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Cellular import and export of free and protein-bound cobalamin (vitamin B12)

Rasmus Beedholm-Ebsen  

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Accepted by: Faculty of Health Sciences Aarhus University
Defended on: February 29, 2008
Official opponents: Roger Alberto, professor, PhD , Uffe Holmskov, professor, dr. med. , Thomas Ledet, prof. dr. med.
Tutors: Søren Kragh Moestrup, Professor, dr.med. , Ebba Nexø, Professor, dr.med.

Published in the PhD Database: October 15, 2009

English abstract
The PhD dissertation ¿Investigation of the cellular import and export of free and protein-bound cobalamin (vitamin B12)¿ is based on experimental work performed at the Department of Medical Biochemistry, University of Aarhus. The work was conducted to gain insight into the cellular endocytosis and export of vitamin B12. The main results of this PhD study are presented in three separate manuscripts.

Vitamin B12 (cobalamin) is an organic molecule which functions as an essential coenzyme for two metabolic reactions in mammalians. Vitamin B12 deficiency is a common condition causing pernicious anemia, which is characterized by megaloblastic anemia and neurological symptoms. Cellular vitamin B12 uptake is a complex process involving different binding proteins (intrinsic factor and transcobalamin) and at least three different receptors.

In the first study the receptor-mediated uptake of vitamin B12 has been studied in cultured cancer cells. These studies included the use of fluorescently labelled vitamin B12 conjugates, which make it possible for the first time to visualize the vitamin by confocal immunofluorescence microscopy. The uptake studies revealed that the endocytosis of transcobalamin bound to fluorescent vitamin B12 conjugate correlated with the cell growth in fast-dividing cancer cells and the uptake of free vitamin B12 was negliable at low as well as high cell division activity.

The second study describes the vitamin B12 export in cultured cells using 57Co-labeled vitamin. In contrast to what has been previously reported, the data evidenced that the vitamin is exported to the cellular environment as a free molecule and the binding to a carrier protein occurs subsequently to export.

In the third study a transporter for cellular export of vitamin B12 was searched for by a ¿bioinformatics¿ approach, whereby a spectrum of candidate membrane proteins were selected by a subset of functional, structural and cellular location criteria. The role of candidate proteins was investigated by selective RNA silencing with small interfering RNA (siRNA) probes specific for the encoding RNAs. Inhibition of vitamin B12 export after inhibitionof the expression of one the candidate proteins by siRNA was followed up by analysis of the vitamin B12 status of mice with targeted disrupted of the encoding gene. These data showed low levels of extracellular vitamin B12 thus providing further evidence of a role of this particular transporter in cellular export of vitamin B12.

In conclusion, this PhD study provides new information on the cellular vitamin B12 import and in particular export by revealed a novel transport mechanism of non-protein bound vitamin B12 and the identification of a transporter protein for this process.

Danish abstract
Det eksperimentelle arbejde, der danner grundlag for denne ph.d.-afhandling blev udført på Institut for Medicinsk Biokemi, Aarhus Universitet. Formålet var at undersøge hvordan vitamin B12 bliver optaget i celler og væv og hvorledes det efterfølgende bliver eksporteret og hvilken transport-mekanisme der ligger til grund for denne eksport. Resultaterne af disse undersøgelser præsenteres i tre separate manuskripter.

I det første studie undersøges den receptor-medierede optagelse af vitamin B12 i cancerceller i kultur. Disse studier blev til blandt andet ved brug af fluorescensmærket vitamin B12 konjugater, der for første gang muliggør visualiseringen af vitaminet vha. konfokal immunofluorescence mikroskopi. Studierne viser at endocytose af transcobalamin bundet fluorescerende vitamin B12 korrelerer med celledelingen i cancerceller.

Det andet studie beskriver i hvilken form vitamin B12 eksporteres ud af celler ved brug af 57Co-mærket vitamin. Til forskel for hvad der hidtil har været antaget, eksporteres vitaminet til ekstracellulærmiljøet som et frit molekyle og bindingen til transportproteiner sker først efter den cellulære eksport.

I det tredje studie identificeres transporteren, som er ansvarlig for vitamin B12 eksporten ved brug af bioinformatik og small interfering RNA (siRNA) prober. Dette fund konfirmeres og udbygges ved undersøgelse af vitamin B12 status i gene ¿knock out¿-mus, hvor den specifikke transporter ikke udtrykkes.