Characterization of a recombinant proteinase 3, the autoantigen in Wegener's granulomatosis and its reactivity with anti-neutrophil cytoplasmic autoantibodies.
Using the baculovirus/insect cells system, we have expressed a recombinant proteinase 3 (PR3) -- the neutrophil-derived serine protease autoantigen in Wegener's granulomatosis -- as a glycosylated intracellular and membrane-associated protein. Oligosaccharides accounted for the difference in molecular weights between recombinant (34 kDa) and neutrophil-PR3 (29 kDa). Whereas rabbit-anti-PR3 IgG recognized both recombinant and neutrophil-derived PR3, autoantibodies from Wegener patient sera recognized only neutrophil-derived PR3. Although oligosaccharides were not involved in PR3 epitope recognition, autoantibodies did not recognize the amino acid primary structure of recombinant PR3. Improper disulfide bond formation and/or lack of post-translational events in insect cells, may affect the conformation and/or lack of post-translational events in insect cells, may affect the conformation of PR3, precluding its reactivity with sera from WG patients.
Witko-Sarsat
V
V
INSERM U25, Hôpital Necker, Paris, France.
Halbwachs-Mecarelli
L
L
Almeida
R P
RP
Nusbaum
P
P
Melchior
M
M
Jamaleddine
G
G
Lesavre
P
P
Descamps-Latscha
B
B
Gabay
J E
JE
D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D019268:Antibodies, Antineutrophil Cytoplasmic; D000937:Antigen-Antibody Reactions; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology*; D002460:Cell Line; D000939:Epitopes / Q000032:analysis; D006031:Glycosylation; D014890:Granulomatosis with Polyangiitis / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D007106:Immune Sera; D008565:Membrane Proteins; D008970:Molecular Weight; D053146:Myeloblastin; D017924:Nucleopolyhedroviruses / Q000235:genetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012697:Serine Endopeptidases / Q000737:chemistry / Q000276:immunology*; D012995:Solubility; D018411:Spodoptera; D014415:Tunicamycin / Q000494:pharmacology
D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D007106:Immune Sera; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; D012697:Serine Endopeptidases; D053146:Myeloblastin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase