Abstract
Tracking cellular 57Co-labelled cobalamin (57Co-Cbl) uptake is a well-established method for studying Cbl homeostasis. Previous studies established that bovine serum is not generally permissive for cellular Cbl uptake when used as a supplement in cell culture medium, whereas supplementation with human serum promotes cellular Cbl uptake. The underlying reasons for these differences are not fully defined. In the current study we address this question. We extend earlier observations by showing that fetal calf serum inhibits cellular 57Co-Cbl uptake by HT1080 cells (a fibrosarcoma-derived fibroblast cell line). Furthermore, we discovered that a simple heat-treatment protocol (95°C for 10 min) ameliorates this inhibitory activity for HT1080 cell 57Co-Cbl uptake. We provide evidence that the very high level of haptocorrin in bovine serum (as compared to human serum) is responsible for this inhibitory activity. We suggest that bovine haptocorrin competes with cell-derived transcobalamin for Cbl binding, and that cellular Cbl uptake may be minimised in the presence of large amounts of bovine haptocorrin that are present under routine in vitro cell culture conditions. In experiments conducted with AG01518 cells (a neonatal foreskin-derived fibroblast cell line), overall cellular 57Co-Cbl uptake was 86% lower than for HT1080 cells, cellular TC production was below levels detectable by western blotting, and heat treatment of fetal calf serum resulted in only a modest increase in cellular 57Co-Cbl uptake. We recommend a careful assessment of cell culture protocols should be conducted in order to determine the potential benefits that heat-treated bovine serum may provide for in vitro studies of mammalian cell lines.
Highlights
Cobalamin (Cbl), which is commonly referred to as vitamin B12, is required for erythrocyte formation, DNA synthesis, and the maintenance of neurological function [1,2,3,4]
We first examined the cellular uptake of 57Co-Cbl when HT1080 cells were cultured for up to 72 h in growth medium containing either 10% fetal calf serum (FCS) or 10% human serum (HS)
When assessed at the 48 h time point, the amount of 57Co-Cbl detected in cells grown in FCS was 5.5% of the level detected in the cells grown in HS (Fig 1A)
Summary
Cobalamin (Cbl), which is commonly referred to as vitamin B12, is required for erythrocyte formation, DNA synthesis, and the maintenance of neurological function [1,2,3,4]. Several Cbl deficiency states exist in humans, some of which are caused by a loss of function in proteins that transport Cbl either to cells or within subcellular compartments [3, 6, 7]. HC-Cbl (and HC bound corrinoids) are thought to be taken up by the liver asialoglycoprotein receptor [3] Another specialised route for TC-Cbl uptake is via megalin expressed in the kidney [18]. This facilitates TC-Cbl reabsorption, delivering filtered TC-Cbl back to the bloodstream Both megalin and cubilin are expressed in other cell types, including mammary cells, where these receptors may play a specialised role in Cbl homeostasis [19, 20]. HC does not share the same high degree of specificity for Cbl binding as TC, HC has greater affinity for Cbl than TC [3]
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