An Ancient MHC-Linked Gene Encodes a Nonrearranging Shark Antibody, UrIg, Convergent with IgG.

Gnathostome adaptive immunity is defined by the Ag receptors, Igs and TCRs, and the MHC. Cartilaginous fish are the oldest vertebrates with these adaptive hallmarks. We and others have unearthed nonrearranging Ag receptor-like genes in several vertebrates, some of which are encoded in the MHC or in MHC paralogous regions. One of these genes, named UrIg, was detected in the class III region of the shark MHC that encodes a protein with typical V and C domains such as those found in conventional Igs and TCRs. As no transmembrane region was detected in gene models or cDNAs, the protein does not appear to act as a receptor. Unlike some other shark Ig genes, the UrIg V region shows no evidence of RAG-mediated rearrangement, and thus it is likely related to other V genes that predated the invasion of the RAG transposon. The UrIg gene is present in all elasmobranchs and evolves conservatively, unlike Igs and TCRs. Also, unlike Ig/TCR, the gene is not expressed in secondary lymphoid tissues, but mainly in the liver. Recombinant forms of the molecule form disulfide-linked homodimers, which is the form also detected in many shark tissues by Western blotting. mAbs specific for UrIg identify the protein in the extracellular matrix of several shark tissues by immunohistochemistry. We propose that UrIg is related to the V gene invaded by the RAG transposon, consistent with the speculation of emergence of Ig/TCR within the MHC or proto-MHC.

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 IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.

Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

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.

Post-translational modification of type IV collagen with 3-hydroxyproline affects its interactions with glycoprotein VI and nidogens 1 and 2.

Type IV collagen is a major component of the basement membrane and interacts with numerous other basement membrane proteins. Many of these interactions are poorly characterized. Type IV collagen is abundantly post-translationally modified with 3-hydroxyproline (3-Hyp), but 3-Hyp's biochemical role in type IV collagen's interactions with other proteins is not well established. In this work, we present binding data consistent with a major role of 3-Hyp in interactions of collagen IV with glycoprotein VI and nidogens 1 and 2. The increased binding interaction between type IV collagen without 3-Hyp and glycoprotein VI has been the subject of some controversy, which we sought to explore, whereas the lack of binding of nidogens to type IV collagen without 3-Hyp is novel. Using tandem MS, we show that the putative glycoprotein VI-binding site is 3-Hyp-modified in WT PFHR-9 type IV collagen, but not in PFHR-9 cells in which prolyl-3-hydroxylase 2 (P3H2) has been knocked out (KO). Moreover, we observed altered 3-Hyp occupancy across many other sites. Using amino acid analysis of type IV collagen from the WT and P3H2 KO cell lines, we confirm that P3H2 is the major, but not the only 3-Hyp-modifying enzyme of type IV collagen. These findings underscore the importance of post-translational modifications of type IV collagen for interactions with other proteins.

Collagen IV of basement membranes: I. Origin and diversification of COL4 genes enabling animal evolution.

Collagen IV is a primordial component of basement membranes, a specialized form of extracellular matrix that enabled multi-cellular epithelial tissues. In mammals, collagen IV assembles from a family of six α-chains (α1 to α6), encoded by six genes (COL4A1 to COL4A6), into three distinct scaffolds: the α121, the α345 and a mixed scaffold containing both α121 and α565. The six mammalian COL4A genes occur in pairs that occur in a head-to-head arrangement on three distinct chromosomes. In Alport syndrome, variants in the COL4A3, 4 or 5 genes cause either loss or defective assembly of the collagen IV α345 scaffold which results in a dysfunctional glomerular basement membrane, proteinuria and progression to renal failure in millions of people worldwide. Here, we determine the evolutionary emergence and diversification of the COL4A genes using comparative genomics and biochemical analyses. Using syntenic relationships to genes closely linked to the COL4A genes, we determine that the COL4A3 and COL4A4 gene pair appeared in cyclostomes (hagfish and lampreys) while the COL4A5 and COL4A6 gene pair emerged in gnathostomes, jawed vertebrates. The more basal chordate species, lancelets and tunicates, do not have discrete kidneys and have a single COL4A gene pair, though often with single isolated COL4 genes similar to those found in C elegans . Remarkably, while the six COL4A genes are conserved in vertebrates, amphibians have lost the COL4A3 and COL4A4 genes. Our findings of the evolutionary emergence of these genes, together with the amphibian double-knockout, opens an experimental window to gain insights into functionality of the Col IV α345 scaffold.