Edited by Ruma Banerjee In humans, transport of food-derived cobalamin (vitamin B 12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an ␣-domain and a ␤-domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the ␣-domain. These ␤-domainonly proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2 ؊/؊ zebrafish. Vitamin B 12 , also referred to as cobalamin, is a micronutrient that acts as a cofactor for two enzymes: methionine synthase, This article contains Figs. S1-S4 and Table S1.

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