Structure and organization of type IV collagen of renal glomerular basement membrane.
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Amino Acid Sequence↗
Biomedical subjects
B G Hudson
Publications and source records attributed to B G Hudson.
The chain composition of aorta basement membrane collagen IV, localization of the chains in aorta and supramolecular assembly of the collagen IV protomers.
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Amino Acid Sequence↗
Identification of the membrane remnants of transferrin receptor with domain-specific antibodies.
Tissue culture studies with K562 and HL60 cells have demonstrated the production of a soluble form of transferrin receptor identical to that identified in human serum. The present study was undertaken to search for membrane remnants of the truncated receptor with peptide antibodies specific for the extracellular and cytoplasmic domain of transferrin receptor. In cell membranes, a 105K remnant was identified that is consistent with truncation of one extracellular domain monomer of the transferrin receptor. In the exosomal fraction of the culture supernatant, a smaller 20K remnant consistent with truncation of both extracellular domains was also demonstrated. These findings provide evidence that soluble receptor is the product of proteolytic cleavage of intact membrane-bound transferrin receptor. Prior studies showing that the concentration of the extracellular domain in exosomes remained stable during incubation in culture supernatant suggest that this cleavage possibly occurs intracellularly.
Amino Acid Sequence↗
Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.
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Anti-Glomerular Basement Membrane Disease↗
Structure and composition of type IV collagen of bovine aorta.
To determine the chain composition of type IV collagen of bovine thoracic aorta, we analyzed collagenase-solubilized carboxyl-terminal noncollagenous (NC1)-domains by high-pressure liquid chromatography, two-dimensional electrophoresis, immunoblotting and enzyme-linked immunoassay. In addition to the classical alpha 1- and alpha 2-chains, we found small amounts of the recently discovered alpha 3-, alpha 4- and alpha 5-chains. The alpha 3- and alpha 4-chains were, collectively, 7-13% of the total, and the alpha 5-chain was present in a low amount. Seventy-nine percent of the NC1-domains were dimerized. Immunolocalization studies on sections of aorta showed that the alpha 3- and alpha 5-chains were present, along with alpha 1- and alpha 2-chains, in the subendothelium and media. In capillaries of the media, the alpha 3-chain was found at relatively high levels and was co-localized with alpha 1- and alpha 2-chains. Digestion of aorta with Pseudomonas aeruginosa elastase yielded soluble multimolecular assemblies of type IV collagen. Electron microscopy results provided a direct demonstration of the supramolecular structure, in which the collagen molecules were tetramerized at the amino-terminal end and dimerized at the carboxyl-terminal end.
Animals↗
Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen.
Type IV collagen has recently emerged as a family composed of five known chains (alpha 1-alpha 5), each of which contains a carboxyl-terminal noncollagenous domain (NC1) of approximately 230 amino acids. The NC1 domain of the alpha 3(IV) chain is the probable target for autoantibodies in patients with Goodpasture syndrome (GP), as evidenced from studies employing bovine type IV collagen. In the present experiments, the specificity of GP antibodies for the five NC1 domains of human type IV collagen was determined by using recombinant NC1 domains as the antigen. cDNAs encoding each NC1 domain were expressed in E. coli as fusion proteins with a 6-histidine amino-terminal leader. The recombinant NC1 monomers r alpha 1(IV), r alpha 2(IV), r alpha 3(IV), r alpha 4(IV), and r alpha 5(IV) were purified by affinity chromatography to the fusion protein using a nickel resin column, and then characterized by electrophoresis and immunoblot analysis using chain-specific peptide antibodies. The specificity of GP antibodies from four patients to these recombinant proteins was then further evaluated by immunoblot analysis and enzyme-linked immunosorbent assay measurements. The GP antibodies reacted strongly with the r alpha 3(IV) NC1 domain but were not reactive when tested against the other four recombinant monomers. In contrast, neither antisera from patients with two other forms of autoimmune disease (anti-tubular basement membrane disease and Wegener's syndrome) nor normal control sera bound to any of the recombinant NC1 moieties. These results unambiguously establish that GP antibodies are specifically targeted to the NC1 domain of the alpha 3(IV) chain of human type IV collagen. The findings also establish a methodology for large scale preparation of r alpha 3(IV) NC1 domain for use in diagnostic tests and development of therapeutic procedures and offer a strategy for the elucidation of a more complete GP epitope by site-directed mutagenesis.
Amino Acid Sequence↗
Reductive cleavage of the disulfide bonds of the collagen IV noncollagenous domain in aqueous sodium dodecyl sulfate: absence of intermolecular nondisulfide cross-links.
The subunits of the collagen IV hexameric, noncollagenous NC1 domain obtained from bovine aorta, glomerular basement membrane, alveolar basement membrane and placental basement membrane are predominantly dimers. A large fraction of the dimers had been thought to be linked by nondisulfide bonds because they were resistant to cleavage by 5% (v/v) 2-mercaptoethanol, 2% (w/v) SDS, at 100 degrees C. However, if an unusually high concentration of 2-mercaptoethanol, e.g., 40% (v/v), is used, complete conversion of dimers into monomers is achieved, indicating the lack of intersubunit nondisulfide cross-links. Electrophoresis patterns indicate that some of the intermolecular disulfide bonds of the dimers are more resistant to reduction in aqueous SDS than are some of the intramolecular disulfide bonds.
Alkylation↗
Characterization and quantitation of the circulating forms of serum transferrin receptor using domain-specific antibodies.
To characterize the nature of the immunoreactive transferrin receptor in human serum, antisera were developed to peptide sequences of the extracellular domain of human transferrin receptor between amino acids 107 and 120 and the intracellular domain between amino acids 40 and 54. Antisera against the extracellular domain exhibited reactivity against both purified intact receptor and immunopurified circulating receptor, whereas antisera against the intracellular domain reacted only with intact receptor. Using competitive binding enzyme-linked immunosorbent assays, transferrin receptor in ultracentrifuged sera from normal subjects and patients with sickle cell anemia could be detected with antisera against the extracellular but not the intracellular domain. When the pellet obtained by ultracentrifugation of these sera was assayed after solubilization in 1% teric (polyoxyethylene-9-lauryl ether), only 0.6% of total serum receptor was detected in normal subjects and 3.8% in subjects with sickle cell disease. Roughly equal amounts of this pelleted immunoactivity were detected with antibodies against the extracellular and intracellular domains. These results indicate that less than 1% of transferrin receptor in normal human sera is intact receptor consistent with an exosomal origin and that virtually all circulating transferrin receptor is in the form of a truncated extracellular domain.
Amino Acid Sequence↗
Molecular characteristics of the Goodpasture autoantigen.
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.
Amino Acid Sequence↗
Production of the serum form of the transferrin receptor by a cell membrane-associated serine protease.
Recent investigations have demonstrated that the soluble form of the transferrin receptor in human serum is an 85-kDa fragment of intact receptor containing most of the extracellular domain. The recent demonstration of a remnant of the truncated receptor in cell membranes suggests that the soluble form arises from proteolytic cleavage of intact receptor. In the present investigation, domain-specific antibodies were used to further examine the subcellular location and nature of this proteolysis. HL60 cells were used in the investigation because the cells release an 85-kDa soluble form of the receptor to the culture supernatant that is identical to that found in serum. When intact, purified transferrin receptor from human placenta was incubated with the culture supernatant, no proteolytic activity could be demonstrated. However, when purified membrane fractions from HL60 cells were used in this incubation system, the 85-kDa fragment was produced. This activity was inhibited by serine protease inhibitors indicating that cell membrane fractions contain a serine protease capable of producing the serum form of the transferrin receptor.
Amino Acid Sequence↗
The alpha 4(IV) chain of basement membrane collagen. Isolation of cDNAs encoding bovine alpha 4(IV) and comparison with other type IV collagens.
Renal basement membranes are believed to contain five distinct type IV collagens. An understanding of the specific roles of these collagens and the specificities of their interactions will be aided by knowledge of their comparative structures. Genes for alpha 1(IV), alpha 2(IV), alpha 3(IV), and alpha 5(IV) have been cloned and the deduced peptide sequences compared. A fifth chain, alpha 4(IV), has been identified in glomerular and other basement membranes. Using a polymerase chain reaction-based strategy and short known peptide sequences from the noncollagenous domain (NC1), we have cloned and characterized partial bovine cDNAs of alpha 4(IV). Sequence analysis shows that this molecule has characteristic features of type IV collagens including an NH2-terminal Gly-X-Y domain which is interrupted at several points and a COOH-terminal NC1 domain with 12 cysteine residues in positions identical to those of other type IV collagens. Within the NC1 domain bovine alpha 4(IV) has 70, 59, 58, and 53% amino acid identity with human alpha 2(IV), alpha 1(IV), alpha 5(IV), and alpha 3(IV), respectively. Alignment of the peptides also shows that alpha 4(IV) is most closely related to alpha 2(IV). Nevertheless, in the extreme COOH-terminal region of the NC1 domain there are structural features that are unique to alpha 4(IV). Cloning of the region of alpha 4(IV) that encodes the NC1 domain allows comparison of all five type IV collagens and highlights certain regions that are likely to be important in the specificities of NC1-NC1 interactions and in other discriminant functions of these molecules.
Amino Acid Sequence↗
The pathogenesis of Alport syndrome involves type IV collagen molecules containing the alpha 3(IV) chain: evidence from anti-GBM nephritis after renal transplantation.
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.
Adult↗
Type IV collagen of Engelbreth-Holm-Swarm tumor matrix: identification of constituent chains.
The noncollagenous hexamer (NC1) domain of type IV collagen from Engelbreth-Holm-Swarm (EHS) sarcoma matrix was subjected to electrophoretic, amino-terminal amino acid sequence, and immunochemical analysis to determine which of the five known kinds of alpha(IV) chains are present. Electrophoretic analysis, whether by one-dimensional or two-dimensional electrophoresis, showed that nonlathyritic and lathyritic hexamer gave nearly identical patterns. Amino-terminal amino acid sequence analysis of hexamer subunits, transblotted from two-dimensional gels, revealed that the hexamer subunits were derived exclusively from the alpha 1 and alpha 2 chains. Western blots of hexamer subunits confirmed the sequence results, as the subunits. identified as alpha 1(IV) and alpha 2(IV) NC1 domains reacted with antibodies directed specifically against those subunits. Conversely, no reactivity of NC1 hexamer subunits was seen with Goodpasture serum, or with antibodies directed specifically against the alpha 3, alpha 4, and alpha 5 NC1 domains, confirming the lack of alpha 3, alpha 4, and alpha 5 chains. These results revealed that the type IV collagen component of the EHS sarcoma matrix is comprised exclusively of alpha 1 and alpha 2 chains. Its relative homogeneity simplifies, but restricts, interpretation of studies that employ it as a model type IV collagen because the studies would be based only on alpha 1 and alpha 2 chains.
Amino Acid Sequence↗
Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the alpha 3(IV) chain of basement membrane collagen.
The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.
Amino Acid Sequence↗
Glomerular basement membrane. Identification of dimeric subunits of the noncollagenous domain (hexamer) of collagen IV and the Goodpasture antigen.
The noncollagenous (NC1) domain hexamer of glomerular basement membrane (GBM) collagen is composed of a multiplicity of monomeric and dimeric subunits, and specific subunits are the targets for anti-GBM autoantibodies of patients with Goodpasture (GP) syndrome. The identity of GBM monomers has been established and the alpha 3(IV)NC1 monomer identified as the one that binds GP antibodies (Gunwar, S., Saus, J., Noelken, M. E., and Hudson, B. G. (1990) J. Biol. Chem. 265, 5466-5469). In the present study, the chain origin of 25 dimeric components and the identity of those that bound the anti-GBM antibodies from two GP patients were determined. This was accomplished by NH2-terminal sequence analysis and immunoblotting analysis of dimeric components that were resolved by two-dimensional electrophoresis in combination with high pressure liquid chromatography. The results revealed that (a) the components are mainly homodimers of the NC1 domains of alpha 1, alpha 2, alpha 3, alpha 4, and probably alpha 5 chains of collagen IV, reflecting a specificity of promoter-promoter association and (b) each homodimer had several size and charge isoforms. The GP antibodies bound exclusively to both alpha 3(IV)NC1 monomers and dimers and not to other basement membrane constituents. These findings provided new insights about the structure of GBM collagen and together with our previous findings firmly established the alpha 3(IV) chain as the target for the anti-GBM antibodies that mediate glomerulonephritis and pulmonary hemorrhage in patients with Goodpasture syndrome.
Adult↗
Properties of the collagenous domain of the alpha 3(IV) chain, the Goodpasture antigen, of lens basement membrane collagen. Selective cleavage of alpha (IV) chains with retention of their triple helical structure and noncollagenous domain.
A third chain, alpha 3(IV), of basement membrane collagen was recently discovered and was identified as the primary target for the autoantibodies of patients with Goodpasture syndrome (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, this chain was excised in the form of a truncated promoter by cleavage of basement membrane with Pseudomonas aeruginosa elastase and characterized. The triple helical structure and NC1 domain were retained. Elastase selectively cleaved at a site within the triple helical domain of the alpha 3 chain that is distinct from the cleavage site of the alpha 1 and alpha 2 chains. The truncated alpha 3 chain was found to contain 1460 residues, of which 1225 comprise the collagenous domain, and is cross-linked within this domain by disulfide bonds, forming a high Mr complex (greater than 300,000). Truncated protomers with a length of 340 nm corresponding to the theoretical length for the truncated alpha 3 chain were observed by electron microscopy as suprastructures in which the triple helical domains of three protomers were interwined. These protomers were also connected to each other and to the 140-nm protomers that appear to be comprised of the alpha 1 and alpha 2 chains. These results extended the known length of the alpha 3 chain by about 1000 residues and suggested that protomers of this chain self-associate through interactions between their triple helical domains and between their NC1 domains.
Amino Acid Sequence↗
Bovine glomerular basement membrane. Location and structure of the asparagine-linked oligosaccharide units and their potential role in the assembly of the 7 S collagen IV tetramer.
Collagen IV contains an amino-terminal tetramerization domain (7 S) that is involved in aggregation and cross-linking as part of the process of self-assembly of the collagen IV matrix of basement membranes. We determined the structure and location of the Asn-linked oligosaccharides of the 7 S tetramer. Two glycopeptides, GP-1 and GP-2, were isolated from tryptic digests of the 7 S tetramer and were characterized. GP-1 and GP-2 are derived from the alpha 1(IV) chain and the alpha 2(IV) chain, respectively. Each glycopeptide contained one sequence, -Asn-Xaa-Thr-, which was shown to be N-glycosylated at Asn, corresponding to position 126 of the alpha 1 chains and 138 of the alpha 2 chain. 1H NMR spectroscopic analysis of the oligosaccharide is a biantennary N-acetyllactosamine type of N-linked oligosaccharide with a broad heterogeneity in the presence of the sugar residues at their nonreducing termini as indicated. [formula: see text] The location of the Asn-linked oligosaccharide units and Hyl-linked disaccharide units and their orientation with respect to the surface of the triple helix were calculated using two models. We conclude that both units are important determinants in the assembly of the 7 S tetramer.
Amino Acid Sequence↗
Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.
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.
Amino Acids↗