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.

journal_name

Autoimmunity

journal_title

Autoimmunity

authors

Farrag L,Pendergraft WF 3rd,Yang JJ,Jennette JC,Falk RJ,Preston GA

doi

10.1080/08916930701680104

subject

Has Abstract

pub_date

2007-11-01 00:00:00

pages

503-11

issue

7

eissn

0891-6934

issn

1607-842X

pii

783538973

journal_volume

40

pub_type

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