The identification and characterization of a novel protein, c19orf10, in the synovium

Abstract

Joint inflammation and destruction have been linked to the deregulation of the highly synthetic fibroblast-like synoviocytes (FLSs), and much of our current understanding of the mechanisms that underlie synovitis has been collected from studies of FLSs. During a proteomic analysis of FLS cells, we identified a novel protein, c19orf10 (chromosome 19 open reading frame 10), that was produced in significant amounts by these cells. The present study provides a partial characterization of c19orf10 in FLSs, synovial fluid, and the synovium. Murine monoclonal and chicken polyclonal antibodies were produced against recombinant human c19orf10 protein and used to examine the distribution of c19orf10 in cultured FLSs and in synovial tissue sections from patients with rheumatoid arthritis or osteoarthritis. The intracellular staining pattern of c19orf10 is consistent with localization in the endoplasmic reticulum/Golgi distribution. Sections of rheumatoid arthritis and osteoarthritis synovia expressed similar patterns of c19orf10 distribution with perivascular and synovial lining staining. Double-staining in situ analysis suggests that fibroblast-like synovial cells produced c19orf10, whereas macrophages, B cells, or T cells produced little or none of this protein. There is evidence of secretion into the vascular space and the extracellular matrix surrounding the synovial lining. A competitive enzyme-linked immunosorbent assay confirmed the presence of microgram levels of c19orf10 in the synovial fluids of patients with one of various arthropathies. Collectively, these results suggest that c19orf10 is an FLS-derived protein that is secreted into the synovial fluid. However, the significance of this protein in synovial biology remains to be determined. The absence of known structural motifs or domains and its relatively late evolutionary appearance raise interesting questions about its function.

Figure 1

Proteomic identification of c19orf10. (a) Mass spectra of in-gel digest of c19orf10 with six peptides mapping to the c19orf10 protein labeled. (b) Tandem mass spectrum of c19orf10 parent ion 1,154 Da, 146-TAVAHRPGAFK-156. (c) Tandem mass spectrum of c19orf10 parent ion 1,196 Da, 101-SYLYFTQFK-109. (d) Tandem mass spectrum of c19orf10 parent ion 1,750 Da, 131-ESDVPLKTEEFEVTK-145. Peaks contributing to the score are labeled. The mass and sequence of each parent ion are indicated on the appropriate spectrum. c19orf10, chromosome 19 open reading frame 10.

Figure 2

Genomic organization, alternative splicing, and protein sequence of c19orf10. (a) The chromosomal localization of the region containing the c19orf10 gene is indicated on the ideogram. The area containing the c19orf10 gene is expanded and the base-pair positions are indicated. The location of a microsatellite marker linked to juvenile rheumatoid arthritis, D19S216, is also indicated. (b) The expanded region of chromosome 19 containing the c19orf10 gene. Three putative splicing variants are indicated (c19orf10.a, c19orf10.b, and c19orf10.c). Thick blocks indicate translated exons, open blocks indicate untranslated exons, horizontal lines indicate introns, and the arrows indicate the direction of transcription. (c) Alignment of protein products of c19orf10 splicing variants. Variants a and b seem to be complete sequences starting with an N-terminal methionine. Variant c does not start with an N-terminal methionine and is probably incomplete at the N-terminus. Lines above the sequence map the exons to the protein sequence. Shaded sequences indicate peptides observed by mass spectrometry. The putative N-terminal signal peptide is underlined with a solid black line. C63 and 92 are indicated by rectangles. c19orf10, chromosome 19 open reading frame 10.

Figure 3

c19orf10 immunofluorescence staining of fibroblast-like synoviocytes (FLSs). (a) FLSs were labeled with anti-c19orf10 monoclonal antibody, 1B6, and visualized using red fluorescent cyanine 3 (Cy3) goat anti-mouse immunoglobulin G (IgG) (heavy and light chain reactive [H&L]) antibody. The cells were counterstained with green fluorescent Oregon Green phalloidin to visualize the F-actin. (b) Negative control with no primary antibody, stained as above. (c) FLSs were labeled with anti-c19orf10 monoclonal antibody, 1B6, and visualized using red fluorescent Cy3 goat anti-mouse IgG (H&L) antibody. The cells were counterstained with green fluorescent Oregon Green phalloidin to visualize the F-actin (arrow). (d) Negative control with no primary antibody, stained as above. c19orf10, chromosome 19 open reading frame 10.

Figure 4

Expression of c19orf10 in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium. (a,d,f,h) Expression of c19orf10 in RA synovium. (b,c,e,g) Expression of c19orf10 in OA synovium. (a) Intense staining of the synovial lining layer and perivascular regions of RA (OCT section) tissue.(b) Intense staining of the synovial lining layer and perivascular regions of OA (paraffin section) tissue. (c) An area demonstrating a thin lining layer and perivascular region populated with c19orf10-positive cells. Note that the sublining stroma in this area is virtually devoid of c19orf10 staining. This pattern of staining is typical of that seen in both RA and OA sections. (d) In most lymphocytic aggregates, there was minimal staining of the lymphocytes although some mononuclear cells in the aggregates stained positively (arrow). (e) Intense staining of individual cells in the lining layer of a typical OA synovium. (f) An area of an OA synovium demonstrates a lining layer completely devoid of c19orf10 staining. (g) Intense staining of a hyperplastic RA synovial lining cell layer. This staining was typical of most areas of RA synovium where the lining was hyperplastic. (h) An area of an RA synovial lining layer that is not positive for c19orf10 staining. c19orf10, chromosome 19 open reading frame 10.

Figure 5

The cellular origins of c19orf10 in the synovium. Sections of rheumatoid arthritis synovial tissue stained with anti-c19orf10 stained red. (a) Sections stained with anti-CD68 to detect macrophage-like cells stained brown (arrow). (b) Sections stained with anti-CD55 to detect fibroblasts stained brown (arrows). There was a clear colocalization with CD55-staining cells, whereas the association with CD63-positive cells was much less apparent. c19orf10, chromosome 19 open reading frame 10.

Figure 6

Demonstration of c19orf10 in synovial fluids. Synovial fluid c19orf10 levels were determined by competitive enzyme-linked immunosorbent assay for five patients with the indicated arthropathies. Each fluid was measured at two different dilutions. The concentrations for each are indicated in the table. A representative standard curve is presented in the graph. c19orf10, chromosome 19 open reading frame 10; GST-orf10, glutathione S transferase-open reading frame 10; SF, synovial fluid.

Figure 7

The presence of c19orf10 homologues is predicted in multiple vertebrate species. All sequences were obtained from GenBank [25]. Homo sapiens, NP_061980; Mus musculus, NP_543027; Rattus norvegicus, XM_347126; Sus scrofa, SSC.4092; Bos taurus, NP_01001164; Canis familiaris, ENSCAFT30192; Gallus gallus, NP_001006342; Danio rerio, NP_001002480; Tetraodon nigroviridis, CAG08012; and Leucoraja erinacea, CV067465 (translated in reading frame +3); Squalus acanthias, CX789984 (translated in reading frame +2). c19orf10, chromosome 19 open reading frame 10.