Collagen IV of basement membranes: IV. Adaptive mechanism of collagen IV scaffold assembly in Drosophila.

Collagen IV is an essential structural protein in all metazoans. It provides a scaffold for the assembly of basement membranes, a specialized form of extracellular matrix, which anchors and signals cells and provides microscale tensile strength. Defective scaffolds cause basement membrane destabilization and tissue dysfunction. Scaffolds are composed of α-chains that coassemble into triple-helical protomers of distinct chain compositions, which in turn oligomerize into supramolecular scaffolds. Chloride ions mediate the oligomerization via NC1 trimeric domains, forming an NC1 hexamer at the protomer-protomer interface. The chloride concentration-"chloride pressure"-on the outside of cells is a primordial innovation that drives the assembly and dynamic stabilization of collagen IV scaffolds. However, a Cl-independent mechanism is operative in Ctenophora, Ecdysozoa, and Rotifera, which suggests evolutionary adaptations to environmental or tissue conditions. An understanding of these exceptions, such as the example of Drosophila, could shed light on the fundamentals of how NC1 trimers direct the oligomerization of protomers into scaffolds. Here, we investigated the NC1 assembly of Drosophila. We solved the crystal structure of the NC1 hexamer, determined the chain composition of protomers, and found that Drosophila adapted an evolutionarily unique mechanism of scaffold assembly that requires divalent cations. By studying the Drosophila case we highlighted the mechanistic role of chloride pressure for maintaining functionality of the NC1 domain in humans. Moreover, we discovered that the NC1 trimers encode information for homing protomers to distant tissue locations, providing clues for the development of protein replacement therapy for collagen IV genetic diseases.

Collagen IV of basement membranes: II. Emergence of collagen IVα345 enabled the assembly of a compact GBM as an ultrafilter in mammalian kidneys.

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.

Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

Collagen IV Exploits a Cl- Step Gradient for Scaffold Assembly.

Collagen molecules are crucial extracellular players in animal tissue development and in functions ranging from ultrafiltration to organism locomotion. Among the 28 types of collagen found in human, type IV collagen stands out as a primordial type found in all species of the animal kingdom. Collagen IV forms smart scaffolds for basement membranes, sheet-like acellular structures that isolate, coordinate, and direct cells during morphogenesis. Collagen IV is also involved in multiple functions in developed tissues. As part of the basement membrane, collagen IV scaffolds provide mechanical strength, spatially tether extracellular macromolecules and directly signal to cells via receptor binding sites. Proper assembly and structure of the scaffolds are critical for development and function of multiple types of basement membranes. Within last 5 years it was established that Cl- concentration is a key factor for initiating collagen IV scaffold assembly. The biological role of Cl- in multiple physiological processes and detailed mechanisms for its signaling and structural impacts are well established. Cl- gradients are generated across the plasma and intracellular organelle membranes. As collagen IV molecules are secreted outside the cell, they experience a switch from low to high Cl- concentration. This transition works as a trigger for collagen IV scaffold assembly. Within the scaffold, collagen IV remains to be a Cl- sensor as its structural integrity continues to depend on Cl- concentration. Here, we review recent findings and set future directions for studies on the role of Cl- in type IV collagen assembly, function, and disease.

A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.

Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimeric NC1 domains leading to NC1 hexamer formation. Here, we investigated the mechanisms, kinetics, and functionality of the chloride ion-mediated protomer assembly by using a single-chain technology to produce a stable NC1 trimer comprising α1, α2, and α1 NC1 monomers. We observed that in the presence of chloride, the single-chain NC1-trimer self-assembles into a hexamer, for which the crystal structure was determined. We discovered that a chloride ring, comprising 12 ions, induces the assembly of and stabilizes the NC1 hexamer. Furthermore, we found that the chloride ring is evolutionarily conserved across all animals, first appearing in cnidarians. These findings reveal a fundamental role for the chloride ring in the assembly of collagen IV scaffolds of BMs, a critical event enabling tissue evolution and development. Moreover, the single-chain technology is foundational for generating trimeric NC1 domains of other α-chain compositions to investigate the α121, α345, and α565 collagen IV scaffolds and to develop therapies for managing Alport syndrome, Goodpasture's disease, and cancerous tumor growth.

Complexity of type IV collagens: from network assembly to function.

Collagens form complex networks in the extracellular space that provide structural support and signaling cues to cells. Network-forming type IV collagens are the key structural components of basement membranes. In this review, we discuss how the complexity of type IV collagen networks is established, focusing on collagen α chain selection in type IV collagen protomer and network formation; covalent crosslinking in type IV collagen network stabilization; and the differences between solid-state type IV collagen in the extracellular matrix and soluble type IV collagen fragments. We further discuss how complex type IV collagen networks exert their physiological and pathological functions through cell surface integrin and nonintegrin receptors.

Regulation of spermatogenesis by a local functional axis in the testis: role of the basement membrane-derived noncollagenous 1 domain peptide.

Spermatogenesis takes place in the epithelium of the seminiferous tubules of the testes, producing millions of spermatozoa per day in an adult male in rodents and humans. Thus, multiple cellular events that are regulated by an array of signaling molecules and pathways are tightly coordinated to support spermatogenesis. Here, we report findings of a local regulatory axis between the basement membrane (BM), the blood-testis barrier (BTB), and the apical ectoplasmic specialization (apical ES; a testis-specific, actin-rich adherens junction at the Sertoli cell-spermatid interface) to coordinate cellular events across the seminiferous epithelium during the epithelial cycle. In short, a biologically active fragment, noncollagenous 1 (NC1) domain that is derived from collagen chains in the BM, was found to modulate cell junction dynamics at the BTB and apical ES. NC1 domain from the collagen α3(IV) chain was cloned into a mammalian expression vector, pCI-neo, with and without a collagen signal peptide. We also prepared a specific Ab against the purified recombinant NC1 domain peptide. These reagents were used to examine whether overexpression of NC1 domain with high transfection efficacy would perturb spermatogenesis, in particular, spermatid adhesion (i.e., inducing apical ES degeneration) and BTB function (i.e., basal ES and tight junction disruption, making the barrier leaky), in the testis in vivo We report our findings that NC1 domain derived from collagen α3(IV) chain-a major structural component of the BM-was capable of inducing BTB remodeling, making the BTB leaky in studies in vivo Furthermore, NC1 domain peptide was transported across the epithelium via a microtubule-dependent mechanism and is capable of inducing apical ES degeneration, which leads to germ cell exfoliation from the seminiferous epithelium. Of more importance, we show that NC1 domain peptide exerted its regulatory effect by disorganizing actin microfilaments and microtubules in Sertoli cells so that they failed to support cell adhesion and transport of germ cells and organelles (e.g., residual bodies, phagosomes) across the seminiferous epithelium. This local regulatory axis between the BM, BTB, and the apical ES thus coordinates cellular events that take place across the seminiferous epithelium during the epithelial cycle of spermatogenesis.-Chen, H., Mruk, D. D., Lee, W. M., Cheng, C. Y. Regulation of spermatogenesis by a local functional axis in the testis: role of the basement membrane-derived noncollagenous 1 domain peptide.

Antibodies to α5 chain of collagen IV are pathogenic in Goodpasture's disease.

Autoantibody against glomerular basement membrane (GBM) plays a direct role in the initiation and development of Goodpasture's (GP) disease. The principal autoantigen is the non-collagenous domain 1 (NC1) of α3 chain of collagen IV, with two immunodominant epitopes, EA-α3 and EB-α3. We recently demonstrated that antibodies targeting α5NC1 are bound to kidneys in GP patients, suggesting their pathogenic relevance. In the present study, we sought to assess the pathogenicity of the α5 autoantibody with clinical and animal studies. Herein, we present a special case of GP disease with circulating autoantibody reactive exclusively to the α5NC1 domain. This autoantibody reacted with conformational epitopes within GBM collagen IV hexamer and produced a linear IgG staining on frozen sections of human kidney. The antibody binds to the two regions within α5NC1 domain, EA and EB, and inhibition ELISA indicates that they are targeted by distinct sub-populations of autoantibodies. Sequence analysis highlights five residues that determine specificity of antibody targeting EA and EB epitopes of α5NC1 over homologous regions in α3NC1. Furthermore, immunization with recombinant α5NC1 domain induced crescentic glomerulonephritis and alveolar hemorrhage in Wistar-Kyoto rats. Thus, patient data and animal studies together reveal the pathogenicity of α5 antibodies. Given previously documented cases of GP disease with antibodies selectively targeting α3NC1 domain, our data presents a conundrum of why α3-specific antibodies developing in majority of GP patients, with α5-specific antibodies emerged in isolated cases, the answer for which is critical for understanding of etiology and progression of the GP disease.

Collagen at the maternal-fetal interface in human pregnancy.

The survival and development of a semi-allogenic fetus during pregnancy require special immune tolerance microenvironment at the maternal fetal interface. During the establishment of a successful pregnancy, the endometrium undergoes a series of changes, and the extracellular matrix (ECM) breaks down and remodels. Collagen is one of the most abundant ECM. Emerging evidence has shown that collagen and its fragment are expressed at the maternal fetal interface. The regulation of expression of collagen is quite complex, and this process involves a multitude of factors. Collagen exerts a critical role during the successful pregnancy. In addition, the abnormal expressions of collagen and its fragments are associated with certain pathological states associated with pregnancy, including recurrent miscarriage, diabetes mellitus with pregnancy, preeclampsia and so on. In this review, the expression and potential roles of collagen under conditions of physiological and pathological pregnancy are systematically discussed.

Type XIX collagen: a promising biomarker from the basement membranes.

Among collagen members in the collagen superfamily, type XIX collagen has raised increasing interest in relation to its structural and biological roles. Type XIX collagen is a Fibril-Associated Collagen with Interrupted Triple helices member, one main subclass of collagens in this superfamily. This collagen contains a triple helix composed of three polypeptide segments aligned in parallel and it is associated with the basement membrane zone in different tissues. The molecular structure of type XIX collagen consists of five collagenous domains, COL1 to COL5, interrupted by six non-collagenous domains, NC1 to NC6. The most relevant domain by which this collagen exerts its biological roles is NC1 domain that can be cleavage enzymatically to release matricryptins, exerting anti-tumor and anti-angiogenic effect in murine and human models of cancer. Under physiological conditions, type XIX collagen expression decreases after birth in different tissues although it is necessary to keep its basal levels, mainly in skeletal muscle and hippocampal and telencephalic interneurons in brain. Notwithstanding, in amyotrophic lateral sclerosis, altered transcript expression levels show a novel biological effect of this collagen beyond its structural role in basement membranes and its anti-tumor and anti-angiogenic properties. Type XIX collagen can exert a compensatory effect to ameliorate the disease progression under neurodegenerative conditions specific to amyotrophic lateral sclerosis in transgenic SOD1G93A mice and amyotrophic lateral sclerosis patients. This novel biological role highlights its nature as prognostic biomarker of disease progression in and as promising therapeutic target, paving the way to a more precise prognosis of amyotrophic lateral sclerosis.

Basement membrane collagen IV: Isolation of functional domains.

Collagen IV is a major constituent of basement membranes, specialized form of extracellular matrix that provides a mechanical support for tissues, serves as a polyvalent ligand for cell adhesion receptors and as a scaffold for other proteins, and plays a key role in tissue genesis, differentiation, homeostasis, and remodeling. Collagen IV underlies the pathogenesis of several human disorders including Goodpasture's disease, Alport's syndrome, diabetic nephropathy, angiopathy, and porencephaly. While the isolation of the collagen IV molecules from tissues is an ultimate prerequisite for structural and functional studies, it has been always hampered by the protein insolubility due to extensive intermolecular crosslinking and noncovalent associations with other components of basement membranes. In this chapter, we present methods for the isolation of collagen IV fragments from basement membranes or from extracellular matrix deposited by cultured cells, and the recombinant expression alternative. These methods are useful to address the fundamental questions on the role of collagen IV in tissue genesis under the normal and pathological conditions.

Type XIX collagen: A new partner in the interactions between tumor cells and their microenvironment.

Type XIX collagen is a minor collagen that is associated with the basement membrane zone that belongs to the FACIT family (Fibril-Associated Collagens with Interrupted Triple helices). The FACIT family is composed of type IX, XII, XIV, XVI, XX, XXI, XXII and XIX collagens, which share many highly conserved structural motifs: a short NC1 domain, a thrombospondin-like N-terminal domain (TSPN), and numerous cysteine residues. The main role of FACITs is to ensure the integrity and stability of the extracellular matrix and its fibrillar collagen network by regulating the formation and size of the collagen fibrils. Type XIX collagen was discovered in a human rhabdomyosarcoma cell line. The collagen α1(XIX) chain is composed of 5 triple-helical domains (COL) interrupted by 6 non-triple-helical (NC) domains with a short, C-terminal, 19 amino acid non-collagenous domain (NC1). This collagen is involved in the differentiation of muscle cells, central nervous system development, and formation of the esophagus. Type XIX collagen is associated with the basement membrane zone, like type XVIII and XV collagens. Its short NC1(XIX) C-terminal domain inhibits the migration and invasion of melanoma cells. It also exerts a strong anti-angiogenic effect by inhibiting MMP-14 and VEGF expression. NC1(XIX) binding to αvß3 integrin decreases the phosphorylation of proteins involved in the FAK (Focal Adhesion Kinase)/PI3K (PhosphoInositide 3-Kinase)/Akt (protein kinase B)/mTOR (Mammalian Target Of Rapamycin) pathway. On the other hand, NC1(XIX) induces an increase in GSK3ß activity by decreasing its level of phosphorylation. The inhibition of this pathway could explain the anti-tumor properties of the NC1(XIX) domain.

The anti-tumor NC1 domain of collagen XIX inhibits the FAK/ PI3K/Akt/mTOR signaling pathway through αvß3 integrin interaction.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvß3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3ß activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.