Expression of the Human Autoantigen of Wegener's Granulomatosis (PR3) in a Baculovirus Expression System

Szymkowiak, C; Johnston, Thomas W; Csernok, Elena; Gross, Wolfgang L.

doi:10.1006/bbrc.1996.0224

Cited by 21 publications

(17 citation statements)

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“…The primary rPR3 protein expressed in insect cells using baculovirus expression systems is N-terminally unprocessed and needs to be activated in vitro after partial purification [10]. The only rPR3 variants expressed in insect cells that were tested for reactivity with PR3-ANCA were not recognized by the majority of PR3-ANCA þ sera [8,9]. In contrast, native PMN-PR3 or Nterminally processed rPR3 expressed in HMC-1 cells, displayed in a conformationally intact fashion, represent the most sensitive substrates for PR3-ANCA testing [13,14,20].…”

Section: Discussionmentioning

confidence: 99%

“…Recently, different expression systems have been used to express recombinant PR3 (rPR3) variants [8][9][10][11][12]. Full length cDNA constructs encoding the PR3 zymogen cloned into baculovirus vectors have been expressed in Sf9 insect cells [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…Full length cDNA constructs encoding the PR3 zymogen cloned into baculovirus vectors have been expressed in Sf9 insect cells [8][9][10]. It has been reported that the N-terminal activation dipeptide needs to be removed in vitro from the expressed protein in order to obtain the active enzyme with the N-terminal amino acid sequence Ile-ValGly-Gly [10].…”

Section: Introductionmentioning

confidence: 99%

“…It has been reported that the N-terminal activation dipeptide needs to be removed in vitro from the expressed protein in order to obtain the active enzyme with the N-terminal amino acid sequence Ile-ValGly-Gly [10]. The N-terminally unprocessed rPR3 zymogen from Sf9 cells is recognized only by a small number of PR3-ANCAcontaining sera [8,9]. In contrast, enzymatically active, N-terminally processed rPR3 expressed in the human mast cell line, HMC-1, appears to be recognized by all PR3-ANCA [13,14].…”

Section: Introductionmentioning

confidence: 99%

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A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

Sun

Fass

Viss³

et al. 1998

Clinical and Experimental Immunology

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SUMMARYANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and D-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, D-rPR3-S176A does not. Culture supernatants of rPR3-S176A and D-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA þ sera were tested. With D-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the Nterminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

Sun

Fass

Viss³

et al. 1998

Clinical and Experimental Immunology

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“…The expression of recombinant PR3 (rPR3) as a fusion protein in E. coli and in baculovirus systems failed to generate an enzymatically active recombinant product that is recognized by the majority of c-ANCA sera [11][12][13]. Thereby, these reports have confirmed that most c-ANCA recognize conformational epitopes of PR3.…”

Section: Introductionmentioning

confidence: 99%

Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC‐1 cells is enzymatically active and recognized by c‐ANCA

Specks¹,

Fass²,

Fautsch³

et al. 1996

FEBS Letters

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We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-I. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the snbstrate N-methoxysuccinylAla-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by al-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PRYs targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intraceHular processing, structure-function relationships and interaction with autoantibodies.

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Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines

Garwicz

Lindmark

Hellmark

et al. 1997

Journal of Leukocyte Biology

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Expression of the Human Autoantigen of Wegener's Granulomatosis (PR3) in a Baculovirus Expression System

Cited by 21 publications

References 0 publications

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC‐1 cells is enzymatically active and recognized by c‐ANCA

Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines

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