Vadim Pedchenko

Distinct target-derived signals organize formation, maturation, and maintenance of motor nerve terminals

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

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.

αvβ3 and αvβ5 Integrins Bind Both the Proximal RGD Site and Non-RGD Motifs within Noncollagenous (NC1) Domain of the α3 Chain of Type IV Collagen IMPLICATION FOR THE MECHANISM OF ENDOTHELIAL CELL ADHESION

The NC1 domains of human type IV collagen, in particular α3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051–8061). The recombinant α3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-α3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to αvβ3 integrin interactions with non-RGD motifs of the RGD-α3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745–23750). This nonfunctionality is surprising given that RGD is a binding site for αvβ3 integrin in several proteins. In the present study, we used the α3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of αvβ3 and αvβ5 integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the αvβ3 integrin-binding sites of the α3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the α3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-α3NC1 domain.

MT2-MMP-Dependent Release of Collagen IV NC1 Domains Regulates Submandibular Gland Branching Morphogenesis

Proteolysis is essential during branching morphogenesis, but the roles of MT-MMPs and their proteolytic products are not clearly understood. Here, we discover that decreasing MT-MMP activity during submandibular gland branching morphogenesis decreases proliferation and increases collagen IV and MT-MMP expression. Specifically, reducing epithelial MT2-MMP profoundly decreases proliferation and morphogenesis, increases Col4a2 and intracellular accumulation of collagen IV, and decreases the proteolytic release of collagen IV NC1 domains. Importantly, we demonstrate the presence of collagen IV NC1 domains in developing tissue. Furthermore, recombinant collagen IV NC1 domains rescue branching morphogenesis after MT2-siRNA treatment, increasing MT-MMP and proproliferative gene expression via beta1 integrin and PI3K-AKT signaling. Additionally, HBEGF also rescues MT2-siRNA treatment, increasing NC1 domain release, proliferation, and MT2-MMP and Hbegf expression. Our studies provide mechanistic insight into how MT2-MMP-dependent release of bioactive NC1 domains from collagen IV is critical for integrating collagen IV synthesis and proteolysis with epithelial proliferation during branching morphogenesis.

Hormone/growth factor interactions mediating epithelial/stromal communication in mammary gland development and carcinogenesis

Epithelial/mesenchymal interactions begin during embryonic development of the mammary gland and continue throughout mammary gland development into adult life. Stromal and epithelial growth factors that may mediate interactions between these compartments of the mammary gland are reviewed. Since mammogenic hormones are the primary regulators of mammary gland development, special consideration is given to hormonal regulation of growth factors in order to explore the integration of hormones and growth factors in the regulation of mammary gland growth and neoplasia. Examination of hormonal regulation of the fibroblast growth factor (FGF)-7/FGFR2-IIIb receptor system in the mammary gland reveals that mammogenic hormones differentially regulate the synthesis of stromal growth factors and their epithelial receptors. These effects serve to optimize the action of estrogen and progesterone on mammary gland development and illustrate that the ratio of these two hormones is critical in regulating this growth factor axis. The role of stromal/epithelial mitogenic microenvironments in modulating the genotype and phenotype of preneoplastic and neoplastic lesions by chemical carcinogens is discussed. Finally, changes in growth factor expression during mammary tumor progression are described to illustrate the relative roles that stromally-derived and epithelial-derived growth factors may play during progression to hormone independent tumor growth.

Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.

Collagen IV is the major protein found in basement membranes. It comprises three heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity. We constructed linear maps of the collagen IV heterotrimers (“interactomes”) that indicated major structural landmarks, known and predicted ligand‐binding sites, and missense mutations, in order to identify functional and disease‐associated domains, potential interactions between ligands, and genotype–phenotype relationships. The maps documented more than 30 known ligand‐binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin, and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection, and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases, and heparin. The maps indicated that mutations affecting major ligand‐binding sites, for example, for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms, and muscle Cramps [HANAC] syndrome, and early‐onset Alport syndrome, respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets. Hum Mutat 32:127–143, 2011.

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.

Integrin α3β1, a Novel Receptor for α3(IV) Noncollagenous Domain and a Trans-dominant Inhibitor for Integrin αvβ3

Exogenous soluble human α3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of α3NC1 to integrin αvβ3. However, in some tumor cells that express integrin αvβ3, the α3NC1 domain does not inhibit proliferation, suggesting that integrin αvβ3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble α3NC1 domain in cells lacking αvβ3 integrin. In these cells, soluble α3NC1 bound integrin α3β1; however, unlike αvβ3, α3β1 integrin did not mediate cell adhesion to immobilized α3NC1 domain. Interestingly, in cells lacking integrin α3β1, adhesion to the α3NC1 domain was enhanced due to activation of integrin αvβ3. These findings indicate that integrin α3β1 is a receptor for the α3NC1 domain and transdominantly inhibits integrin αvβ3 activation. Thus integrin α3β1, in conjunction with integrin αvβ3, modulates cellular responses to the α3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.

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.

A Nephritogenic Peptide Induces Intermolecular Epitope Spreading on Collagen IV in Experimental Autoimmune Glomerulonephritis

This group previously identified a peptide p13 of alpha3(IV)NC1 domain of type IV collagen that induces experimental autoimmune glomerulonephritis (EAG) in rats with generation of antibodies to sites on alpha3(IV)NC1 external to the peptide as a result of intramolecular epitope spreading. It was hypothesized that intermolecular epitope spreading to other collagen IV chains also was induced. Rats were immunized with nephritogenic peptide that was derived from the amino terminal part of rat alpha3(IV)NC1 domain, and serum and kidney eluate were examined for antibodies to both native and recombinant NC1 domains of collagen IV. Peptide induced EAG with proteinuria and decreased renal function and glomerular basement membrane IgG deposits. Sera from these rats were examined by ELISA, which revealed reactivity not only to immunizing peptide but also to human and rat alpha3(IV)NC1 and to human alpha4(IV)NC1 domains. Kidney eluate that was depleted of alpha3(IV)NC1 antibodies still reacted to alpha4(IV)NC1, and alpha3(IV)NC1 column-bound antibody reacted with alpha3(IV)NC1. There was minimal reactivity to other collagen chains. Eluate that was adsorbed to NC1 hexamer from rat glomerular basement membrane lost all reactivity to glomerular constituents, and the eluted antibodies reacted to alpha3(IV)NC1 and alpha4(IV)NC1 domains. These studies show that a T cell epitope of alpha3(IV)NC1 induces EAG, intramolecular epitope spreading along alpha3(IV)NC1, and intermolecular epitope spreading to alpha4(IV)NC1 domain with minimal or no reactivity to other collagen chains or glomerular constituents. This is the first demonstration in EAG of intermolecular epitope spreading and identification of the spread epitopes.