extracts showed that pp75 was phosphorylated and associated with 2 other phosphoproteins of 64 kDa. In Northern blot analysis, pp75 was expressed in all tissues analyzed; the highest expression was in the human heart. Based on immunofluorescence of transfected HeLa cells, pp75 is localized predominantly in the cytoplasm, an observation confirmed by immunohistochemistry in untransfected cells. Based on Western blot and ELISA assays, sera from 14 of 84 mothers of children with NLE recognized pp75, including 1 mother in whom anti–SS-A/Ro antibodies were not detected. In addition, sera from 5 of 80 patients with SS were positive for anti-pp75 antibody. Identification of molecular partners is a first step toward elucidating the functions and possible involvement in pathogenesis of long-recognized autoantigens such as 60-kDa SS-A/Ro, which are at present poorly understood.

Authors

Dunrui Wang, Jill P. Buyon, Weiguo Zhu, Edward K.L. Chan

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Figure 1

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Cloning of human 60-kDa SS-A/Ro–associated protein pp75. (a) Schematic r...
Cloning of human 60-kDa SS-A/Ro–associated protein pp75. (a) Schematic representation of cDNAs pINP1 derived from yeast 2-hybrid cloning, 5′ RACE, WI38 cDNA library screen, and clone KIAA0857. The shaded boxes represent the open reading frame (ORF). (b) Nucleotide and deduced amino acid sequence of pp75/KIAA0857. Nucleotide sequence of 3,017 bases for the WI38 cDNA was submitted to GenBank under accession number AF153085. The nucleotide sequences corresponding to the ORF between sequence submitted under AF153085 and clone KIAA0857 are identical; 4 single base substitutions and insertion/deletion are observed in the 3′ untranslated regions. The methionine start site (Kozak’s sequence CCGCCATGG) and poly-A signal AATAAA are underlined. The GC-rich 5′-untranslated region is highlighted by a thick underline of 5 or more consecutive GC residues. The protein has relatively high percentage of serine residues (13.94%; boldface and italics). A leucine zipper motif is double-underlined, and the 4 leucine residues are circled. RT-PCR primers and antisense RNA probe for the analysis of mRNA expression in different cells and tissues are indicated by arrows in both panels. The BamHI site used to generate antisense RNA probe is boxed in b.