Autoantibodies recognizing citrullinated rat filaggrin in an ELISA using citrullinated and non-citrullinated recombinant proteins as antigens are highly diagnostic for rheumatoid arthritis.

The objective of the study was to determine the diagnostic value for rheumatoid arthritis (RA) of anti-filaggrin autoantibodies (autoAb) recognizing citrullinated recombinant rat filaggrin (ACRF) in community cases of very early arthritis. To evaluate the diagnostic value of ACRF, were studied sera from patients with different classified rheumatic diseases and healthy subjects (group 1, n= 422) and 314 community cases of very early arthritis (group 2) that were classified as RA (n = 176), non-RA (n = 63) and undifferentiated (n = 75) arthritides after 1 years of follow-up. ACRF were measured using a new ELISA, with results expressed as the difference between the OD value obtained on citrullinated minus that on noncitrullinated rat filaggrin (differential ACRF; dACRF). For both groups, rheumatoid factors (RF), anti-keratin autoAb (AKA) and anti-perinuclear factor (APF) were tested; for group 2, anti-CCP autoAb were also tested. Different reactivity patterns against citrullinated and noncitrullinated filaggrin were observed. Almost all sera reacting with citrullinated but not noncitrullinated filaggrin were from RA patients. Among RA and non-RA sera that recognized both forms of filaggrin, a positive result was obtained only with RA sera. For groups 1 and 2, dACRF sensitivity was 58.4% and 30.7%, and specificity for RA was 99.5% and 98.4%, respectively. In group 2, dACRF specificity for RA was better than that of RF (92.1%), APF (95.2%), AKA (96.8%) and anti-CCP (95.2%). dACRF positive predictive value was high (98.2) and close to that given by the concomitant positivity of RF and anti-CCP autoAb. Despite a high positive correlation between AKA, APF, anti-CCP and dACRF test results, they were complementary since some sera were positive for only one test. Thus, in a community setting, anti-citrullinated rat filaggrin reactivity detected by a new ELISA, whose originality is based on the difference between serum's reactivities on the citrullinated and native forms of filaggrin, had a higher diagnostic value for RA than other autoAb.

Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation.

Hepatitis B virus core protein (HBc) is an important serology marker of hepatitis B infection and patient follow-up. It is an M, 21,000 protein, which has the intrinsic capacity to self-assemble as a capsid-like particle. The hepatitis B core protein has been expressed in Escherichia coli and Pichia pastoris (three different constructions) in order to select a HBc recombinant antigen suitable for serodiagnosis requirements with a cost effective downstream strategy. The expression and purification of the different forms of recombinant HBc have been described. For the last step, ultracentrifugation and size-exclusion chromatography were compared. The morphology of these capsids was observed using an electron microscope. Our data shows that HBc antigen is produced in large quantities in E. coli but some contaminants remained which were associated with the E. coli HBc protein after ultracentrifugation or size-exclusion chromatography. The ultracentrifugation enables a higher purity of HBc antigen to be obtained than size-exclusion chromatography but the latter enables a higher recovery rate. P. pastoris enables the expression and extraction of a highly purified HBc antigen suitable for diagnostic purposes.

Characterization of Toxoplasma gondii surface antigen 1 (SAG1) secreted from Pichia pastoris: evidence of hyper O-glycosylation.

A truncated form of surface antigen 1 (SAG1t), the immunodominant surface antigen of Toxoplasma gondii, was expressed in the methylotrophic yeast, Pichia pastoris. The truncated protein lacked the C-terminal residues which, in native SAG1, encompass a glycosylphosphatidylinositol anchorage site. The single potential N-glycosylation site was mutated and a sequence encoding a hexahistidine tag was introduced at the C-terminal of the construction to aid purification by immobilized metal-chelate chromatography. Recombinant SAG1t was efficiently secreted into the culture medium as three protein species having molecular masses of 29, 38/45 and 50/60 kDa. This heterogeneity was dependent upon the composition of the medium used to grow the yeast transformants. Mass spectrometric analyses, chemical deglycosylation, lectin recognition and sensitivity to mannosidase treatments showed that SAG1t heterogeneity was related to the presence of O-linked oligosaccharides containing alpha1-2-, alpha1-3- or alpha1-6-linked mannoses. The glycosylated and deglycosylated recombinant SAG1t were recognized by monoclonal and human-serum-derived antibodies, specific for the native SAG1, which suggested that the O-glycosylations had no major effect on the protein conformation. However, ELISA and Western-blot analysis with human sera showed that the O-carbohydrates added by P. pastoris could be recognized as antigenic structures. As a consequence, purification of the unglycosylated 29 kDa recombinant SAG1t species or deglycosylation is required in order to use recombinant SAG1t as a diagnostic reagent. Moreover, the presence of carbohydrates, not found on the native protein, suggests that addition of unnatural glycan structures by P. pastoris is a potential drawback that should be considered when using this expression system.

The unglycosylated extracellular domain of type-II receptor for transforming growth factor-beta. A novel assay for characterizing ligand affinity and specificity.

The activation of the human transforming growth factor (TGF-beta) system begins with the cytokine-induced association of the extracellular domains of two structurally related receptor subunits. To study the protein-protein interactions between TGF-beta and the ligand-specific receptor subunit, the extracellular domain of the human TGF-beta receptor type II (T beta R-II) has been expressed as an intracellular protein in insect cells using the baculovirus expression system. The cDNA construct was engineered to encode amino acids 24-159 (the signal sequence 1-23 was lacking) preceded by one initiator methionine residue and six histidine residues added at the carboxy terminus. The soluble receptor accumulated in the cytoplasm of infected cells and was purified by one-step nickel-chelate affinity chromatography. The purified protein was not glycosylated; it migrated as a single band of apparent mass 19.5 kDa in SDS/polyacrylamide gels, and had a homogeneous N-terminal sequence. We have established a solid-phase binding assay using radioiodinated TGF-beta 3 and capture antibodies to immobilize the soluble receptor. In this assay, the apparent dissociation constant of the TGF-beta type-II receptor ectodomain for TGF-beta 3 was approximately 150 nM (this value is approximately 1000-fold higher than that of the cell-membrane receptor complex of living cells). The affinity of TGF-beta 3 for the unglycosylated ectodomain of T beta R-II from insect cells was lower than the affinity for the recombinant glycosylated ectodomain T beta R-II from mouse cells. The novel assay has been used to characterize affinities and specificities of TGF-beta 3, TGF-beta 2, corresponding mutants and hybrid proteins, as well as a related protein, BMP-2. The assay could also be used to search for inhibitors.

Ligand-induced dimerization of the extracellular domain of the TGF-beta receptor type II.

Transforming growth factor-beta (TGF-beta) signals by mediating the association of two distinct transmembrane serine/threonine kinase receptors, the type I (T beta RI) and II (T beta RII). Here, we took advantage of recombinant human T beta RII extracellular domain (T beta RII-ED) to analyze TGF-beta/T beta RII complex formation which is the initial event in the construction of a signaling complex. We found that recombinant T beta RII-ED binds TGF-beta 3 more efficiently than TGF-beta 2 and therefore maintains the native T beta RII binding selectivity for the different TGF-beta isoforms. Biochemical analysis showed that free T beta RII-ED is expressed as a monomer. Upon ligand binding, both TGF-beta 3 and -beta 2 isoforms induce homodimerization of T beta RII-ED, each TGF-beta subunit being able to bind one T beta RII-ED molecule. These results suggested that ligand dependent receptor dimerization may be an important early step in the TGF-beta signaling complex formation.

Glycosylation of human truncated Fc epsilon RI alpha chain is necessary for efficient folding in the endoplasmic reticulum.

The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex. The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t. All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t. Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t. In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t. Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells.

Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor.

Nonreceptor tyrosine kinases such as the newly described 70-kDa (ZAP-70/Syk) and Src-related tyrosine kinases are coupled to a variety of receptors, including the antigen receptors on B- and T-cells and the Fc receptors for IgE (Fc epsilon RI) and IgG (Fc gamma RI, Fc gamma RIII/CD16). Various subunits of these receptors contain homologous activation motifs which appear capable of autonomously triggering cell activation. Two forms of this motif are present in the Fc epsilon RI multimeric complex: one in the beta chain and one in the gamma chain. Here we show that each of the two tyrosine kinases known to be involved in Fc epsilon RI signaling is controlled by a distinct motif-containing chain. Lyn associates with the nonactivated beta chain, whereas gamma promotes the activation of Syk. We also show that neither the beta nor the gamma motif alone can account for the full signaling capacity of the entire receptor. We propose that, upon triggering of the tetrameric receptor, Lyn already bound to beta becomes activated and phosphorylates beta and gamma; the phosphorylation of gamma induces the association of Syk with gamma and also the activation of Syk, resulting in the phosphorylation and activation of phospholipase C gamma 1. Cooperative recruitment of specific kinases by the various signaling chains found in this family of antigen receptors could represent a way to achieve the full signaling capacity of the multimeric complexes.

Characterization of the family of dimers associated with Fc receptors (Fc epsilon RI and Fc gamma RIII).

The receptor for IgE (Fc epsilon RI) is a multimeric complex containing one alpha chain, one beta chain with four transmembrane domains and one homodimer of disulfide-linked gamma-chains. The Fc epsilon RI gamma-chains form additional disulfide-linked dimers with the homologous zeta- and eta-chains, as part of the TCR complex. The low affinity receptor for IgG (Fc gamma RIII)2 on NK cells is also associated with zeta-chains. Here we show that the gamma-chain is expressed in NK cells both as a group of heterogenous gamma gamma homodimers and also as a heterodimer bound to zeta. Fc gamma RIIIA is associated with three types of dimers zeta zeta, gamma zeta, and notably gamma gamma as well. In fact, gamma gamma appears to be the predominant species associating with Fc gamma RIIIA. The surface expressed Fc epsilon RI also associates with the same group of heterogenous gamma gamma homodimers. We also show that there is no C-terminal posttranslational cleavage of gamma occurring before its insertion into the plasma membrane as previously suggested. Thus, like the TCR, Fc gamma RIIIA may form a variety of receptor isoforms, though at present we do not understand the functional implications of these structures.