title: A study of conformational restraints on reactivity of human PR3-specific autoantibodies (ANCA) facilitated through protein folding manipulations of a new recombinant proteinase 3 protein.
abstract: Proteinase 3 (PR3)-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) recognize conformational epitopes on PR3. This study evaluates PR3-ANCA target epitopes utilizing a novel recombinant PR3 (rPR3) produced to accommodate manipulations of the N-terminal domain. The rPR3 molecule contains an N-terminus six histidine tag, which can be removed by enterokinase (EK) cleavage of an adjacent EK cleavage site. Once cleaved the remaining amino acids correspond to the mature N-terminus of PR3. This rPR3 can be manipulated to produce three variant forms: tagged rPR3(+his), EK-cleaved (his-tag removed) rPR3(- his), and EK-cleaved, denatured/refolded rPR3(- his/dr) (the proteolytically active form). Patients with clinically positive PR3-ANCA titers (n = 40) were confirmed for reactivity against purchased native PR3 in our system. Controls included 29 healthy volunteers and 34 MPO-ANCA patients. All PR3-ANCA sera samples tested were reactive with one or more forms of the recombinant protein (greater than mean ELISA OD 405 + 2 SDs of controls). Of significance, three sera were reactive with non-active forms only and three others were more reactive with rPR3(- his/dr) than with native PR3. The results of our evaluation of PR3-ANCA sera for reactivity against the three forms of our rPR3 protein uniquely exemplify the diverse array of epitopes within the PR3-ANCA population. This new recombinant form of PR3 should provide a suitable approach to mapping ANCA epitopes using site-directed mutagenesis.
authors.affiliation: UNC Kidney Center, University of North Carolina at Chapel Hill, NC 27599, USA.
authors.forename: Lila
authors.initials: L
authors.lastname: Farrag
authors.forename: William F
authors.initials: WF
authors.lastname: Pendergraft
authors.forename: J J
authors.initials: JJ
authors.lastname: Yang
authors.forename: J Charles
authors.initials: JC
authors.lastname: Jennette
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Falk
authors.forename: Gloria A
authors.initials: GA
authors.lastname: Preston
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000737:chemistry* / Q000276:immunology; D004765:Enteropeptidase / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D005260:Female; D014890:Granulomatosis with Polyangiitis / Q000276:immunology; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008297:Male; D016297:Mutagenesis, Site-Directed; D053146:Myeloblastin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D000939:Epitopes; D011994:Recombinant Proteins; C033223:polyhistidine; D006639:Histidine; D053146:Myeloblastin; D004765:Enteropeptidase