Sripad Gunwar

Circulating Factor Associated with Increased Glomerular Permeability to Albumin in Recurrent Focal Segmental Glomerulosclerosis

BACKGROUND Heavy proteinuria and progressive renal injury recur after transplantation in up to 40 percent of patients with renal failure caused by idiopathic focal segmental glomerulosclerosis. A circulating factor may be responsible for this recurrence. METHODS To determine whether patients with focal segmental glomerulosclerosis have a circulating factor capable of causing glomerular injury, we tested serum samples from 100 patients with the disorder in an in vitro assay of glomerular permeability to albumin. Of the 56 patients who had undergone renal transplantation, 33 had recurrences. Sixty-four patients, many of whom had undergone transplantation, were being treated with dialysis. Thirty-one patients with other renal diseases and nine normal subjects were also studied. RESULTS The 33 patients with recurrent focal segmental glomerulosclerosis after transplantation had a higher mean (+/-SE) value for permeability to albumin (0.47+/-0.06) than the normal subjects (0.06+/-0.07) or the patients who did not have recurrences (0.14+/-0.06). After plasmapheresis in six patients with recurrences, the permeability was reduced (from 0.79+/-0.06 to 0.10+/-0.05, P = 0.008), and proteinuria was significantly decreased. Patients with corticosteroid-sensitive nephrotic syndrome or with membranous nephropathy after transplantation had low levels of serum activity. The circulating factor bound to protein A and hydrophobic-interaction columns and had an apparent molecular mass of about 50 kd. CONCLUSIONS A circulating factor found in some patients with focal segmental glomerulosclerosis is associated with recurrent disease after renal transplantation and may be responsible for initiating the renal injury.

Type IV Collagen of the Glomerular Basement Membrane EVIDENCE THAT THE CHAIN SPECIFICITY OF NETWORK ASSEMBLY IS ENCODED BY THE NONCOLLAGENOUS NC1 DOMAINS

The ultrafiltration function of the glomerular basement membrane (GBM) of the kidney is impaired in genetic and acquired diseases that affect type IV collagen. The GBM is composed of five (α1 to α5) of the six chains of type IV collagen, organized into an α1·α2(IV) and an α3·α4·α5(IV) network. In Alport syndrome, mutations in any of the genes encoding the α3(IV), α4(IV), and α5(IV) chains cause the absence of the α3·α4·α5 network, which leads to progressive renal failure. In the present study, the molecular mechanism underlying the network defect was explored by further characterization of the chain organization and elucidation of the discriminatory interactions that govern network assembly. The existence of the two networks was further established by analysis of the hexameric complex of the noncollagenous (NC1) domains, and the α5 chain was shown to be linked to the α3 and α4 chains by interaction through their respective NC1 domains. The potential recognition function of the NC1 domains in network assembly was investigated by comparing the composition of native NC1 hexamers with hexamers that were dissociated and reconstituted in vitro and with hexamers assembled in vitro from purified α1-α5(IV) NC1 monomers. The results showed that NC1 monomers associate to form native-like hexamers characterized by two distinct populations, an α1·α2 and α3·α4·α5 heterohexamer. These findings indicate that the NC1 monomers contain recognition sequences for selection of chains and protomers that are sufficient to encode the assembly of the α1·α2 and α3·α4·α5 networks of GBM. Moreover, hexamer formation from the α3, α4, and α5 NC1 monomers required co-assembly of all three monomers, suggesting that mutations in the NC1 domain in Alport syndrome may disrupt the assembly of the α3·α4·α5 network by interfering with the assembly of the α3·α4·α5 NC1 hexamer.

Glomerular Basement Membrane IDENTIFICATION OF A NOVEL DISULFIDE-CROSS-LINKED NETWORK OF α3, α4, AND α5 CHAINS OF TYPE IV COLLAGEN AND ITS IMPLICATIONS FOR THE PATHOGENESIS OF ALPORT SYNDROME

Glomerular basement membrane (GBM) plays a crucial function in the ultrafiltration of blood plasma by the kidney. This function is impaired in Alport syndrome, a hereditary disorder that is caused by mutations in the gene encoding type IV collagen, but it is not known how the mutations lead to a defective GBM. In the present study, the supramolecular organization of type IV collagen of GBM was investigated. This was accomplished by using pseudolysin (EC3.4.24.26) digestion to excise truncated triple-helical protomers for structural studies. Two distinct sets of truncated protomers were solubilized, one at 4 °C and the other at 25 °C, and their chain composition was determined by use of monoclonal antibodies. The 4 °C protomers comprise the α1(IV) and α2(IV) chains, whereas the 25 °C protomers comprised mainly α3(IV), α4(IV), and α5(IV) chains along with some α1(IV) and α2(IV) chains. The structure of the 25 °C protomers was examined by electron microscopy and was found to be characterized by a network containing loops and supercoiled triple helices, which are stabilized by disulfide cross-links between α3(IV), α4(IV), and α5(IV) chains. These results establish a conceptual framework to explain several features of the GBM abnormalities of Alport syndrome. In particular, the α3(IV)·α4(IV)·α5(IV) network, involving a covalent linkage between these chains, suggests a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of not only α5(IV) chain but also the α3(IV) and α4(IV) chains in the GBM of patients with Alport syndrome.

The goodpastiure autoantigen; mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC-1 domain

The Goodpasture (GP) autoantigen has been identified as the α3(IV) collagen chain, one of six homologous chains designated α1–α6 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 α3(IV) chain. Fourteen α1/α3 NC1 chimeras were constructed by substituting one or more short sequences of α3(IV)NC1 at the corresponding positions in the non-immunoreactive α1(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 α3(IV)NC1 and C6 containing residues 127–141 of α3(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 α3(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 α1- and α3(IV)NC1 sequences. The amino acid sequences 17–31 and 127–141 in α3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.

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.

Seminiferous Tubule Basement Membrane COMPOSITION AND ORGANIZATION OF TYPE IV COLLAGEN CHAINS, AND THE LINKAGE OF α3(IV) AND α5(IV) CHAINS

Seminiferous tubule basement membrane (STBM) plays an important role in spermatogenesis. In the present study, the composition and structural organization of type IV collagen of bovine STBM was investigated. STBM was found to be composed of all six α-chains of type IV collagen based upon immunocytochemical and biochemical analysis. The content of α3(IV) chain (40%) and the α4(IV) chain (18%) was substantially higher than in any other basement membrane collagen. The supramolecular structure of the six α(IV) chains was investigated using pseudolysin (EC 3.4.24.26) digestion to excise triple-helical molecules, subsequent collagenase digestion to produce NC1 hexamers and antibody affinity chromatography to resolve populations of NC1 hexamers. The hexamers, which reflect specific arrangements of α(IV) chains, were characterized for their α(IV) chain composition using high performance liquid chromatography, two-dimensional electrophoresis, and immunoblotting with α(IV) chain-specific antibodies. Three major hexamer populations were found that represent the classical network of the α1(IV) and α2(IV) chains and two novel networks, one composed of the α1(IV)-α6(IV) chains and the other composed of the α3(IV)-α6(IV) chains. The results establish a structural linkage between the α3(IV) and α5(IV) chains, suggesting a molecular basis for the conundrum in which mutations in the gene encoding the α5(IV) chain cause defective assembly of the α3(IV) chain in the glomerular basement membrane of patients with Alport syndrome.

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.

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.

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli

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.

Unusual dissociative behavior of the noncollagenous domain (hexamer) of basement membrane collagen during electrophoresis and chromatofocusing.

The noncollagenous domain of basement membrane collagen exists as a hexamer upon excision with bacterial collagenase. Two hexamer subtypes, differing in subunit composition, have been identified and several additional subtypes are possible because at least two, and possibly more, triple-helical molecules, differing in chain composition, exist in authentic basement membranes (Saus J, Wieslander J, Langeveld JPM, Quinones S, and Hudson BG. (1988) Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV. J. Biol. Chem. 263:13374-13380). In the present study, the physiochemical behavior of hexamer during two-dimensional electrophoresis was evaluated. The hexamers from three different membranes of bovine origin (lens capsule, glomerular, and placenta) were found to exhibit an unusual dissociative property during the pH gradient electrophoresis used in the first dimension; namely, the hexamers dissociate under nondenaturing conditions into monomer and dimer subunits concomitant with the resolution of subunits. This dissociative property provided the basis for a new procedure using chromatofocusing for the preparative resolution of hexamer subunits with retention of their native structure and capacity to associate into a hexamer configuration. Associative studies revealed that the capacity for hexamer assembly is contained within the monomer subunit, a property which may be of fundamental importance in the mechanism of the assembly of collagen IV protomers and the association of protomers forming a supramolecular structure.