Collagen IV of basement membranes: I. Origin and diversification of COL4 genes enabling metazoan multicellularity, evolution, and adaptation

Collagen IV (Col-IV) is a major component of basement membranes, a specialized form of extracellular matrix that enabled the assembly of multicellular epithelial tissues. In mammals, Col-IV assembles from a family of six α-chains (α1-α6), forming three supramolecular scaffolds: Col-IV^α121, Col-IV^α345, and Col-IV^α121-α556. The α-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-IV^α345 scaffold in glomerular basement membrane (GBM), the kidney ultrafilter, cause progressive renal failure in millions of people worldwide. The molecular mechanisms of how variants cause dysfunction remain obscure. Here, we gained insights into Col-IV^α345 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-IV^α121 is the progenitor scaffold, expressed ubiquitously in metazoan basement membranes, and which evolved into vertebrate Col-IV^α345 and expressed in GBM. The Col-IV^α345 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.