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Arginine 383 is a crucial residue in ABCG2 biogenesis.

ABSTRACT: ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by functional assay, while the R383A mutant exhibited detectable but significantly decreased levels compared to wild-type, partial retention in the ER and altered glycosylation. Efflux of the ABCG2-substrates mitoxantrone and pheophorbide a was observed. Our experiments suggested rapid degradation of the R383A mutant by the proteasome via a kifunensine-insensitive pathway. Interestingly, overnight treatment of the R383A mutant with mitoxantrone assisted in protein maturation as evidenced by a shift to the N-glycosylated form. The R383A mutant when expressed in insect cells, though detected on the surface, had no measurable ATPase activity. In addition, substitution with the positively charged lysine resulted in significantly decreased protein expression levels in HEK cells, while retaining function. In conclusion, arginine 383 is a crucial residue for ABCG2 biogenesis, where even the most conservative mutations have a large impact.

SUBMITTER: Polgar O

PROVIDER: S-EPMC4163909 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Arginine 383 is a crucial residue in ABCG2 biogenesis.

Polgar Orsolya O Ediriwickrema Lilangi S LS Robey Robert W RW Sharma Ajay A Hegde Ramanujan S RS Li Yongfu Y Xia Di D Ward Yvona Y Dean Michael M Ozvegy-Laczka Csilla C Sarkadi Balazs B Bates Susan E SE

Biochimica et biophysica acta 20090503 7

ABCG2 is an ATP-binding cassette half-transporter initially identified in multidrug-resistant cancer cell lines and recently suggested to play an important role in pharmacokinetics. Here we report studies of a conserved arginine predicted to localize near the cytoplasmic side of TM1. First, we determined the effect of losing charge and bulk at this position via substitutions with glycine and alanine. The R383G mutant when transfected into HEK cells was not detectable on immunoblot or by function ...[more]

PMID: 19406100

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Project description:Heme nitrite reductases reduce NO2- by 1e-/2H+ to NO or by 6e-/8H+ to NH4+ which are key steps in the global nitrogen cycle. Second-sphere residues, such as arginine (with a guanidine head group), are proposed to play a key role in the reaction by assisting substrate binding and hydrogen bonding and by providing protons to the active site for the reaction. The reactivity of an iron porphyrin with a NO2- covalently attached to a guanidinium arm in its 2nd sphere was investigated to understand the role of arginine residues in the 2nd sphere of heme nitrite reductases. The presence of the guanidinium residue allows the synthetic ferrous porphyrin to reduce NO2- and produce a ferrous nitrosyl species ({FeNO}7), where the required protons are provided by the guanidinium group in the 2nd sphere. However, in the presence of additional proton sources in solution, the reaction of ferrous porphyrin with NO2- results in the formation of ferric porphyrin and the release of NO. Spectroscopic and kinetic data indicated that re-protonation of the guanidine group in the 2nd sphere by an external proton source causes NO to dissociate from a ferric nitrosyl species ({FeNO}6) at rates similar to those observed for enzymatic sites. This re-protonation of the guanidine group mimics the proton recharge mechanism in the active site of NiR. DFT calculations indicated that the lability of the Fe-NO bond in the {FeNO}6 species is derived from the greater binding affinity of anions (e.g. NO2-) to the ferric center relative to neutral NO due to hydrogen bonding and electrostatic interaction of these bound anions with the protonated guanidium group in the 2nd sphere. The reduced {FeNO}7 species, once formed, is not affected significantly by the re-protonation of the guanidine residue. These results provide direct insight into the role of the 2nd sphere arginine residue present in the active sites of heme-based NiRs in determining the fate of NO2- reduction. Specifically, the findings using the synthetic model suggest that rapid re-protonation of these arginine residues may trigger the dissociation of NO from the {FeNO}6, which may also be the case in the protein active site.

Dataset Information

Arginine 383 is a crucial residue in ABCG2 biogenesis.

Publications

Arginine 383 is a crucial residue in ABCG2 biogenesis.

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