Analysis of the specificity of anti‐pm‐scl autoantibodies

Abstract

Objective. To compare the specificity of anti-PMScl autoantibodies in serum samples from 43 patients with myositis, scleroderma, or both.Methods. Anti-PM-Scl immunoprecipitates from HeLa cell extract were used as antigen for immunoblot analyses to determine the antigenic components. A series of complementary DNA fragments was expressed in Escheriehiu coli for immunoblot examination of the reaction with the 100-kd protein.Results. The immunoblot against immunoprecipitates was sensitive and specific for detecting reactions with components of the PM-Scl antigen: 42 of 43 sera (97.7%) reacted with the lOO-kd, 27 of 43 (62.8%) with the 70-kd, and 5 of 43 (11.6%) with the 37-kd protein (not previously recognized as antigenic). Forty-one sera reacted with N-terminal protein S1 (amino acids 11-437), 39 with central protein S2 (amino acids 439-749), and 24 with C-terminal protein S3 (amino acids 750-882). Of 42 sera tested, 28 (66.7%) reacted most strongly with S1, and 6 (14.3%) reacted most strongly with S2. Absorption studies implied additional, conformational epitopes not present on the bacterially expressed antigen.Conclusion. There was an overall similarity in reactivity to the PM-Scl antigen, but there were differences in the reactivity to the 70-kd and 37-kd proteins, as well as in the relative strength of the reactivity to the S2 protein.Anti-PM-Scl autoantibody is strongly associated with a clinical picture marked by overlap of

A human aminoacyl-tRNA synthetase as a regulator of angiogenesis

“…Residues 12-65 of the NH 2 -terminal extension of human TrpRS are homologous to the WHEP-TRP conserved domain protein family (pfam00458) (8). This domain has a helix-turn-helix architecture (9) and also is present in the human tRNA synthetases for glycine, histidine, and methionine (10)(11)(12)(13). Three repeats of this domain are inserted into the linker region of the fused glutamyl-prolyl-tRNA synthetase (9,14,15).…”

Epitope analysis of the major reactive region of the 100‐kd protein of PM‐Scl autoantigen

“…All but 1 was known to be Figure 1, were prepared by PCR amplification (performed as previously described [7]), using gene-specific primers and the S1 fragment as template. Each of the 9 deletion fragments was isolated independently by agarose gel electrophoresis, subcloned into pQE plasmid, and transformed into E coZi strain MlS/pREP4 using the Qiaexpressionist system (Qiagen, Chatsworth, CA) as previously described (14). The constructs used produced independent proteins except for a 6-histidine tag.…”

“…A 20-aa peptide (MAP4b) was similarly synthesized, but without the proline at the third peptide position (aa 228 in the whole protein), and was similarly tested. The hydrophilicity and surface probability estimates were performed previously (14), according to the KyteDoolittle and Emini analyses, with the Peptidestructure program of the Sequence Analysis Software Package (Genetics Computer Group, Madison, WI). Correlations were calculated using Microsoft Excel, and probability by t-test.…”

“…Two regions of PM-Scl 100-kd protein that are reactive with autoantibodies have previously been identified by studies of recombinant protein fragments produced in Escherichia coli (12,14), Of 3 Sma I restriction fragments, S1 (including the N-terminal437 aa) reacted by immunoblot (IB) with 41 of 42 sera that had reacted with the whole recombinant protein, and the intensity of reaction of most individual sera was similar to that of whole protein (14). The central S2 fragment (aa 439-749) also reacted with 39 sera by IB, but reaction with S1 was often estimated to be stronger (S1 reaction greater than S2 reaction in 28 sera).…”

“…Some anti-PM-Scl positive sera reacted with the C-terminal S3 portion, but none reacted most strongly with this region. Using proteins expressed from H i m 11, Rsa I, and Pvu I1 restriction fragments, that study localized the major reactivity of the N-terminal S1 fragment to between aa 156 and aa 312 (14). This area included a region of high hydrophilicity and predicted surface probability that was a good candidate for the epitope site.…”