Hudson, J. K. | Search bioRxiv

bioRxiv2026

Collagen IV of basement membrane: V. Bromide-mediated sulfilimine bonds interlock the quaternary structure of NC1-hexamer of scaffolds enabling metazoan evolution.

Collagen-IV (Col-IV) scaffolds, a primordial basement membrane component, enabled animal multicellularity, evolution and adaptation. These scaffolds provide tensile strength and tether macromolecules, forming supramolecular complexes that interact with cell-surface receptors and influence cell-behavior. Triple-helical Col-IV protomers, composed of three -chains, with a trimeric globular NC1-domain at the C-terminus, oligomerize forming a NC1-hexamer structure that connects adjoining protomers of Col-IV121, Col-IV556--121, and Col-IV345 scaffolds. Hexamer formation and stability are driven by the extracellular chloride concentration-"chloride pressure". Hexamer structure is reinforced by six sulfilimine bonds forming covalent crosslinks that weld together trimeric NC1-domains of adjoining protomers. We recently found evidence that sulfilimine bonds, independent of chloride, stabilize the quaternary structure of the Col-IV345 hexamer of the Col-IV345 scaffold. Here, we sought to determine whether this function also pertains to the Col-IV121 scaffold that occurs ubiquitously across the animal kingdom, and whether bromine, a cofactor of peroxidasin in bond formation, are evolutionary conserved. We found that sulfilimine bonds stabilized the quaternary structure of the Col-IV121 hexamer of bovine, mouse and a basal cnidarian, Nematostella vectensis, and that the mechanism of bond formation mediated by peroxidasin and bromide is evolutionary conserved. Analyses of the crystal structure of the NC1-hexamer revealed that sulfilimine bonds covalently fasten a clasp-motif across the trimer-trimer interface, interlocking the domain-swapping region of neighboring subunits, which reinforces the hexamer quaternary structure imposed by chloride conformational constraints. Collectively, our findings reveal that the sulfilimine-bond reinforcement is a critical event in Col-IV scaffold assembly enabling multicellularity, evolution and adaptation of metazoans, beginning with ancient cnidarians.

molecular biology↗

bioRxiv2023

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

Collagen IV is a major component of basement membranes, a specialized form of extracellular matrix that enabled the assembly of multicellular epithelial tissues. In mammals, collagen IV assembles from a family of six -chains (1 to 6), forming three supramolecular scaffolds: Col-IV121, Col-IV345 and Col-IV121-556. The -chains are encoded by six genes (COL4A1-6) that occur in pairs in a head-to-head arrangement. In Alport syndrome, variants in COL4A3, 4 or 5 genes, encoding Col-IV345 scaffold in glomerular basement membrane (GBM), the kidney ultrafilter, cause progressive renal failure in millions of people worldwide. How variants cause dysfunction remains obscure. Here, we gained insights into Col-IV345 function by determining its evolutionary lineage, as revealed from phylogenetic analyses and tissue expression of COL4 gene-pairs. We found that the COL4A[<]1|2[>] gene-pair emerged in basal Ctenophores and Cnidaria phyla and is highly conserved across metazoans. The COL4A[<]1|2[>] duplicated and arose as the progenitor to the COL4A[<]3|4[>] gene-pair in cyclostomes, coinciding with emergence of kidney GBM, and expressed and conserved in jawed-vertebrates, except for amphibians, and a second duplication as the progenitor to the COL4A[<]5|6[>] gene-pair and conserved in jawed-vertebrates. These findings revealed that Col-IV121 is the progenitor scaffold, expressed ubiquitously in metazoan basement membranes, and which evolved into vertebrate Col-IV345 and expressed in GBM. The Col-IV345 scaffold, in comparison, has an increased number of cysteine residues, varying in number with osmolarity of the environment. Cysteines mediate disulfide crosslinks between protomers, an adaptation enabling a compact GBM that withstands the high hydrostatic pressure associated with glomerular ultrafiltration.

genomics↗