Cellular uptake of vitamin B12: Role and fate of TCblR/CD320, the transcobalamin receptor

https://doi.org/10.1016/j.yexcr.2020.112256Get rights and content

Abstract

Cellular uptake of vitamin B12 (cobalamin, Cbl) is mediated by a cell surface receptor (TCblR/CD320) that binds transcobalamin (TC) saturated with Cbl. TC is secreted by the vascular endothelium, has a relatively short half-life, binds Cbl with high affinity and presents the vitamin to the receptor for cellular uptake. Here we show binding and internalization of the TC-Cbl complex along with its’ receptor (TCblR) in several human cell lines. The expression of TCblR is linked to the cell cycle with highest expression in actively proliferating cells. Upon binding TC-Cbl, the receptors appear to segregate on the plasma membrane and are internalized over the course of 30–60 min. Subsequently, the receptors appear to be destroyed along with the TC, which results in the release of free Cbl in the lysosome. The appearance of TCblR on the cell surface is limited to newly synthesized protein without contribution from recycling of the receptor. Therefore, Cbl uptake into cells is fully dependent on the expression of newly synthesized TCblR that is up-regulated in actively proliferating cells. The cell cycle-associated up-regulation of TCblR in cancers provides a route for targeted drug delivery.

Introduction

Vitamin B12 (cobalamin; Cbl) in the form of methylCbl or 5′-deoxyadenosylCbl is needed as a cofactor for two enzymes, namely methionine synthase and methylmalonyl CoA mutase, respectively, in mammalian cells [1]. The cellular uptake of Cbl requires transcobalamin (TC), a plasma protein secreted by most cells but the vascular endothelium secretes copious amounts of this protein and is the likely source for most of the circulating TC [2]. TC has a relatively short plasma half-life of about 60–90 min binds Cbl with high affinity and carries about a third of the plasma Cbl. This protein is only partially saturated with Cbl (~20–30%) and as holoTC is the source of Cbl for cellular uptake [[2], [3], [4]]. A plasma membrane receptor protein (TCblR) encoded by the CD320 gene that is ubiquitously expressed in all cells is needed for cellular uptake of TC-Cbl. This receptor has two LDL-R type A domains that together bind TC-Cbl with high affinity [5]. Expression of the receptor is cell - cycle associated with highest expression in actively proliferating cells and down-regulation of receptor expression in differentiated resting cells [3, [6], [7], [8]. Many cancer cell lines show higher sustained expression of TCblR and this has the potential for increasing targeting of drugs and toxins to tumors [[9], [10], [11]]. The TCblR-TC-Cbl complex is internalized by receptor-mediated endocytosis involving clathrin-coated pits [[9], [10], [11]]. The binding of TC-Cbl to TCblR requires Ca++ and the process of internalization involves active transport [13]. Following internalization, TC is degraded in the lysosomes to release free Cbl and genetic defects of this process result in the cbl F phenotype [[14], [15], [16]] ; while the fate of endocytosed TCblR has remained unclear [17]. Earlier studies had suggested that the receptor may be recycled in a manner similar to the transferrin receptor [7, 12]. In this report, we provide the profile of receptor expression in several human cell lines and show that following ligand-mediated internalization, TCblR, along with TC is degraded in lysosomes without recycling to the cell surface and discuss its physiologic significance.

Section snippets

Cell culture

All cell lines used in this study were obtained from the American Type Culture Collection (ATCC, Bethesda, MD) except where indicated otherwise. Acute promyeloblastic leukemia HL60 cells (CCL 240), human epidermoid lung carcinoma H292 (CRL 1848), human erythroid leukemia K562 (CCL-243), human colon carcinoma SW48 (CCL-231), human epidermoid carcinoma KB (CCL-17), human breast carcinoma MCF7 (HTB-22), normal human skin fibroblasts RFP3 (primary culture produced from a biopsy) and a human

Results

The expression of TCblR in cells replicating in culture was closely associated with the proliferative phase of the cell cycle with highest expression between 24 and 48 h in culture during the log phase of growth, depending on the initial seeding density (Fig. 1A–D). Subsequently, receptor expression decreased as the cell density increased with lowest expression in confluent cells as shown for HL60 and K562 cells (Fig. 1A and B). Receptor expression in numerous other cell lines was also similar (

Discussion

TCblR, a cell membrane receptor, mediates the uptake of vitamin B12 in mammalian cells with high affinity and specificity for TC-Cbl [5]. TCblR binding of TC saturated with Cbl triggers TCblR segregation to clathrin-coated pits and subsequent endocytosis [12]. It is presumed that following internalization, TC is directed to lysosomes for degradation, free Cbl is released and converted into Cbl cofactors [17]. The content of plasma membrane-associated TCblR is highest during early log phase of

Credit author statement

Gregory G. Gick, designed research, analyzed data, wrote the paper, performed the experiments. Kaveri Arora, analyzed data, performed the experiments. Jeffrey M. Sequeira, analyzed data, performed the experiments. Yasumi Nakayama, performed the experiments. Shao-Chiang Lai, performed the experiments. Edward V. Quadros, designed research, analyzed data, wrote the paper, All authors have reviewed the final manuscript and approved it.

Declaration of competing interest

EVQ and JMS are listed as inventors in a patent to target the TCblR receptor in drug delivery and cancer therapy issued to the Research Foundation of SUNY. All other authors declare no conflicts.

Acknowledgements

This work was supported in part by grant # DK064732 (to EVQ) from the NIH, USA.

References (28)

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