Roberto M. Vanacore

A Sulfilimine Bond Identified in Collagen IV

Toughing It Out in Membranes The formation of crosslinks in the triple-helical structure of collagen is crucial to its function. In the case of collagen IV, which provides structural integrity to the basement membrane of animal tissues, the chemical nature of the crosslinks has been difficult to pin down. Vanacore et al. (p. 1230) report a combination of high-resolution mass spectrometric and nuclear magnetic resonance studies that establish the crosslink between hydroxylysine-211 and methionine-93 is a sulfimine group (-S=N-). This type of bond appears to have evolved to withstand mechanical stress as animals evolved into more structurally complex organisms. An unusual sulfilimine bond provides a reinforcing cross-link of an extracellular matrix protein, collagen IV. Collagen IV networks are ancient proteins of basement membranes that underlie epithelia in metazoa from sponge to human. The networks provide structural integrity to tissues and serve as ligands for integrin cell-surface receptors. They are assembled by oligomerization of triple-helical protomers and are covalently crosslinked, a key reinforcement that stabilizes networks. We used Fourier-transform ion cyclotron resonance mass spectrometry and nuclear magnetic resonance spectroscopy to show that a sulfilimine bond (-S=N-) crosslinks hydroxylysine-211 and methionine-93 of adjoining protomers, a bond not previously found in biomolecules. This bond, the nitrogen analog of a sulfoxide, appears to have arisen at the divergence of sponge and cnidaria, an adaptation of the extracellular matrix in response to mechanical stress in metazoan evolution.

Bromine Is an Essential Trace Element for Assembly of Collagen IV Scaffolds in Tissue Development and Architecture

Bromine is ubiquitously present in animals as ionic bromide (Br(-)) yet has no known essential function. Herein, we demonstrate that Br(-) is a required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks, a posttranslational modification essential for tissue development and architecture found within the collagen IV scaffold of basement membranes (BMs). Bromide, converted to hypobromous acid, forms a bromosulfonium-ion intermediate that energetically selects for sulfilimine formation. Dietary Br deficiency is lethal in Drosophila, whereas Br replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br(-), collagen IV, and peroxidasin. We establish that Br(-) is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Thus, bromine is an essential trace element for all animals, and its deficiency may be relevant to BM alterations observed in nutritional and smoking-related disease. PAPERFLICK:

Peroxidasin forms sulfilimine chemical bonds using hypohalous acids in tissue genesis

Collagen IV comprises the predominant protein network of basement membranes, a specialized extracellular matrix, which underlie epithelia and endothelia. These networks assemble through oligomerization and covalent crosslinking to endow mechanical strength and shape cell behavior through interactions with cell-surface receptors. A recently discovered sulfilimine (S=N) bond between a methionine sulfur and hydroxylysine nitrogen reinforces the collagen IV network. We demonstrate that peroxidasin, an enzyme found in basement membranes, catalyzes formation of the sulfilimine bond. Drosophila peroxidasin mutants have disorganized collagen IV networks and torn visceral muscle basement membranes, pointing to a critical role for the enzyme in tissue biogenesis. Peroxidasin generates hypohalous acids as reaction intermediates, suggesting a paradoxically anabolic role for these usually destructive oxidants. This work highlights sulfilimine bond formation as what is to our knowledge the first known physiologic function for peroxidasin, a role for hypohalous oxidants in tissue biogenesis, and a possible role for peroxidasin in inflammatory diseases.

Cellular Protection Mechanisms against Extracellular Heme HEME-HEMOPEXIN, BUT NOT FREE HEME, ACTIVATES THE N-TERMINAL c-Jun KINASE

Hemopexin protects cells lacking hemopexin receptors by tightly binding heme abrogating its deleterious effects and preventing nonspecific heme uptake, whereas cells with hemopexin receptors undergo a series of cellular events upon encountering heme-hemopexin. The biochemical responses to heme-hemopexin depend on its extracellular concentration and range from stimulation of cell growth at low levels to cell survival at otherwise toxic levels of heme. High (2–10 μm) but not low (0.01–1 μm) concentrations of heme-hemopexin increase, albeit transiently, the protein carbonyl content of mouse hepatoma (Hepa) cells. This is due to events associated with heme transport since cobalt-protoporphyrin IX-hemopexin, which binds to the receptor and activates signaling pathways without tetrapyrrole transport, does not increase carbonyl content. The N-terminal c-Jun kinase (JNK) is rapidly activated by 2–10 μm heme-hemopexin, yet the increased intracellular heme levels are neither toxic nor apoptotic. After 24 h exposure to 10 μm heme-hemopexin, Hepa cells become refractory to the growth stimulation seen with 0.1–0.75 μm heme-hemopexin but HO-1 remains responsive to induction by heme-hemopexin. Since free heme does not induce JNK, the signaling events, like phosphorylation of c-Jun via activation of JNK as well as the nuclear translocation of NFκB, G2/M arrest, and increased expression of p53 and of the cell cycle inhibitor p21 WAF1/CIP1/SDI1 generated by heme-hemopexin appear to be of paramount importance in cellular protection by heme-hemopexin.

A unique covalent bond in basement membrane is a primordial innovation for tissue evolution

Significance The evolution of multicellular animals from single-celled ancestors was one of the most significant transitions of life on earth. The emergence of larger, more complex animals able to resist predation and colonize new environments was enabled, in part, by a collagen scaffold, which anchors cells together to form tissues and organs. Here, we show that a unique chemical bond, a link between sulfur and nitrogen atoms called a sulfilimine bond, arose over 500 Mya, binding this scaffold together and enabling tissues to withstand mechanical forces. Peroxidasin forms the bond by generating hypohalous acids as strong oxidants, a form of bleach, which normally function as antimicrobial agents. These understandings may lead to approaches for targeting tumors and treatment of other diseases. Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad—a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids—is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Identification of S-Hydroxylysyl-methionine as the Covalent Cross-link of the Noncollagenous (NC1) Hexamer of the α1α1α2 Collagen IV Network A ROLE FOR THE POST-TRANSLATIONAL MODIFICATION OF LYSINE 211 TO HYDROXYLYSINE 211 IN HEXAMER ASSEMBLY

Collagen IV networks are present in all metazoans as components of basement membranes that underlie epithelia. They are assembled by the oligomerization of triple-helical protomers, composed of three α-chains. The trimeric noncollagenous domains (NC1) of each protomer interact forming a hexamer structure. Upon exposure to acidic pH or denaturants, the hexamer dissociates into monomer and dimer subunits, the latter reflect distinct interactions that reinforce/cross-link the quaternary structure of hexamer. Recently, the cross-link site of the α1α1α2 network was identified, on the basis of x-ray crystal structures at 1.9-Å resolution, in which the side chains of Met93 and Lys211 were proposed to be connected by a novel thioether bond (Than, M. E., Henrich, S., Huber, R., Ries, A., Mann, K., Kuhn, K., Timpl, R., Bourenkov, G. P., Bartunik, H. D., and Bode, W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6607-6612); however, at the higher resolution of 1.5 Å, we found no evidence for this cross-link (Vanacore, R. M., Shanmugasundararaj, S., Friedman, D. B., Bondar, O., Hudson, B. G., and Sundaramoorthy, M. (2004) J. Biol. Chem. 279, 44723-44730). Given this discrepancy in crystallographic findings, we sought chemical evidence for the location and nature of the reinforcement/cross-link site. Trypsin digestion of monomer and dimer subunits excised a ∼5,000-Da complex that distinguished dimers from monomers; the complex was characterized by mass spectrometry, Edman degradation, and amino acid composition analyses. The tryptic complex, composed of two peptides of 44 residues derived from two α1 NC1 monomers, contained Met93 and Lys211 post-translationally modified to hydroxylysine (Hyl211). Truncation of the tryptic complex with post-proline endopeptidase reduced its size to 14 residues to facilitate characterization by tandem mass spectrometry, which revealed a covalent linkage between Met93 and Hyl211. The novel cross-link, termed S-hydroxylysyl-methionine, reflects at least two post-translational events in its formation: the hydroxylation of Lys211 to Hyl211 within the NC1 domain during the biosynthesis of α-chains and the connection of Hyl211 to Met93 between the trimeric NC1 domains of two adjoining triple-helical protomers, reinforcing the stability of collagen IV networks.

Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-β mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-β receptor (TβRII), which in turn promotes a TβRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TβRII is considered the principal regulatory mechanism of TβRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TβRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TβRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TβRII tail tyrosine residues, resulting in inhibition of TβR-dependent fibrotic signaling. The collagen-binding receptor integrin α1β1 was required for recruitment of TCPTP to the TβRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TβRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1β1 and TβRII that is essential for TβRII-mediated SMAD activation and fibrotic signaling pathways.

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.

Extracellular chloride signals collagen IV network assembly during basement membrane formation

Chloride is ubiquitous in physiology but understood to provide ionic strength for tissue function. The authors discover a molecular function of chloride whereby the ion signals the assembly of collagen IV, establishing a microenvironment on the outside of cells.

Goodpasture's disease: molecular architecture of the autoantigen provides clues to etiology and pathogenesis.

Purpose of reviewGoodpastures disease is an autoimmune disorder characterized by the deposition of pathogenic autoantibodies in basement membranes of kidney and lung, which induces rapidly progressive glomerulonephritis and pulmonary hemorrhage. The target antigen is the α3NC1 domain of collagen IV, which is expressed in target organs as an α345 network. Recent studies of specificity and epitopes of Goodpastures autoantibodies and discovery of novel posttranslational modification of the antigen, a sulfilimine bond, provide further insight into mechanisms of initiation and progression of Goodpastures disease. Recent findingsAnalysis of the specificity of Goodpastures autoantibodies revealed a distinct subset of circulating and kidney-bound antiα5NC1 antibody, which is associated with loss of kidney function. Structural integrity of the α345NC1 hexamer is stabilized by the novel sulfilimine crosslinks conferring immune privilege to the Goodpastures autoantigen. Native antibodies may contribute to establishment of immune tolerance to autoantigen. Structural analysis of epitopes for autoantibodies and alloantibodies indicates a critical role of conformational change in the α345NC1 hexamer in eliciting an autoimmune response in Goodpastures disease. SummaryUnderstanding of the quaternary structure of the Goodpastures autoantigen continues to provide insights into autoimmune mechanisms that serve as a basis for development of novel diagnostic tools and therapies for Goodpastures disease.