ABCG2 Transports and Transfers Heme to Albumin through Its Large Extracellular Loop*

Elodie Desuzinges-Mandon,Ophélie Arnaud,Lorena Martinez,Frédéric Huché,Attilio Di Pietro,Pierre Falson

doi:10.1074/jbc.m110.139170

Abstract

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K(d) values are in the range 0.5-3.5 μm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K(d) of about 3 μm.

Highlights

As HIV-1 nucleoside reverse transcriptase inhibitors [5]
zinc mesoporphyrin (ZnMP) Transport by ABCG2 in K562 Cells and Modulation by the 5D3 mAb of in Vitro ABCG2 Binding to Hemin-Agarose— Because ABCG2 was shown to transport protoporhyrin IX (PPIX) out of stem cells submitted to hypoxia [26], we hypothesized that the transporter could be able to transport heme or hemin, which correspond to ferrous and ferric PPIX
By revealing an antagonist effect between 5D3 binding onto ECL3 and ABCG2 binding onto hemin-agarose, this experiment suggested a hemebinding capacity of ECL3

Summary

NdeI BamHI

Contains a conformational epitope recognized by the 5D3 monoclonal antibody [24], the interaction of which is prevented upon reduction of the intramolecular disulfide bridge Cys603Cys603 [35], making 5D3 a powerful conformation-sensing tool. We hypothesized that ABCG2 should transport hemin out of the cell, which could be critical in the early stages of hematopoiesis when it is overexpressed, and hemin plays a role in differentiation. We found that ABCG2 transports zinc mesoporphyrin (ZnMP), a validated fluorescent heme analog [36, 37]. We hypothesized that hemin, once exported by ABCG2, should be transferred to an extracellular protein through interaction with an accessible domain of ABCG2 such as ECL3, which could transitorily interact with hemin after membrane translocation. ECL3 displayed a high affinity for some, but not all, porphyrins, especially for heme and hemin, whereas it does not bind any of the known nonporphyrin substrates and inhibitors of ABCG2. We found that hemin, when bound onto ECL3, can be rapidly transferred to human serum albumin (HSA)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION