Parvin Todd

Isoform switching of type IV collagen is developmentally arrested in X-linked Alport syndrome leading to increased susceptibility of renal basement membranes to endoproteolysis.

Normal glomerular capillaries filter plasma through a basement membrane (GBM) rich in alpha3(IV), alpha4(IV), and alpha5(IV) chains of type IV collagen. We now show that these latter isoforms are absent biochemically from the glomeruli in patients with X-linked Alport syndrome (XAS). Their GBM instead retain a fetal distribution of alpha1(IV) and alpha2(IV) isoforms because they fail to developmentally switch their alpha-chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS also confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins. The incorporation of cysteine-rich alpha3(IV), alpha4(IV), and alpha5(IV) chains into specialized basement membranes like the GBM may have normally evolved to protectively enhance their resistance to proteolytic degradation at the site of glomerular filtration. The relative absence of these potentially protective collagen IV isoforms in GBM from XAS may explain the progressive basement membrane splitting and increased damage as these kidneys deteriorate.

In Vitro Kinetic Studies of Formation of Antigenic Advanced Glycation End Products (AGEs) NOVEL INHIBITION OF POST-AMADORI GLYCATION PATHWAYS

Nonenzymatic protein glycation (Maillard reaction) leads to heterogeneous, toxic, and antigenic advanced glycation end products (“AGEs”) and reactive precursors that have been implicated in the pathogenesis of diabetes, Alzheimers disease, and normal aging. In vitro inhibition studies of AGE formation in the presence of high sugar concentrations are difficult to interpret, since AGE-forming intermediates may oxidatively arise from free sugar or from Schiff base condensation products with protein amino groups, rather than from just their classical Amadori rearrangement products. We recently succeeded in isolating an Amadori intermediate in the reaction of ribonuclease A (RNase) with ribose (Khalifah, R. G., Todd, P., Booth, A. A., Yang, S. X., Mott, J. D., and Hudson, B. G. (1996) Biochemistry 35, 4645-4654) for rapid studies of post-Amadori AGE formation in absence of free sugar or reversibly formed Schiff base precursors to Amadori products. This provides a new strategy for a better understanding of the mechanism of AGE inhibition by established inhibitors, such as aminoguanidine, and for searching for novel inhibitors specifically acting on post-Amadori pathways of AGE formation. Aminoguanidine shows little inhibition of post-Amadori AGE formation in RNase and bovine serum albumin, in contrast to its apparently effective inhibition of initial (although not late) stages of glycation in the presence of high concentrations of sugar. Of several derivatives of vitamins B1 and B6 recently studied for possible AGE inhibition in the presence of glucose (Booth, A. A., Khalifah, R. G., and Hudson, B. G. (1996) Biochem. Biophys. Res. Commun. 220, 113-119), pyridoxamine and, to a lesser extent, thiamine pyrophosphate proved to be novel and effective post-Amadori inhibitors that decrease the final levels of AGEs formed. Our mechanism-based approach to the study of AGE inhibition appears promising for the design and discovery of novel post-Amadori AGE inhibitors of therapeutic potential that may complement others, such as aminoguanidine, known to either prevent initial sugar attachment or to scavenge highly reactive dicarbonyl intermediates.

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.

Quaternary Organization of the Goodpasture Autoantigen, the α3(IV) Collagen Chain SEQUESTRATION OF TWO CRYPTIC AUTOEPITOPES BY INTRAPROTOMER INTERACTIONS WITH THE α4 AND α5 NC1 DOMAINS

Goodpastures (GP) disease is caused by autoantibodies that target the α3(IV) collagen chain in the glomerular basement membrane (GBM). Goodpasture autoantibodies bind two conformational epitopes (EA and EB) located within the non-collagenous (NC1) domain of this chain, which are sequestered within the NC1 hexamer of the type IV collagen network containing the α3(IV), α4(IV), and α5(IV) chains. In this study, the quaternary organization of these chains and the molecular basis for the sequestration of the epitopes were investigated. This was accomplished by physicochemical and immunochemical characterization of the NC1 hexamers using chain-specific antibodies. The hexamers were found to have a molecular composition of (α3)2(α4)2(α5)2 and to contain cross-linked α3-α5 heterodimers and α4-α4 homodimers. Together with association studies of individual NC1 domains, these findings indicate that the α3, α4, and α5 chains occur together in the same triple-helical protomer. In the GBM, this protomer dimerizes through NC1-NC1 domain interactions such that the α3, α4, and α5 chains of one protomer connect with the α5, α4, and α3 chains of the opposite protomer, respectively. The immunodominant Goodpasture autoepitope, located within the EA region, is sequestered within the α3α4α5 protomer near the triple-helical junction, at the interface between the α3NC1 and α5NC1 domains, whereas the EB epitope is sequestered at the interface between the α3NC1 and α4NC1 domains. The results also reveal the network distribution of the six chains of collagen IV in the renal glomerulus and provide a molecular explanation for the absence of the α3, α4, α5, and α6 chains in Alport syndrome.

Unusual susceptibility of heme proteins to damage by glucose during non-enzymatic glycation

Glucose modifies the amino groups of proteins by a process of non-enzymatic glycation, leading to potentially deleterious effects on structure and function that have been implicated in the pathogenesis of diabetic complications. These changes are extremely complex and occur very slowly. We demonstrate here that hemoglobin and myoglobin are extremely susceptible to damage by glucose in vitro through a process that leads to complete destruction of the essential heme group. This process appears in addition to the expected formation of so-called advanced glycation end products (AGEs) on lysine and other side-chains. AGE formation is enhanced by the iron released. In contrast, the heme group is not destroyed during glycation of cytochrome c, where the sixth coordination position of the heme iron is not accessible to solvent ligands. Glycation leads to reduction of ferricytochrome c in this case. Since hydrogen peroxide is known to destroy heme, and the destruction observed during glycation of hemoglobin and myoglobin is sensitive to catalase, we propose that the degradation process is initiated by hydrogen peroxide formation. Damage may then occur through reaction with superoxide generated (a reductant of ferricytochrome c), or hydroxyl radicals, or with both.

A Role for Collagen IV Cross-links in Conferring Immune Privilege to the Goodpasture Autoantigen : STRUCTURAL BASIS FOR THE CRYPTICITY OF B CELL EPITOPES

The detailed structural basis for the cryptic nature (crypticity) of a B cell epitope harbored by an autoantigen is unknown. Because the immune system may be ignorant of the existence of such “cryptic” epitopes, their exposure could be an important feature in autoimmunity. Here we investigated the structural basis for the crypticity of the epitopes of the Goodpasture autoantigen, the α3α4α5 noncollagenous-1 (NC1) hexamer, a globular domain that connects two triple-helical molecules of the α3α4α5 collagen IV network. The NC1 hexamer occurs in two isoforms as follows: the M-isoform composed of monomer subunits in which the epitopes are accessible to autoantibodies, and the D-isoform composed of both monomer and dimer subunits in which the epitopes are cryptic. The D-isoform was characterized with respect to quaternary structure, as revealed by mass spectrometry of dimer subunits, homology modeling, and molecular dynamics simulation. The results revealed that the D-isoform contains two kinds of cross-links as follows: S-hydroxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the α3α5-heterodimers and α4α4-homodimers, respectively. Construction and analysis of a three-dimensional model of the D-isoform of the α3α4α5 NC1 hexamer revealed that crypticity is a consequence of the following: (a) sequestration of key residues between neighboring subunits that are stabilized by domain-swapping interactions, and (b) by cross-linking of subunits at the trimer-trimer interface, which stabilizes the structural integrity of the NC1 hexamer and protects against binding of autoantibodies. The sequestrated epitopes and cross-linked subunits represent a novel structural mechanism for conferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.