The majority of asparagine-linked glycosylation isoforms of rPR3 from Sf9 cells have a molecular mass of 34 kDa, whereas the majority of the native PR3 glycosylation isoforms purified from azurophil granules of neutrophils have a mass of about 29 kDa [26]. undamaged disulfide bonds are crucial for PR3’s antigenicity. Additional post-translational modifications of native PR3 not happening in may also become prerequisites for appropriate acknowledgement by PR3-ANCA. Another rPR3 product indicated in was also not identified by PR3-ANCA [24]: this particular variant did contain the amino-terminal propeptide extension. When the same construct was indicated in the candida the indicated rPR3 product was identified by 7 of 10 Phenytoin sodium (Dilantin) PR3-ANCA-positive sera Phenytoin sodium (Dilantin) when the rPR3 was used as target antigen inside a capture enzyme-linked immunosorbent assay (ELISA), but only by 1 of 10 when it was used in a direct ELISA [24]. This suggests that not all conformational epitopes identified by PR3-ANCA are displayed on rPR3 indicated in after purification of the proenzyme. Disulfide bonds are created appropriately in Sf9 cells. Other post-translational modifications of rPR3 are either lacking or improper for the generation of the active enzyme identified by all PR3-ANCA [25,26]. The amino-terminal propeptide is not cleaved intracellularly [21*], and asparagine-linked glycosylation of rPR3 in Sf9 cells is different from that of PR3 purified from neutrophils [26]. The majority of asparagine-linked glycosylation isoforms of rPR3 from Sf9 cells have a molecular mass of 34 kDa, whereas the majority of the native PR3 glycosylation isoforms purified from azurophil granules of neutrophils have a mass of about 29 kDa [26]. In addition, in the reported crystal structure of PR3, which is based on rPR3 indicated in Sf9 cells, only one of the two potential asparagine-linked glycosylation sites appeared occupied [21*], whereas both sites are used in rPR3 indicated in hematopoietic cells and in native PR3 from neutrophils [31]. When rPR3 is definitely indicated in hematopoietic cells, it is processed to an active enzyme and stored in granules [27,28]. All PR3-ANCA recognize rPR3 expressed in the human mast cell line HMC-1 [32]. As indicated by immunofluorescence and capture ELISA data, the Phenytoin sodium (Dilantin) affinity of PR3-ANCA to HMC-1 cell rPR3 is similar to that of neutrophil PR3 [32,33]. This similarity suggests that the conformational epitopes recognized by PR3-ANCA are fully accessible on rPR3 expressed in HMC-1 cells. Furthermore, the recognition of rPR3 by PR3-ANCA is not affected by the substitution of the active site serine by an alanine residue (S176A), indicating that the conformational epitopes recognized by PR3-ANCA are not affected by this mutation even though it alters the active site sufficiently to render the molecule enzymatically inactive [27,33]. This mutation subsequently allowed us to express rPR3 variants in the epithelial cell line 293 [29]. The overwhelming majority of rPR3 expressed in 293 cells is usually secreted into the media supernatant in an unprocessed form [29]. Two rPR3 variants representing the amino-terminally unprocessed pro-form of PR3 (rPR3-S176A) and the amino-terminally processed mature form of PR3 (-rPR3-S176A) were expressed in 293 cells [29]. In the capture ELISA, PR3-ANCA recognize mature rPR3 contained in HMC-1/PR3-S176A cells just as well as they recognize that contained in media supernatants of-rPR3-S176A expressing 293 cells [29,33]. In contrast, the proform variant of rPR3 is not recognized by all PR3-ANCA [29]. While some PR3-ANCA sera show the same reactivity with both mature and pro-rPR3, most of them bind less well to pro-PR3 than to mature rPR3 [29]. This indicates that some PR3-ANCA epitopes are equally accessible on mature and pro-PR3, whereas other epitopes are less or not at all accessible on pro-PR3. The clinical relevance of the differential recognition of these rPR3 variants with different conformations is currently under investigation. Preliminary evidence obtained using our capture ELISA suggests that the correlation of clinical disease activity with PR3-ANCA reacting with pro-PR3 is usually more readily apparent than that of the reactivity with mature PR3 [34]. While preservation of proper disulfide bonds in the PR3 molecule is crucial for recognition of PR3-ANCA, Mouse monoclonal to Influenza A virus Nucleoprotein and more PR3-ANCA appear to recognize the mature form of the enzyme than the amino-terminally unprocessed proform, the carboxy-terminal processing and asparagine-linked glycosylation appear less relevant. The fact that rPR3 secreted into the 293 cell media supernatant is usually carboxy-terminally unprocessed (my unpublished data) implies that carboxy-terminal processing of PR3 has little effect on the antigenicity of PR3 [29]. This interpretation is usually supported by the observation that an rPR3 construct carrying.