Abstract
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.
Publication types
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, P.H.S.
MeSH terms
-
Amidohydrolases / pharmacology
-
Animals
-
Antibodies, Antineutrophil Cytoplasmic
-
Antigen-Antibody Reactions
-
Autoantibodies / immunology*
-
Autoantigens / chemistry
-
Autoantigens / immunology*
-
Cell Line
-
Epitopes / analysis
-
Glycosylation
-
Granulomatosis with Polyangiitis / enzymology
-
Granulomatosis with Polyangiitis / immunology*
-
Humans
-
Immune Sera
-
Membrane Proteins
-
Molecular Weight
-
Myeloblastin
-
Nucleopolyhedroviruses / genetics
-
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
-
Recombinant Fusion Proteins / biosynthesis
-
Serine Endopeptidases / chemistry
-
Serine Endopeptidases / immunology*
-
Solubility
-
Spodoptera
-
Tunicamycin / pharmacology
Substances
-
Antibodies, Antineutrophil Cytoplasmic
-
Autoantibodies
-
Autoantigens
-
Epitopes
-
Immune Sera
-
Membrane Proteins
-
Recombinant Fusion Proteins
-
Tunicamycin
-
Serine Endopeptidases
-
Myeloblastin
-
Amidohydrolases
-
Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase