The Alloantigenic Sites of α3α4α5(IV) Collagen

Jeong Suk Kang, Clifford E. Kashtan, A. Neil Turner, Laurence Heidet, Billy G. Hudson, Dorin-Bogdan Borza
2007 Journal of Biological Chemistry  
and the ʈ INSERM U574, Hôpital Necker-Enfants Malades, 75743 Paris Cedex 15, France Anti-glomerular basement membrane (GBM) antibody nephritis is caused by an autoimmune or alloimmune reaction to the NC1 domains of ␣3␣4␣5(IV) collagen. Some patients with X-linked Alport syndrome (XLAS) develop post-transplant nephritis mediated by pathogenic anti-GBM alloantibodies to collagen IV chains present in the renal allograft but absent from the tissues of the patient. In this work, the epitopes
more » ... by alloantibodies from these patients were identified and characterized. All XLAS alloantibodies recognized conformational epitopes in the NC1 domain of ␣5(IV) collagen, which were mapped using chimeric ␣1/␣5 NC1 domains expressed in mammalian cells. Allograft-eluted alloantibodies mainly targeted two conformational alloepitopes mapping to ␣5NC1 residues 1-45 and 114 -168. These regions also encompassed the major epitopes of circulating XLAS alloantibodies, which in some patients additionally targeted ␣5NC1 residues 169 -229. Both kidney-eluted and circulating alloantibodies to ␣5NC1 distinctively targeted epitopes accessible in the ␣3␣4␣5NC1 hexamers of human GBM, unlike anti-GBM autoantibodies, which targeted sequestered ␣3NC1 epitopes. The results identify two immunodominant ␣5NC1 epitopes as major alloantigenic sites of ␣3␣4␣5(IV) collagen specifically implicated in the pathogenesis of post-transplant nephritis in XLAS patients. The contrast between the accessibility of these alloepitopes and the crypticity of autoepitopes indicates that distinct molecular forms of antigen may initiate the immunopathogenic processes in the two forms of anti-GBM disease. . 2 The abbreviations used are: GBM, glomerular basement membrane; APTN, Alport post-transplant nephritis; NC1, the non-collagenous domain of type IV collagen; XLAS, X-linked Alport syndrome; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney. http://www.jbc.org/ Downloaded from FIGURE 2. Design of ␣1/␣5 NC1 chimeras. A, comparison of amino acid sequences of human ␣5NC1 and ␣1NC1. Conserved amino acid residues are indicated by asterisks. Among residues specific for ␣5NC1, those indicated in bold are predicted to be accessible in the NC1 hexamer based on the analysis of the x-ray structure of homologous ␣1␣2NC1 hexamers. Arrowheads indicate the boundaries of regions swapped in ␣1/␣5NC1 chimeras. The location of ␣3NC1 epitopes E A and E B is indicated by solid and interrupted lines, respectively. B, schematic representation of cloning strategy for constructing human ␣1/␣5NC1 chimeras. The ApaI, KpnI, NarI, and XbaI cleavage sites were introduced into the ␣1NC1 and ␣5NC1, and then quarter-domains of ␣5NC1 were substituted into the corresponding position within NC1 to produce four chimeric constructs, designated 5111, 1511, 1151, and 1115. The chimeric constructs consisted of FLAG peptide followed by two or four Gly-X-Y repeats (for ␣1NC1 or ␣5NC1-derived amino termini, respectively) and the full NC1 sequence cloned into the pRC-X expression vector. UTR, untranslated region; aa, amino acids.
doi:10.1074/jbc.m611892200 pmid:17293596 fatcat:3qg6iawrcje57lwpb5fc5odsjm