Goodpasture antigen: expression of the full-length alpha3(IV) chain of collagen IV and localization of epitopes exclusively to the noncollagenous domain.
BACKGROUND: Tissue injury in Goodpasture (GP) syndrome (rapidly progressive glomerular nephritis and pulmonary hemorrhage) is mediated by antibasement membrane antibodies that are targeted to the alpha3(IV) chain of type IV collagen, one of five alpha(IV) chains that occur in the glomerular basement membrane. GP antibodies are known to bind epitopes within the carboxyl terminal noncollagenous domain (NC1) of the alpha3(IV) chain, termed the GP autoantigen. Whether epitopes also exist in the 1400-residue collagenous domain is unknown because studies to date have focused solely on the NC1 domain. A knowledge of GP epitopes is important for the understanding of the etiology and pathogenesis of the disease and for the development of therapeutic strategies. METHODS: A cDNA construct was prepared for the full-length human alpha3(IV) chain. The construct was stably transfected into human embryonic kidney 293 cells. The purified full-length r-alpha3(IV) chain was characterized by electrophoresis and electron microscopy. The capacity of this chain for binding of GP antibodies from five patients was compared with that of the human r-alpha3(IV)NC1 domain by competitive enzyme-linked immunosorbent assay. RESULTS: The r-alpha3(IV) chain was secreted from 293 cells as a single polypeptide chain that did not spontaneously undergo assembly into a triple-helical molecule. An analysis of GP-antibody binding to the full-length r-alpha3(IV) chain showed binding exclusively to the globular NC1 domain. CONCLUSION: The full-length human alpha3(IV) chain possesses the capacity to bind GP autoantibodies. The epitope(s) is found exclusively on the nontriple-helical NC1 domain of the alpha3(IV) chain, indicating the presence of specific immunogenic properties. The alpha3(IV) chain alone does not spontaneously undergo assembly into a triple-helical homotrimeric molecule, suggesting that coassembly with either the alpha4(IV) and/or the alpha5(IV) chain may be required for triple-helix formation. (+info)
Identification of a clinically relevant immunodominant region of collagen IV in Goodpasture disease.
BACKGROUND: The characteristic feature of Goodpasture disease is the occurrence of an autoantibody response to the noncollagenous domain of the alpha3 chain of type IV collagen [alpha3(IV)NC1] in the alveolar and glomerular basement membrane. These antibodies are associated with the development of a rapidly progressive glomerulonephritis, with or without lung hemorrhage, whereas autoantibodies specific for the other alpha chains of the heterotrimeric type IV collagen probably do not cause disease. In this study, we have investigated whether differences in fine specificity of autoimmune recognition of the alpha3(IV)NC1 correlate with clinical outcome. METHODS: For mapping of antibody binding to type IV collagen, chimeric collagen constructs were generated in which parts of the alpha3(IV)NC1 domain were replaced by the corresponding sequences of homologous nonreactive alpha1(IV). The different recombinant collagen chimeras allowed the analysis of antibody specificities in 77 sera from well-documented patients. RESULTS: One construct that harbors the aminoterminal third of the alpha3(IV)NC1 was recognized by all sera, indicating that it represents the dominant target of the B-cell response in Goodpasture disease. Seventy percent of the samples recognized other parts of the molecule as well. However, only reactivity to the N-terminus of the alpha3(IV)NC1 correlated with prognosis, that is, kidney survival after six months of follow-up. CONCLUSION: The results indicate the crucial importance of antibody recognition of this particular domain for the pathogenesis of Goodpasture disease, thereby opening new avenues for the development of better diagnostic and therapeutic procedures. (+info)
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. (+info)
Characterization of a novel type of serine/threonine kinase that specifically phosphorylates the human goodpasture antigen.
Goodpasture disease is an autoimmune disorder that occurs naturally only in humans. Also exclusive to humans is the phosphorylation process that targets the unique N-terminal region of the Goodpasture antigen. Here we report the molecular cloning of GPBP (Goodpasture antigen-binding protein), a previously unknown 624-residue polypeptide. Although the predicted sequence does not meet the conventional structural requirements for a protein kinase, its recombinant counterpart specifically binds to and phosphorylates the exclusive N-terminal region of the human Goodpasture antigen in vitro. This novel kinase is widely expressed in human tissues but shows preferential expression in the histological structures that are targets of common autoimmune responses. The work presented in this report highlights a novel gene to be explored in human autoimmunity. (+info)
The goodpasture autoantigen. Identification of multiple cryptic epitopes on the NC1 domain of the alpha3(IV) collagen chain.
Goodpasture (GP) disease is an autoimmune disorder in which autoantibodies against the alpha3(IV) chain of type IV collagen bind to the glomerular and alveolar basement membranes, causing progressive glomerulonephritis and pulmonary hemorrhage. Two major conformational epitope regions have been identified on the noncollagenous domain of type IV collagen (NC1 domain) of the alpha3(IV) chain as residues 17-31 (E(A)) and 127-141 (E(B)) (Netzer, K.-O. et al. (1999) J. Biol. Chem. 274, 11267-11274). To determine whether these regions are two distinct epitopes or form a single epitope, three GP sera were fractionated by affinity chromatography on immobilized NC1 chimeras containing the E(A) and/or the E(B) region. Four subpopulations of GP antibodies with distinct epitope specificity for the alpha3(IV)NC1 domain were thus separated and characterized. They were designated GP(A), GP(B), GP(AB), and GP(X), to reflect their reactivity with E(A) only, E(B) only, both regions, and neither, respectively. Hence, regions E(A) and E(B) encompass critical amino acids that constitute three distinct epitopes for GP(A), GP(B), and GP(AB) antibodies, respectively, whereas the epitope for GP(X) antibodies is located in a different unknown region. The GP(A) antibodies were consistently immunodominant, accounting for 60-65% of the total immunoreactivity to alpha3(IV)NC1; thus, they probably play a major role in pathogenesis. Regions E(A) and E(B) are held in close proximity because they jointly form the epitope for Mab3, a monoclonal antibody that competes for binding with GP autoantibodies. All GP epitopes are sequestered in the hexamer configuration of the NC1 domain found in tissues and are inaccessible for antibody binding unless dissociation of the hexamer occurs, suggesting a possible mechanism for etiology of GP disease. GP antibodies have the capacity to extract alpha3(IV)NC1 monomers, but not dimers, from native human glomerular basement membrane hexamers, a property that may be of fundamental importance for the pathogenesis of the disease. (+info)
Distinct antitumor properties of a type IV collagen domain derived from basement membrane.
Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth. (+info)
Two RGD-independent alpha vbeta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties.
Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (alpha3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54-132) anti-angiogenic and a C-terminal (amino acids 185-203) anti-tumor cell activity (Maeshima, Y., et al. 2000) J. Biol. Chem. 275, 21340-21348). Previous studies have identified the 185-203 amino acid sequence as a ligand for alpha(v)beta(3) integrin (Shahan, T. A., et al. (1999) Cancer Res. 59, 4584-4590). In the present study, we found distinct additional RGD-independent alpha(v)beta(3) integrin binding site within 54-132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54-132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185-203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the alpha(v)beta(3) integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique alpha(v)beta(3) integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin. (+info)
High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to alpha3(IV)NC1 collagen.
BACKGROUND: Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha3(IV)NC1 were evaluated using an optical biosensor interaction analysis. METHODS: Polyclonal anti-GBM Abs were purified by alpha3(IV)NC1 affinity chromatography from the sera of patients with anti-GBM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha3(IV)NC1 dimers. RESULTS: All of the autoantibody preparations bound to alpha3(IV)NC1, whereas none bound to alpha1(IV)NC1 (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s-l (GS) and 8. 90E-05 +/- 2.80E-05 s-l (AL). Their estimated association constants (Ka) were 2.67E+04 +/- 1.8E+04 (M-ls-l) and 2.76E+04 +/- 1. 70E+04(M-ls-l) for GS and AL patients, respectively. By comparison with other Ab interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. CONCLUSIONS: The results suggest that anti-GBM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation, despite removal of circulating Abs and adequate immunosuppression. (+info)