The Cobalamin-Binding Protein in Zebrafish Is an Intermediate between the Three Cobalamin-Binding Proteins in Human

Abstract

In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

“…The presence of just one soluble cobalamin-binding protein in trout is in accord with our previous study on zebrafish (7) and the database prediction for salmon. Trout HIT shares sequence identity with the zebrafish HIT protein (58.2%) and even more with its close relative, the salmon (91.3%).…”

“…Blood samples were collected from the caudal tail vein into lithium heparin tubes, and plasma was removed after centrifugation at 2000 ϫ g for 10 min and stored at Ϫ20°C until analysis. Protein extractions of roe and tissue samples (ϳ1 ml of buffer per 1 g of tissue) were carried out by homogenization on ice as described previously (7).…”

“…Protein extracts of roe, roe fluid, human HC, IF, and TC were investigated. The study was carried out as described previously (7).…”

New B vitamin recommendations in fish when fed plant-based diets

“…Zebrafish lack intrinsic factor, the main vitamin B 12 binding protein needed for absorption in humans. Greibe et al (2012) found that zebrafish and rainbow trout have only one vitamin B 12 binding protein, a structurally intermediate between the three vitamin B 12 binding proteins identified in human.…”

Structural Basis for Universal Corrinoid Recognition by the Cobalamin Transport Protein Haptocorrin

“…Interestingly, in species lacking HC, including mice and lower vertebrates, a TC-like protein has been shown to be less specific for Cbl and to recognize Cbi and other corrinoids, similar to human HC (32)(33)(34). According to the sequence alignment (Figs.…”