The goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain.
The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.
Netzer
K O
KO
Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Leinonen
A
A
Boutaud
A
A
Borza
D B
DB
Todd
P
P
Gunwar
S
S
Langeveld
J P
JP
Hudson
B G
BG
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003094:Collagen / Q000737:chemistry* / Q000235:genetics; D024141:Collagen Type IV; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry*; D006801:Humans; D007106:Immune Sera; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
D001324:Autoantigens; D024141:Collagen Type IV; D017931:DNA Primers; D000939:Epitopes; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; C057112:type IV collagen alpha3 chain; D003094:Collagen