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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.

Goodpasture syndrome is an autoimmune disease causing rapidly progressive glomerulonephritis and pulmonary hemorrhage. The clinical manifestations are caused by autoantibodies that bind to a constituent, termed the Goodpasture autoantigen, of alveolar and glomerular basement membranes. Searches for the identity of this constituent have recently culminated in the discovery of two new chains (alpha 3 and alpha 4) of type IV collagen and the identification of the alpha 3 chain as the Goodpasture autoantigen. The gene, COL4A3, encoding this autoantigen was recently cloned and localized to the q35-37 region of chromosome 2. The major protomeric form of the alpha 3 chain is a homotrimer. The alpha 3-protomers associate through NC1-to-NC1 interactions mainly with each other to form a suprastructure, although some associate with protomers containing the alpha 1(IV) and alpha 2(IV) chains. The alpha 3-protomers also form suprastructures involving triple helical interactions of three or more protomers. The Goodpasture epitope is localized to the carboxylterminal region of the alpha 3(IV) chain, encompassing the last 36 residues of the chain, as the primary interaction site, and its structure is discontinuous.

Mutations in the COL4A5 collagen gene have been implicated as the primary defect in Alport syndrome, a heritable disorder characterized by sensorineural deafness and glomerulonephritis that progresses to end-stage renal failure. In the present study, the molecular nature of the defect in Alport glomerular basement membrane (GBM) was explored using anti-GBM alloantibodies (tissue-bound and circulating) produced in three Alport patients subsequent to renal transplantation. The alloantibodies bound to the alpha 3(IV)NC1 domain of type IV collagen and not to any other basement membrane component. In tissue sections, the alloantibodies bound specifically to peripheral GBM in normal kidney and the affected renal transplant but not to that of Alport kidney. These results establish that: the alpha 3 chain in type IV collagen molecules, the Goodpasture autoantigen, is the target alloantigen in post-transplant anti-GBM nephritis in patients with Alport syndrome, and that a molecular commonality exists in the pathogenesis of anti-GBM nephritis causing loss of renal allografts in patients with Alport syndrome and renal failure in patients with Goodpasture syndrome. These findings implicate: (1) defective assembly of type IV collagen molecules containing the alpha 3(IV) chain in Alport GBM; and (2) the existence of a mechanism linking the assembly of molecules containing the alpha 3(IV) chain with those containing the alpha 5(IV) chain.

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of fibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (alpha 1 subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM.