Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tubular basement membrane (TBM) was prepared from normal human kidneys and solubilized with various enzymes. Collagenase digestion released antigenic moieties from the TBM. All four anti-TBM antibodies we studied, three from patients with idiopathic tubulo-interstitial nephritis (TIN) and one from a renal allograft recipient, distinctively reacted with collagenase-digested (CD) TBM during enzyme-linked immunoassay and could discriminate among sera of normal controls or of other nephritis patients, including anti-glomerular basement membrane (anti-GBM) nephritis. When digested with pronase, trypsin, or pepsin, antigenicity of the TBM decreased. We studied the TBM antigens with immunoprecipitation and immunoblotting. After incubation of radio-iodinated CDTBM with anti-TBM sera, immunoprecipitates were identified by single-dimension SDS polyacrylamide gel electrophoresis or two-dimension gel electrophoresis, followed by autoradiography. All four antibodies had identical results on immunoprecipitation; under nonreducing conditions, they gave two protein bands with m.w. of 54,000 and 48,000 and with pI 7.0 to 8.0 and 6.5 to 7.0. Electrophoresis performed under reducing conditions disclosed only one band at the m.w. of 48,000 and pI of 6.5, suggesting that the 54-kDa component is composed of peptides linked by interchain disulfide bonds. Immunoblot analysis showed that the anti-TBM antibodies were heterogeneous; three antibodies from the idiopathic TIN patients reacted with the 54-kDa band, but the one from the renal allograft recipient reacted with neither band. This finding suggests that there are two antigenic determinants on the 54-kDa component. One such determinant that was resistant to denaturation with SDS was detected by the first three antibodies, and the other that was sensitive to such denaturation bound to the last antibody. The 48-kDa component seemed not to be immunoreactive after incubation with SDS. We studied TBM antigens reactive with anti-GBM antibodies. By immunoblotting, all four sera from patients with anti-GBM nephritis stained TBM proteins of 45 to 50 kDa and 25 to 27 kDa at pH 8.0 to 9.0; this was similar to the staining pattern of CDGBM with the same sera, but the highly cationic (pH greater than 9.0) components were specifically detected in the CDGBM. By inhibition ELISA, the binding of the anti-GBM sera to denatured CDTBM decreased with preincubation of the sera with CDGBM, suggesting that the anti-GBM antibodies recognize the same epitope(s) on the GBM and the TBM.
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PMID:Characterization of tubular basement membrane antigens in human kidney. 300 98

The sera of 21 patients positive for antibodies against GBM in indirect immunofluorescence tests were examined by immunoblotting. We demonstrated antibodies against 50, 48, 43 and 29 kD molecular weight peptides in 20 of 21 sera using collagenase-digested GBM, in 19 of 21 using trypsin-digested GBM, and in 10 of 21 using elastase-digested GBM. Although the spectrum of molecular weights of the antigenic proteins was similar in all three digests, they differed with respect to preservation of antigenicity upon reduction with mercaptoethanol. Many of the sera of patients and controls reacted with proteins unrelated to GBM, e.g. albumin and prealbumin. Furthermore, some control sera reacted with one single peptide of the above-mentioned specific GBM peptides. Our results suggest that the highly purified 29 kD peptide of the collagenase digest or the 50 kD peptide of the trypsin digest provide the best antigens to develop a screening test for antibodies against GBM. However, serum antibodies against these antigens will not be absolutely specific for anti-GBM antibody-mediated nephritis, as shown by the immunoblot experiments.
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PMID:Characterisation and specificity of glomerular basement membrane antigens identified by sera of patients with anti-GBM nephritis. 311 Jun 69

The globular domain NC1 of human basement membrane collagen IV was isolated from glomerular basement membrane after collagenase digestion by chromatographic purification. The human NC1 appears as a hexamer of 160 kD by molecular sieve chromatography which migrates as a single molecule at gel electrophoresis without sodium dodecyl sulphate (SDS). Reversible dissociation of the hexamer into monomers and dimers was achieved by 6 M guanidine-HC1, SDS, or at pH values less than 4.0. All the subunits of 26 kD, 28 kD, 44 kD, and 50 kD showed reactivity with anti-GBM antibodies on immunoblotting. Inhibition-ELISA demonstrated that the intact hexamer also binds to anti-GBM antibodies at higher NC1 concentrations. However, dose-response curves indicated an approximately 20-50-fold increase in reactivity after dissociation of the hexamer in 6 M guanidine-HC1. Analysis of thermostability demonstrated that heating for 24 h at 37 degrees C or 56 degrees C did not alter the reactivity to anti-GBM antibodies, while reactivity was lost after heating for more than 120 min at 95 degrees C. In contrast to bovine NC1 unfolding of the antigen occurs immediately and does not require elevated temperature. Rotary shadowing of human NC1 at neutral pH revealed homogeneous globules. Distinct but incomplete dissociation into monomers and dimers could be observed at pH 2.5. These in vitro data of the human NC1 domain give further evidence that most of the Goodpasture epitopes are sequestered within the NC1 hexamer and support the hypothesis that production of anti-GBM autoantibodies may be initiated after dissociation of the hexamer has been induced, possibly by a toxic or infective episode.
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PMID:Immunological properties of the human Goodpasture target antigen. 322 45

NC1 subunits were purified from gel filtration pools of acid-extracted, collagenase-digested human glomerular basement membranes (hGBM). This methodology, which enriches 28-kDa monomers (M28) in the total digest, allowed purification of these monomers and 24-kDa (M24) and 26-kDa (M26) monomers free from dimers. Reactivity of these subunits with Goodpasture autoantibodies using immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional nonequilibrium pH gradient electrophoresis gels showed strong reactivity with the purified M28 subunits. Inhibition enzyme-linked immunosorbent assay, used to quantitate the reactivity of the purified NC1 subunits, indicated that M28 had a greater than 10-fold increase in ability to inhibit binding to NC1 than NC1 itself. Comparison of hGBM NC1 components were made with those obtained from collagenase digests of salt and acid-extracted bovine and sheep GBM and Englebreth-Holm-Swarm tumor similarly purified by gel filtration and reverse-phase high performance liquid chromatography. Two-dimensional gel analysis of these NC1 isolates revealed absence of the very cationic M28 monomers. Reactivity with antibodies eluted from diseased kidneys of sheep immunized with hGBM (Steblay nephritis) was compared with Goodpasture autoantibody reactivity by immunoblotting two-dimensional gels of hGBM NC1. We conclude that a very cationic M28 monomer (M28 ) found only in hGBM is the probable target in Goodpasture syndrome, that the epitope is present on most NC1 components from extracted and unextracted hGBM, and is exposed by urea denaturation which is enhanced by acid treatment. A weakly cationic M28 monomer (M28+) is present in GBM from other species and is the probable target in Steblay nephritis. Differential recognition of the two M28 components by these antibodies points to different genetic origins or possibly distinct post-translational modifications for these components. This is supported by their presence or absence in different species and tissues, as well as biochemical differences from the M24/26 monomers which presumably are derived from alpha 1(IV) and alpha 2(IV) collagen chains.
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PMID:Antibody specificity of human glomerular basement membrane type IV collagen NC1 subunits. Species variation in subunit composition. 378 34

Components were solubilized from human glomerular basement membrane by digestion with collagenase and pepsin or by extraction with guanidine-HCl either directly or after previous digestion with the enzyme. The diverse preparations were used as antigens in the enzyme-linked immunosorbent assay (ELISA) of antibody titers in sera from patients with Goodpasture syndrome and patients with other forms of glomerulonephritis, that is, systemic lupus erythematosus, periarteritis nodosa, and IgA-related nephropathy. Patients with Goodpasture syndrome had high titers of IgG antibodies reacting most strongly with collagenase digests. The antigen(s) was only partly solubilized by guanidine-HCl extraction, was destroyed by pepsin digestion as well as reduction, and partly destroyed by trypsin digestion. The antigen(s) is most likely noncollagenous protein. Antibodies from patients with other forms of nephritis were directed primarily against antigens in guanidine-HCl extracts, while the antigen(s) was not solubilized by collagenase digestion. Pepsin digestion destroyed the antigen(s). The antibodies were of a different class, that is, the patients with systemic lupus erythematosus had IgG and IgA as well as IgM antibodies; the patients with periarteritis nodosa had IgM or IgG and IgA antibodies, while the patients with IgA-related nephritis had the highest recorded titers of IgA but also had IgG as well as IgM antibodies. None of the patients had antibodies directed against triple helical collagen. The antibody response in anti-GBM antibody-related nephritis, then, is different both with respect to antigen and antibody class and depends on the underlying disease syndrome.
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PMID:Antiglomerular basement membrane antibody: antibody specificity in different forms of glomerulonephritis. 613 25

To clarify the immunological mechanisms in poststreptococcal acute glomeulonephritis (PSAGN) and anaphylactoid purpura (AP), anti-streptococcal cell membrane (anti-SCM) and anti-human glomerular basement membrane (anti-GBM) titers in the sera of patients with PSAGN and AP were determined by passive hemagglutination with chromic chloride-treated sheep erythrocytes. Sodium lauryl sulfate (SLS) soluble SCM and collagenase soluble GBM were used as soluble antigens. Positive anti-SCM titers (greater than 1:8) were demonstrated in 10 of 14 patients (71.4%) with PSAGN and in 4 of 9 patients (44.4%) with AP with evidence of antecedent streptococcal infection. Two of 4 patients with AP without evidence of antecedent streptococcal infection had positive anti-SCM titers. No correlation was noted between anti-streptolysin O (ASO) titers and anti-SCM titers in patients with PSAGN or AP, but many patients with high ASO titers also had high anti-SCM titers. No positive anti-GBM reactions were detected in patients with PSAGN or AP. No cross-reactions were noted between SLS soluble SCM and collagenase soluble GBM.
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PMID:Anti-streptococcal cell membrane and anti-human glomerular basement membrane titers in sera of patients with poststreptococcal acute glomerulonephritis and anaphylactoid purpura. 635 77

Anti-basement membrane antibodies are now being associated with an increasing spectrum of disease, including Goodpasture's syndrome, rapidly progressive and occasionally milder forms of glomerulonephritis (GN), tubulointerstitial nephritis, pulmonary damage, and potentially other forms of tissue injury. We have developed a radioimmunoassay to detect circulating antiglomerular basement membrane (GBM) antibodies. The antigens for this assay are derived from the noncollagenous portion of the GBM remaining after collagenase digestion. After immunoabsorptive purification, the major antigens precipitated by human anti-GBM antibodies can be characterized by polyacrylamide gel electrophoresis (PAGE) into an unresolved high molecular weight fraction and two antigenic peaks of 54,000 and 27,000 daltons. The noncollagenous nature of the antigenic material has been confirmed by amino acid analysis. The radiolabelled antigen has proven useful in detecting circulating anti-GBM antibodies in over 500 patients. The assay is of use in monitoring the activity of disease and judging the patient's response to therapy. It is also useful in determining the timing of renal transplantation, if required. Differences in antigenic content of glomerular and tubular basement membranes (TBM) have been noted between individuals. These antigenic differences, under certain circumstances, can lead to the induction of anti-basement membrane antibody responses after transplantation.
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PMID:Anti-basement membrane antibodies in immunologic renal disease. 702 Jun 72

The Goodpasture's epitope has been mapped to the alpha 3 non-collagenous chain (NC1) of type (IV) collagen [alpha 3col(IV)]. We have developed a model of experimental autoimmune glomerulonephritis (EAG) in rats immunized once with collagenase solubilized GBM (csGBM). Engelbreth-Holm-Swarm (EHS) tumor contains abundant col(IV) with little or no alpha 3col(IV). To test the hypothesis that antigens related to Goodpasture epitope are required to produce EAG in our model, we immunized rats once with 40 micrograms csEHS. Positive controls immunized with csGBM developed typical EAG with GBM bound antibody, proteinuria, and glomerulonephritis. EHS rats developed circulating and bound antibody to mesangium and tubular basement membrane with minimal GBM deposits, but did not develop proteinuria or glomerulonephritis. Although circulating antibody in EHS rats bound to csGBM by ELISA, there was no binding in ELISA to M2 antigen containing the Goodpasture epitope while EAG rat's serum did bind. By Western blot with antisera to Goodpasture epitope, EHS antigen was less complex than GBM in the monomer/dimer regions and appeared to lack NC1 corresponding to alpha 3col(IV). Blotting with sera from EHS rats demonstrated reactivity to various components of GBM but not to alpha 3col(IV). EAG sera and renal eluates bound to alpha 3col(IV). EAG rats evidenced cell mediated immunity while EHS rats did not (stimulation index EHS 1.1, EAG rats 8.0).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Study of EHS type IV collagen lacking Goodpasture's epitope in glomerulonephritis in rats. 753 54

A high titre of antibodies to collagenase-solubilised human glomerular basement membrane (CS-GBM) is almost pathognomic of Goodpasture's (anti-GBM) disease. In order to develop an assay independent of scarce human material, a molecule of approximately 26 kD corresponding to the C-terminal NC1 domain of the alpha 3 chain of type IV collagen was purified from sheep GBM by gel filtration and reverse phase HPLC. This molecule was antigenic when assessed by inhibition studies, by immunoblotting, and as a ligand on ELISA plates. An ELISA using this sheep alpha 3(IV)NC1 preparation to detect circulating anti-GBM antibodies gave comparable results to the standard RIA using crude CS-GBM. Sera from patients with a variety of nephropathies other than Goodpasture's disease gave negative results. In a prospective study, 170 consecutive sera were analysed by both the ELISA and the RIA. Twenty seven specimens gave positive results in one or both of the assays. Eleven of these were confirmed as true positive results and all were correctly identified by the RIA. Two false negative results in the ELISA occurred in previously treated cases, and both sera were only weakly positive by RIA. The RIA gave 13 false positive results compared with five by ELISA. The ELISA using highly purified sheep antigen is a robust, reliable, and more specific alternative to immunoassays based on crude human antigen preparations.
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PMID:Development and application of an ELISA for Goodpasture's disease based on sheep alpha 3(IV)NC1 domains. 873 32

X-linked Alport syndrome (AS) is a heritable disorder which is associated with mutations in the type IV collagen alpha 5 (IV) chain gene (COL4A5) located on chromosome X. Following renal transplantation, an average of 6% of male AS patients develop anti-GBM nephritis. We studied the specificity of the antibodies against type IV collagen in the serum of a patient with COL4A5 partial deletion. The specificity of these alloantibodies was determined against collagenase-digested GBM, as well as against recombinant non-collagenous (NC1) domains of the type IV collagen alpha 1(IV)-alpha 6(IV) chains expressed in escherichia coli. Immunoblotting and ELISA demonstrated that these antibodies bound specifically to the NC1 domain of alpha 5(IV) collagen. There was no binding to the NC1 domain of the other chains, including the Goodpasture antigen. Competitive ELISA confirmed the results obtained by ELISA and immunoblotting. This patient developed alloantibodies directed against antigens present in the grafted kidney, but absent from his Alport kidney. The pathogenesis of post-transplantation glomerulonephritis in the Alport patient studied is thus similar to that of Goodpasture syndrome, with the exception that the pathogenic antibodies are targeted to another alpha chain of type IV collagen.
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PMID:Identification of post-transplant anti-alpha 5 (IV) collagen alloantibodies in X-linked Alport syndrome. 891 11


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