Aldo-keto Reductase 1B15 (AKR1B15): A mitochondrial human aldo-keto reductase with activity toward steroids and 3-keto-acyl-CoA conjugates

JBC Papers in Press. Published on January 10, 2015 as Manuscript M114.610121

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.610121

Aldo-Keto Reductase 1B15 (AKR1B15): a Mitochondrial Human Aldo-Keto Reductase with Activity

towards Steroids and 3-Keto-acyl-CoA Conjugates*

Susanne Weber¹, Joshua K. Salabei², Gabriele Möller¹, Elisabeth Kremmer³, Aruni Bhatnagar²,

Jerzy Adamski^{1,4,5, §}, and Oleg A. Barski^{2, §}

¹Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Institute of

Experimental Genetics, Genome Analysis Center, 85764 Neuherberg, Germany

²Diabetes and Obesity Center, School of Medicine, University of Louisville, Louisville, KY 40202, USA

³Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Institute of

Molecular Immunology, 81377 Muenchen, Germany

⁴Lehrstuhl für Experimentelle Genetik, Technische Universitaet Muenchen, 85356 Freising-

Weihenstephan, Germany

⁵German Center for Diabetes Research, 85764 Neuherberg, Germany

*Running title: Characterization of human AKR1B15

§ To whom correspondence should be addressed:

Oleg A. Barski, NIGMS, NIH, 45 Center Dr. Rm. 2AS55C, Bethesda, MD 20892, USA. Tel: (301)-496-

1511; Fax: (301)-480-2802; E-mail: oleg.barski@nih.gov (present address)^#

or

Jerzy Adamski, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health,

Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstr. 1, 85764 Neuherberg,

Germany. Tel.: 049-89-3187-3155; Fax: 049-89-3187-3225; E-mail: Adamski@helmholtz-muenchen.de

^#Disclaimer: This article was prepared while Oleg A. Barski was employed at the University of

Louisville. The opinions expressed in this article are the author's own and do not reflect the view of the

National Institutes of Health, the Department of Health and Human Services, or the United States

government.

Keywords: Aldo-keto reductase (AKR), gene expression, enzyme kinetics, mitochondria, oxidation‐

reduction, 3-keto-acyl-CoA, steroids

-1-

Characterization of novel human AKR1B15

Background: Aldo-keto reductases (AKRs) are

enzymes involved in the metabolism of carbonyl

substrates.

which is associated with mitochondria and

expressed mainly in steroid-sensitive tissues.

Results: Two alternatively-spliced protein

isoforms encoded by the human AKR gene

AKR1B15 were identified. The AKR1B15.1

isoform catalyzes reduction of steroids and 3-keto-

INTRODUCTION

The Aldo-Keto Reductase (AKR)²superfamily

comprises 15 families containing over 150

members that are present in all phyla (1, 2). AKRs

are multifunctional enzymes that catalyze the

reduction of biogenic and xenobiotic aldehydes

and ketones, as well as the synthesis and

metabolism of sex hormones. The majority of

AKRs catalyze oxidation-reduction reactions

between carbonyl and alcohol groups, whereas

enzymes of the AKR1D family reduce double

bonds in the bile acid biosynthesis pathway acting

as 5β-reductases (3). Some AKR proteins have

very low or no activity and perform predominantly

non-catalytic functions, e.g., structural (lens rho-

crystallines: AKR1C10a and AKR1C10b) or

regulatory and chaperone-like (voltage-gated

potassium channel β-subunits of AKR6 family:

Kvβ) functions (4, 5).

Prior to identification of AKR1B15, 14 human

AKRs have been described. These proteins are

generally cytosolic and monomeric with molecular

weights ranging between 35-40 kDa. These

enzymes catalyze oxidation-reduction reactions in

a variety of cellular pathways such as glucose

metabolism (AKR1B1) (2), vitamin C biosynthesis

(AKR1A1) (6), steroid and prostaglandin

metabolism (AKR1Bs and AKR1Cs) (7, 8), bile

acid synthesis (AKR1D1) (9), neurotransmitter

metabolism (AKR7) (10), as well as the

detoxification of both endogenous oxidation by-

products, such as advanced glycation end-product

precursors or lipid peroxidation-derived aldehydes

(11, 12), and exogenous toxins, such as aflatoxin

B1 (13) or tobacco-derived carcinogen 4-methyl-

nitrosamino-1-(3-pyridyl)-1-butanone (NNK) (14).

Generally, the enzymes of the AKR superfamily

prefer NADPH over NADH as a reducing cofactor

(2, 15, 16).

acyl-CoA

conjugates

and

localizes

to

mitochondria.

Conclusion: AKR1B15.1 is a mitochondrial

carbonyl reductase.

Significance: AKR1B15.1 is a new enzyme with

unique localization and catalytic features.

ABSTRACT

Aldo-Keto Reductases (AKRs) comprise

a

superfamily of proteins involved in the reduction

and oxidation of biogenic and xenobiotic

carbonyls. In humans, at least 15 AKR

superfamily members have been identified so far.

One of these is a newly identified gene locus,

AKR1B15, which clusters on chromosome 7 with

the other human AKR1B subfamily members, i.e.,

AKR1B1 and AKR1B10. We show that alternative

splicing of the AKR1B15 gene transcript gives rise

to two protein isoforms with different N-termini:

AKR1B15.1 is a 316 amino acid protein with

91 % amino acid identity to AKR1B10;

AKR1B15.2 has a prolonged N-terminus and

consists of 344 amino acid residues. The two gene

products differ in their expression level,

subcellular localization, and activity. In contrast

with other AKR enzymes which are mostly

cytosolic, AKR1B15.1 co-localizes with the

mitochondria. Kinetic studies show that

AKR1B15.1 is predominately a reductive enzyme

that catalyzes the reduction of androgens and

estrogens with high positional selectivity (17β-

hydroxysteroid dehydrogenase activity) as well as

3-keto-acyl-CoAs and exhibits strong cofactor

selectivity towards NADP(H). In accordance with

its substrate spectrum, the enzyme is expressed at

highest levels in steroid-sensitive tissues, namely

placenta, testis, and adipose tissue. Placental and

adipose expression could be reproduced in the

BeWo and SGBS cell lines, respectively. In

contrast, AKR1B15.2 localizes to the cytosol and

displays no enzymatic activity with the substrates

tested. Collectively, these results demonstrate the

existence of a novel catalytically active AKR,

The AKR1 family is the most numerous and has

been further divided into 5 subfamilies (A-E). The

AKR1B subfamily has been intensely studied due

to the potential role of its founding member human

aldose reductase (AKR1B1) in the development of

diabetic complications (17, 18). Under hyper-

glycemic conditions, AKR1B1 converts excess

glucose into sorbitol, leading to osmotic and redox

-2-

Characterization of novel human AKR1B15

imbalances and resulting in tissue injury

associated with diabetes (19–21). Inhibition of

AKR1B1 has been shown to prevent, delay or

reverse tissue injury due to hyperglycemia (19,

22). In this context, a large number of studies and

clinical trials have been devoted to find efficient

inhibitors of aldose reductase to prevent the

development of diabetic complications, however,

these efforts have met limited success due to

problems in trials design, low efficacy, and

nonspecific side effects of inhibitors (17, 23). In

addition to glucose, AKR1B1 catalyzes the

reduction of several substrates of physiological

significance including advanced glycation end-

product precursors, 4-hydroxy-trans-2-nonenal,

and oxidized phospholipids (11, 24, 25) and has

been suggested to play important roles in the

development of atherosclerosis (26), ischemic

preconditioning (27), and restenosis (28).

AKR1B15.2. Furthermore, we characterize the

catalytic activity, tissue distribution, and

subcellular localization of both AKR1B15

isoforms.

EXPERIMENTAL PROCEDURES

Chemicals and materials

–

Primers were

synthesized by Integrated DNA Technology (IDT)

or Metabion. Restriction enzymes and T4 DNA

Ligase were obtained from either New England

Biolabs (NEB) or Promega. Total RNA from

human tissues was purchased from Clontech or

ZenBio (adipose). Cofactors were purchased from

Sigma (NAD⁺; NADP⁺; NADH) and Serva

(NADPH). Unlabeled substrates were obtained

3

from Sigma, whereas H-labeled substrates were

synthesized by American Radiolabeled Chemicals

(cortisone [1,2-³H(N)]), Amersham (17α-estradiol

[6,9-³H(N)]), and Perkin Elmer (3α,17β-

androstanediol [9,11-³H(N)]; androsterone [9,11-

³H(N)]; Δ4-androstenedione [1,2,6,7-³H(N)];

dehydroepiandrosterone [1,2,6,7-³H(N)]; dihydro-

testosterone [1,2,4,5,6,7-³H(N)]; 17β-estradiol

[6,7-³H(N)]; estrone [2,4,6,7-³H(N)]; hydro-

cortisone [1,2,6,7-³H(N)]; progesterone [1,2,6,7-

³H(N)]; testosterone [1,2,6,7-³H(N)]). All other

chemicals and solvents were purchased from

Sigma, Merck or AppliChem.

AKR1B1 is closely related to the small intestine

aldose reductase (AKR1B10) (29, 30). In contrast

to AKR1B1, AKR1B10 is expressed mainly in

small intestine, colon, liver, thymus (29), and

adrenal gland (30). AKR1B10 shares 71 % amino

acid sequence identity with AKR1B1 and exhibits

substrate specificity similar to aldose reductase

with the exception that it has significantly higher

catalytic efficiency with all-trans-retinal (31).

AKR1B10 is strongly overexpressed in lung and

hepatic carcinomas (squamous cell and adeno-

carcinomas) (29) as well as in colorectal and

uterine cancers (32) and has been implicated in

conferring resistance to anticancer drugs (33, 34).

Recently, a novel gene, AKR1B15, with 91 %

identity to AKR1B10 has been predicted in the

genetic cluster encompassing AKR1B1 and

AKR1B10 on human chromosome 7. We

previously reported that this gene encodes a

functional protein (35). However, in contrast to

AKR1B1 and AKR1B10, the enzymatic activity of

this newly identified AKR was low, and the

protein expressed with N-terminal His-tag was

found in the microsomal fraction in both the

mammalian and bacterial expression systems (35).

While orthologs of AKR1B1 are known in rodents

(AKR1B3 in mouse and AKR1B4 in rat), direct

orthology between AKR1B15 and AKR1B10 and

rodent AKR1Bs has not been established so far.

In the present study we show that the AKR1B15

gene gives rise to two alternatively spliced mRNA

products, each coding for a unique protein,

hereafter referred to as AKR1B15.1 and

Cloning of AKR1B15 – The protein encoding

sequences of the AKR1B15 splice variants

AKR1B15.1 (Ensembl entry: AKR1B15-201,

ENST00000423958) and AKR1B15.2 (Ensembl

entry: AKR1B15-001, ENST00000457545) were

amplified by PCR from cDNA libraries of testis

and thymus, respectively, using Phusion High

Fidelity polymerase (NEB) and transcript specific

primers with restriction enzyme sites (Table 1).

The PCR products were cloned into pET28a(+)

(Novagen) via Nde I/Xho I, into pcDNA3.1(+)

(Invitrogen) via Not I/Xho I, into N-myc-pcDNA3

(modified pcDNA3, with N-terminal myc-tag) via

Not I/Xho I, into pcDNA4-myc/HisB (Invitrogen)

via Hind III/Not I, and into pIRES-hrGFP1α

(Stratagene) via Not I/Xho I restriction sites, using

Hind III, Hind III-HF, Nde I, Not I-HF, and Xho I

restriction enzymes and T4 DNA Ligase (NEB).

The complete sequence of the inserts was verified

by Sanger sequencing. The sequences obtained

were identical to the sequences deposited in the

Ensembl database.

-3-

Characterization of novel human AKR1B15

0.05 % Trypsin-EDTA (Gibco). Transfections of

HEK293 or HeLa cells were carried out using

Xtreme DNA 9 transfection reagent (Roche)

according to the manufacturer’s protocols.

Expression and purification of AKR1B15 isoforms

– His₆tagged AKR1B15.1 and AKR1B15.2 were

expressed in E. coli BL21 (DE3), carrying the

respective pET28a(+) expression vectors, by

induction with 0.5 mM IPTG and overnight

incubation at 25 °C. Cell pellets were harvested,

resuspended in lysis buffer (50 mM potassium

phosphate buffer (KP_i) pH 8.0, 300 mM KCl,

5 mM imidazole, 1 % (m/v) N-lauroylsarcosine)

and lysed by four cycles of 30 sec ultra-sonication

pulses and 30 sec ice bath. The lysate was

centrifuged (13000xg, 4 °C, 30 min), the resulting

supernatant supplemented with TritonX100 to a

final concentration of 2 % and applied on a

Profinia Affinity Chromatography Protein

Purification System (BioRad). The proteins were

automatically purified according to the “Native

IMAC Purification Protocol for His tagged

Proteins” given by the manufacturer with modified

buffers (2x wash buffer-1: 100 mM KP_ipH 8.0,

600 mM KCl, 10 mM imidazole, 0.5 % (m/v) N-

lauroylsarcosine; 2x wash buffer-2: 100 mM KP_i

pH 8.0, 600 mM KCl, 20 mM imidazole, 0.5 %

(m/v) N-lauroylsarcosine; 2x elution buffer:

100 mM KP_ipH 8.0, 600 mM KCl, 500 mM

imidazole, 0.1 % (m/v) N-lauroylsarcosine; 1x

desalting buffer: 20 mM KP_ipH 7.4, 1 mM

EDTA) using a 1 ml Bio-Scale Mini Profinity

IMAC cartridge (BioRad) followed by a 10 ml

Bio-Scale Mini Bio-Gel P-6 Desalting Cartridge

(BioRad). The final concentration of eluted

proteins was determined via BioRad DC Protein

Assay Kit (BioRad).

Enrichment of mitochondria from BeWo cells –

Mitochondria were enriched from the BeWo cell

line using a pump-controlled cell rupture system

(PCC), following the protocol published by

Schmitt et al. (37). For cell rupture 2x10⁷freshly

harvested BeWo cells were resuspended in 4 ml

isolation buffer (300 mM sucrose, 5 mM Tes, and

200 µM EGTA, pH 7.2) and passed three times

through a Cell Homogenizer with 10 µm clearance

(Isobiotech) at a constant flow of 700 µl/min.

Ruptured cells were centrifuged at 800xg and 4 °C

for 5 min. The resulting supernatant was

centrifuged once more at 9000xg and 4 °C for

10 min. The concentration of total protein in the

fractions (800xg pellet, 9000xg pellet, and 9000xg

supernatant) was determined via BioRad DC

Protein Assay Kit (BioRad).

Generation of polyclonal and monoclonal

antibodies against AKR1B15 – A peptide sequence

with the most dissimilarity between AKR1B15

and AKR1B10 (Fig. 1B, red box) was used as

antigen for the generation of polyclonal IgG

antibodies against AKR1B15 in rabbit, which was

carried out by 21st Century Biochemicals

(Marlboro,

MA).

The

peptide

Ac-

NWRAFDFKEFSHLC-amide was synthesized,

conjugated to an immune carrier and used for the

immunization of two rabbits to produce polyclonal

antisera. Prior to use the resulting antibodies were

affinity purified via the peptides deployed for

immunization.

Cell culture and transfection of human cells –

HEK293 cells (CRL-1573^TM; ATCC) were

cultured in DMEM (high glucose, stable

glutamine) medium (PAA) and HeLa cells

(ACC57; DSMZ) in MEM (L-glutamine) medium

(PAA), both supplemented with 10 % FBS Gold

(PAA), 100 U/ml penicillin, and 100 µg/ml

streptomycin (Pen Strep; Gibco). BeWo cells

(CCL-98^TM; ATCC) were cultured in F12-K

medium (Invitrogen) supplemented with 10 %

FBS Gold (PAA). SGBS cells were provided by

Prof. M. Wabitsch (36) and cultured in DMEM/F-

12 (1:1) (L-glutamine, 15 mM HEPES) medium

(Gibco) supplemented with 10 % FBS Gold

(PAA), 17 µM pantothenate, and 33 µM biotin.

All cells were maintained at 37 °C, 5 % CO₂in a

humidified incubator and trypsinized for

continuative cultivation or cell harvest using

For the generation of the monoclonal antibody the

peptide comprising the amino acid sequence

QGFKTGDDFFPKDDKGNMISGKGTF from the

human AKR1B15 protein (Fig. 1B, green box)

was synthesized and coupled to ovalbumin (OVA)

(Peps4LS, Heidelberg, Germany). Lou/c rats were

immunized subcutaneously and intraperitoneally

with a mixture of 50 µg peptide-OVA, 5 nmol

CPG oligonucleotide (Tib Molbiol, Berlin), 500 µl

PBS, and 500 µl incomplete Freund's adjuvant. A

boost without adjuvant was given six weeks after

the primary injection. Fusion of the myeloma cell

line P3X63Ag8.653 (ATCC® CRL-1580™) with

rat immune spleen cells was performed using

standard procedures. Hybridoma supernatants

-4-

Characterization of novel human AKR1B15

were tested in a differential ELISA with the

biotinylated AKR1B15 peptide and an irrelevant

biotinylated peptide on avidin coated ELISA

plates. MAbs that reacted specifically with the

AKR1B15 peptide were further analyzed by

Western blot. Hybridoma culture supernatant of

the anti-AKR1B15 clone 9A5 (rat IgG2a subclass)

was used in this study.

(Promega) according to the manufacturer’s

protocols.

RT-PCR and qPCR – End-point and real-time RT-

PCR were carried out both with the same set of

transcript specific primer pairs (Table 1). For the

end-point RT-PCR, DreamTaq Green DNA

Polymerase (Thermo) was used according to the

manufacturer’s protocol, with 38 amplification

cycles for AKR1B transcripts and 24 cycles for

GAPDH controls in a RoboCycler (Stratagene).

PCR products were analyzed on a 2 % agarose gel

containing 0.0025 % Midori Green (Biozym).

QPCR was carried out applying the Perfect CT

SYBR Green master mix (Quanta) and a 3-step-

protocol (95 °C, 15 s; 57 °C, 30 s; 72 °C, 45 s)

with an ABI 7900 HT instrument. Amplification

efficiency was verified for each pair of primers

using a standard curve constructed by serial

dilution of a control template. Resulting C_Tvalues

for AKR1B15.1, AKR1B15.2, and AKR1B10

transcripts were corrected by the average C_Tvalue

of the three housekeeping genes GAPDH, HPRT,

and 18S RNA (ΔC_Tcalculated) and normalized to

the expression level of AKR1B15.1 in placenta.

SDS-PAGE and Western blotting – Denatured

proteins were loaded onto a 12 % Mini-Protean or

Criterion TGX gel (BioRad) and separated by

conventional electrophoresis in running buffer

(25 mM Tris pH 8.3, 192 mM glycine, 0.1 %

(m/v) SDS). Afterwards gels were either stained

via Coomassie Brilliant Blue (0.05 % (m/v)

Coomassie Brilliant Blue R250, 10 % (v/v) acetic

acid, 40 % (v/v) methanol) or blotted onto a PVDF

membrane (Immobilon FL, Millipore) via semi-

dry-blot in transfer-buffer (48 mM Tris, 39 mM

glycine,

0.0375 % (m/v)

SDS,

20 % (v/v)

methanol). For testing antibody specificity,

membranes after transfer were blocked with 5 %

skimmed milk powder in PBS and then incubated

overnight with primary antibodies in 0.5 %

skimmed milk powder in PBS at 4 °C. Membranes

were washed three times with PBS for 10 min,

followed by incubation with HPR-conjugated

secondary antibodies (also in 0.5 % skimmed milk

powder in PBS) and another washing step. Signals

were detected by incubating the membranes in

Pierce ECL Plus Western Blotting Substrate

Activity assays – Catalytic activity was measured

using ³H-labeled steroids either with 10⁶harvested

HEK293 cells (untransfected or transfected with

pIRES-hrGFP-1α-AKR1B15) or with purified

enzymes as described previously (38) with slight

modifications. Reaction assays using HEK293

cells contained 10-40 nM ³H-labeled steroid

(Perkin Elmer, NEN) and generally 350 µM

NAD(P)(H) cofactor (Serva, Sigma) in 500 µl

reaction buffer (100 mM sodium phosphate buffer

(NaP_i) pH 7.4, 1 mM EDTA). For the

determination of Michaelis-Menten parameters

0-10 µM unlabeled steroid (Sigma) was added. In

assay mixtures containing purified proteins (up to

55 nM), 0.05 % (m/v) BSA was added. The

reaction mixtures were incubated with continuous

shaking at 37 °C and the reaction was terminated

by the addition of 20 % stop solution (0.5 M

ascorbic acid, 1 % (v/v) acetic acid in methanol).

Steroids were extracted on StrataC18-E (55 µm,

70 Å, 100 mg/ml) SPE cartridges (Phenomenex)

with methanol as eluent. Substrates and products

were separated by RP-HPLC on a Luna 5 µm

C18(2) 125x4 mm column (Phenomenex) with

43 % ACN in MilliQ-H₂O as mobile phase, using

a Beckman-Coulter system coupled to an online

(Thermo

Scientific)

according

to

the

manufacturer’s protocol and visualized using a

Fusion FX7 system (Vilber Lourmat). A similar

procedure was used to detect endogenous

AKR1B15 isoforms, except that IR-dye labeled

secondary antibodies and an Odyssey infrared

imaging system (LI-COR) were used. Briefly,

membranes were blocked with 50 % Odyssey

Blocking Buffer (PBS) in PBS after transfer,

antibodies were diluted in 50 % Odyssey Blocking

Buffer (PBS) in PBS-T (0.05 % Tween20 in PBS),

and washing steps were performed with PBS-T.

RNA isolation and cDNA synthesis – RNA from

cultured cells was isolated using the RNeasy Mini

Kit (Qiagen) combined with a DNaseI (Qiagen)

digestion treatment. 1 µg RNA was reverse

transcribed using Oligo(dT)₁₈primers and the

AffinityScript QPCR cDNA Synthesis Kit

(Agilent) or AMV Reverse Transcriptase

scintillation

detector

(Berthold

LB506D).

-5-

Characterization of novel human AKR1B15

Conversion of ³H-labeled substrate was

determined from ratios of areas under the peaks by

Karat software (Beckman-Coulter).

the cytoplasm and endoplasmic reticulum, pCMV-

DsRed-Express2 and pDsRed2-ER (Clontech)

vectors were co-transfected, respectively. After

transfection, cells were incubated at 37 °C and

5 % CO₂in a humidified incubator for two

additional days. Mitochondria were counterstained

before the cells were prepared for immuno-

cytochemical analysis by incubating living cells in

serum-free MEM medium containing MitoTracker

Orange CMTM-Ros (Molecular Probes) for

30 min. After staining the mitochondria, the cells

were fixed in 3.7 % formaldehyde in PBS for

10 min at culturing conditions, permeabilized for

5 min using 0.5 % TritonX100 in PBS, and

blocked with 3 % BSA in PBS for 1 h to prevent

unspecific binding of the antibodies. The fixed

cells were consecutively incubated with mouse-

anti-myc (Roche) / goat-anti-rabbit-AlexaFluor

488 (Molecular Probes) antibodies in case of myc-

tagged AKR1B15 isoforms or with rabbit-anti-

AKR1B15 (21st Century Biochemicals) / goat-

anti-rabbit-AlexaFluor 488 (Molecular Probes)

and rat-anti-AKR1B15 clone 9A5 (in-house

Activity assays with unlabeled substrates were

carried out with purified enzyme by measuring the

change in NADPH absorbance at 340 nm using a

Cary50 UV-Visible spectrophotometer (Varian) as

described previously (2). The reaction mixture

contained 150 µM NADPH, 6 µM purified

enzyme, and substrate at variable concentrations

(0-1 mM) in reaction buffer (100 mM NaP_i

pH 7.0, 1 mM EDTA). The reaction was initiated

by the addition of substrate to the pre-warmed

mixture and allowed to run at 37 °C for 10-15 min

with continuous absorbance recording. The initial

velocity was calculated from the linear portion

(0-5 min) of the curve. Kinetic parameters were

calculated using SigmaPlot 12 software (Systat

software).

Fluorescence titrations – The binding affinity of

AKR1B15 isoforms to pyridine nucleotide

cofactors was studied fluorometrically by

following the quenching of the protein

fluorescence (λ_ex= 280 nm, λ_em= 340 nm) upon

addition of cofactors (39), using a Shimadzu

RF5000 instrument. Aliquots containing 14 µg of

production)

/

goat-anti-rat-AlexaFluor 488

(Molecular Probes) in case of untagged proteins

for 1-2 h. Before mounting objects on slides with

VectaShield

mounting

medium

(Vector

Laboratories), nuclei were counterstained with

Hoechst 33342 dye (Molecular Probes) diluted

1:5000 in PBS for 2 min. After each step, cells

were washed twice with PBS. Subcellular

localization data were collected and analyzed

using a Zeiss AxioImager Z1/ApoTome confocal

microscope with an AxioCam MRm camera and

the AxioVision Rel. 4.8 software. In silico

prediction of the localization of AKR1B15

isoforms were carried out using iPSORT

(http://ipsort.hgc.jp) for non-plant proteins (40).

purified protein (corresponding to

a

final

concentration of 180 µM or 170 µM for

AKR1B15.1 or AKR1B15.2, respectively) were

added to 2 ml of 20 mM KP_ipH 7.4 at room

temperature and equilibrated for 15-20 min.

Aliquots of nucleotides were added sequentially

and changes in emission were recorded.

Dissociation constants were calculated by fitting

Morrison equation [1] to the data after correction

for the volume increase due to the addition of

nucleotides and inner filter effect using

SigmaPlot 12 software (Systat software).

RESULTS

2

(

)

푑

퐸+푁+퐾 −� 퐸+푁+퐾

−4∙퐸∙푁

푑

∆F = ∆F_max

∙

[1]

2∙퐸

Two splice variants of AKR1B15 are expressed in

vivo – Previously, we reported the identification

and functional expression of a novel human

member of the AKR1B family with 91 % amino

acid identity to the well characterized human

enzyme AKR1B10 (35). The gene encoding this

protein, AKR1B15, is located on chromosome 7

next to AKR1B10. After our report, a newly

predicted transcript sequence corresponding to the

AKR1B15 gene was deposited in the NCBI and

Ensembl databases. The predicted transcript is

(ΔF, decrease in protein fluorescence; E, total

enzyme concentration; N, total nucleotide

concentration)

Subcellular localization studies – HeLa cells were

grown on glass coverslips on the bottom of a

6-well plate and transiently transfected with

plasmids (pcDNA3.1(+), N-myc-pcDNA3 or

pcDNA4-myc/HisB

AKR1B15.1 or AKR1B15.2. For counterstaining

backbone)

encoding

-6-

Characterization of novel human AKR1B15

1621 bp in length and differs from our reported

1242 bp cDNA sequence in the 5’-end.

Bioinformatics analysis revealed that the two

sequences might result from an alternative use of

the first exons of the AKR1B15 gene, leading to

two transcripts, in the following referred to as

AKR1B15.1 (Ensembl: transcript AKR1B15-201)

for the short and AKR1B15.2 (Ensembl: transcript

AKR1B15-001) for the long transcript (Fig. 1A).

To determine whether the alternative transcript

AKR1B15.2, like AKR1B15.1, is expressed in vivo,

we designed specific primers based on the

distinct distribution patterns (Fig. 2A). The highest

expression levels of both AKR1B15 splice variants

were seen in adipose tissue, skeletal muscle,

thymus, thyroid gland, and reproductive tissues

(ovary,

placenta,

prostate,

and

testis).

Corroborating the results from tissues, the human

placental cell line BeWo and the pre-adipocyte

cell strain SGBS express significant levels of both

AKR1B15 transcripts (Fig. 2A). In order to gain

additional insights into the expression levels of

AKR1B15, we performed qPCR on selected

AKR1B15 expressing tissues, using the same

transcript-specific primers as for the end-point RT-

PCR. We found the highest level of expression of

both AKR1B15 mRNA variants in placenta,

followed by adipose tissue and testes (Fig. 2B). On

the absolute level the abundance of AKR1B15

mRNA was quite low. In placenta, the tissue with

the highest level of expression, the abundance of

AKR1B15.1 transcripts was 100-150 -fold lower

than that of GAPDH. In a majority of tissues the

AKR1B15.1 transcript was more abundant (at least

3-fold) than AKR1B15.2. In the thymus, prostate,

and uterus comparable levels of both transcripts

were found. Skeletal muscle was the only tissue

where AKR1B15.2 showed higher expression than

AKR1B15.1. Only testis and adipose tissue

displayed either of AKR1B15 transcripts at a level

of more than 10 % of that of AKR1B15.1 in

placenta. Skeletal muscle expressed around 2.5 %

and all other tissues tested had less than 1 % of the

level found in placenta. Hence, the expression

pattern of AKR1B15 is specific to a few tissues.

AKR1B10 and AKR1B15, despite high sequence

similarity, display different tissue abundance:

Among the tissues with AKR1B15 expression, the

abundance of AKR1B10 mRNA exceeded that of

AKR1B15 by over 500-fold in the lung, thymus,

and uterus. In contrast, the expression level of

AKR1B10 was only 6 % of that of AKR1B15.1 in

placenta (Fig. 2B).

predicted

sequence,

and

amplified

the

corresponding product by PCR from the cDNA

libraries of human thymus and salivary gland.

Sequencing of the resulting product confirmed its

100 % identity to the sequence reported in the

databases. Thus, AKR1B15.2 mRNA is expressed

in tissues and might be translated into a protein as

it contains an open reading frame corresponding to

a 344 amino acid protein. We conclude that the

AKR1B15 gene gives rise to two splice variants in

vivo, presumably coding for two different protein

isoforms (Fig. 1) and classify these protein

isoforms as AKR1B15.1 (shorter, 316 amino acid

isoform, encoded by transcript AKR1B15.1) and

AKR1B15.2 (longer, 344 amino acid isoform,

encoded by transcript AKR1B15.2) in accordance

with the guidelines for the nomenclature of

alternative splicing in the AKR superfamily (41).

The two AKR1B15 isoforms differ in their N-

termini, but share the same sequence beginning

with the amino acid Ser23 in case of AKR1B15.1

and Ser51 in case of AKR1B15.2 (Fig. 1B). The

four amino acid residues known to comprise the

catalytic tetrad in AKRs are found in both AKR

variants (Asp44, Tyr49, Lys78, and His111 for

AKR1B15.1 and Asp72, Tyr77, Lys106, and

His139 for AKR1B15.2, respectively; Fig. 1B).

Tissue distribution of AKR1B15 – To determine

the tissue abundance of the two AKR1B15

isoforms, we first analyzed the expression of the

two mRNA splice variants in a broad panel of

tissues by RT-PCR, using transcript-specific

primers as depicted in Fig. 1A (sequences listed in

Table 1). For comparison, the expression of the

highly homologous AKR1B10 was analyzed in the

same set of samples. We found that the expression

of AKR1B15.1 and AKR1B15.2 differs completely

from that of AKR1B10. While AKR1B10 is

expressed in a fairly ubiquitous manner across the

tissue panel, the AKR1B15 variants show more

Recombinant expression and purification of

AKR1B15 – To verify that both AKR1B15 mRNA

variants produce functional proteins, we cloned

their coding regions into the vector pET28a(+) and

expressed encoded His-tagged proteins in E. coli.

After induction with IPTG, bands with the

predicted molecular weights were detected in

bacterial

extracts

transformed

with

the

corresponding constructs (Fig. 3A). AKR1B10,

which differs only in 27 amino acid residues from

AKR1B15.1 (Fig. 1B) was also expressed for

-7-

Characterization of novel human AKR1B15

potential substrates of AKR1B15.1 or

AKR1B15.2. In activity assays using HEK293

cells, transiently transfected with pIRES-

comparison. In contrast with AKR1B10, which is

expressed as soluble protein in E. coli, both

AKR1B15 isoforms were found in the insoluble

fraction (Fig. 3B). We were able to solubilize both

N-terminally histidine-tagged AKR1B15 isoforms

using a sarkosyl-triton buffer system and purified

both proteins to apparent homogeneity using a

one-step immobilized metal (Ni²⁺) affinity

chromatography (Fig. 3C). We attributed the

minor low molecular weight bands to the

degradation or truncation products of AKR1B15

isoforms (Fig. 3C). The purification yielded

6-10 mg protein per liter bacterial culture.

hrGFP1α-AKR1B15.1

or

pIRES-hrGFP1α-

AKR1B15.2 and NADP(H) or NAD(H) as

cofactor, we found that neither AKR1B15.1 nor

AKR1B15.2 was able to reduce or oxidize

progesterone and corticosteroids (data not shown).

However, AKR1B15.1 catalyzed oxidation-

reduction reactions with androgens and estrogens,

as shown for the estrone and 17β-estradiol pair

(Fig. 5A). Catalysis was supported by NADPH or

NADP⁺, but not by NADH or NAD⁺(up to a

concentration of 1.5 mM), which agrees with the

binding studies, suggesting that similar to a

majority of other AKRs, AKR1B15 exhibits

strong preference for phosphorylated cofactors. In

addition, we found that AKR1B15.1 possesses

high positional selectivity as it catalyzed only

reactions on C17(β) position (C17) but not on C3

position (C3) of the steroid nucleus (Fig. 5B). In

Nucleotide binding – To determine the affinities of

the two AKR1B15 proteins for nucleotide

cofactors, we determined dissociation constants

(K_d) of AKR1B15.1 and AKR1B15.2 for the four

major pyridine nucleotides NADPH, NADH,

NADP⁺, and NAD⁺using fluorometric titrations.

As shown in Fig. 4A, the addition of incremental

concentrations of NADPH or NADP⁺to

AKR1B15.1 led to a gradual decrease in protein

fluorescence. The maximal degree of fluorescence

quenching was 25-26 % in both cases, and the K_d

values calculated from the concentration

dependence of the decrease in fluorescence were:

59.3 ± 1.9 nM for NADPH and 60.4 ± 3.5 nM for

NADP⁺. In contrast, less than 3 % quenching of

AKR1B15.1 fluorescence was observed with

NADH and NAD⁺in concentrations of up to

40 µM after correction for inner filter effect

(Fig. 4B; titration curve not shown). These results

indicate that like other AKRs, AKR1B15.1 binds

pyridine dinucleotides with high affinity and that it

highly discriminates between phosphorylated and

non-phosphorylated nucleotides. Addition of any

of the four nucleotides to AKR1B15.2 failed to

produce any change in protein fluorescence, in

agreement with the lack of detectable enzymatic

activity of AKR1B15.2 (see below).

time-course

experiments

using

purified

AKR1B15.1 and estrogens as well as androgens in

a final concentration of 20 nM, we found that

AKR1B15.1 prefers reductive over oxidative

reactions and androgens over estrogens (Fig. 5C).

In contrast to AKR1B15.1, AKR1B15.2 did not

exhibit any enzymatic activity with estrogens,

androgens, or other steroids tested. Because

neither the activity assays using solubilized

purified AKR1B15.2 nor the assays using

HEK293 cells, in which AKR1B15.2 was

expressed under physiological conditions, showed

any enzymatic activity, it appears that the protein

is catalytically inactive and the lack of activity of

the protein purified from bacteria could not be

attributed to improper folding.

Because we found that AKR1B15.1 co-localizes

with mitochondria (see below), we tested whether

the enzyme displays catalytic activity with

mitochondrial carbonyls or alcohols such as

acetoacetyl-CoA, oxaloacetic acid, 2-oxobutyric

acid, methylmalonyl-CoA, succinyl-CoA, DL-

3-hydroxy-butyryl-CoA, and DL-3-hydroxy-3-

methylglutaryl-CoA. We found that AKR1B15.1,

but not AKR1B15.2, possessed enzymatic activity

with acetoacetyl-CoA, while all other compounds

were not detectably reduced or oxidized by either

isoform (data not shown). These results indicate

that AKR1B15.1 acts only on hydroxyl or keto

groups of substrates possessing a bulky ring

system like the steroid nucleus or the CoA.

Enzymatic activity of AKR1B15 isoforms – We

performed detailed kinetic characterizations of

both AKR1B15 isoforms, AKR1B15.1 and

AKR1B15.2, using a variety of physiological

substrates.

Because AKR1B15 is abundant in reproductive

organs (the first full-length AKR1B15 transcript

was initially found in testis) we reasoned that the

protein might possess enzymatic activity with sex

steroids. Therefore, we tested estrogens,

androgens, progesterone, and corticosteroids as

-8-

Characterization of novel human AKR1B15

We also determined kinetic parameters of

AKR1B15.1 with different substrate classes using

the purified enzyme (Table 2). The K_Mvalues for

oxidized steroids carrying a keto group on C17

appeared to be in the low micromolar range

(1.9-2.8 µM), while reduced steroids carrying a

hydroxyl group on C17 showed a 4-7 -fold higher

K_M(7.1-19.2 µM). The turnover numbers

(k_catvalues) mirrored the results of the time

course experiments: k_catvalues of androgens

(0.6-3.0 min^-1) were higher than those of estrogens

(0.5-1.0 min^-1) and, with the exception of the k_cat

of 3α,17β-androstandiol, k_catvalues of reductive

reactions were about 2-fold higher than those of

oxidative reactions. Estimates of the catalytic

efficiencies (k_cat/ K_M) support the conclusion that

the the protein has higher reductase than

dehydrogenase activity. With acetoacetyl-CoA, the

enzyme had a K_Mvalue of 63.4 µM and a k_catof

0.5 min^-1. The catalytic activity in the reverse

direction, oxidation of 3-hydroxybutyryl-CoA,

was below our detection limit (0.1 min^-1).

AKR1B15.2 showed no activity with any of the

substrates tested.

molecular weight to the AKR1B15 isoforms

(Fig. 6B, left panel). Because the high cross-

reactivity of the polyclonal antibody could

complicate the analysis of the expression of the

native AKR1B15 proteins and because we found

that antibodies against C-terminal sequences often

cross-react with several other proteins, we

generated

a

monoclonal antibody (rat-anti-

AKR1B15 (9A5) recognizing both AKR1B15

isoforms and targeting a sequence more centrally

located in the protein (AKR1B15.1: amino acids

114-138, AKR1B15.2: amino acids 142-166) with

high divergence compared with AKR1B10

(Fig. 1B). Like the polyclonal antibody, the

monoclonal antibody also recognized both

AKR1B15.1 and AKR1B15.2 and did not cross-

react with other recombinant human AKRs

(Fig. 6A). In contrast with the polyclonal antibody,

the monoclonal antibody displayed no cross-

reactivity with proteins of the HEK293 cell

background, when performing Western blots

(Fig. 6B, right panel). To examine whether the

mRNA of AKR1B15 is translated to a protein in

vivo, we performed Western blots of BeWo cell

extracts using the specific monoclonal antibody

and an IR-dye labeled secondary antibody (goat-

anti-rat-AlexaFluor 790) which allows for the

sensitive detection of low abundance proteins.

Although no endogenous AKR1B15 isoforms

were detectable in total cell lysates (data not

shown), we were able to detect endogenous

AKR1B15.1 and AKR1B15.2 in both the 800xg

and the 9000xg pellet fraction of BeWo

homogenates processed for the enrichment of

mitochondria (Fig. 6C). Whereas AKR1B15.2

appeared as a single protein band at the expected

molecular weight of 39.5 kDa, endogenous as well

as overexpressed AKR1B15.1 was present as a

double band corresponding to molecular weights

of 36.5 kDa (expected) and 35.5-36 kDa, which

could be proteolyzed or post-translationally

modified form of the protein.

Generation of AKR1B15-specific antibodies and

Western blot analysis – To examine the expression

and the subcellular localization of AKR1B15

proteins, we first generated a specific polyclonal

antibody that recognizes both AKR1B15 isoforms

and distinguishes them from other AKR family

members. Although the amino acid sequence of

AKR1B15.1 is 91 % identical to that of

AKR1B10, there is a single stretch of six

consecutive amino acids at the C-terminus of the

proteins (amino acids 299-304) that is different

between the two proteins. Using a peptide

corresponding to this area (AKR1B15.1: amino

acids 295-307, AKR1B15.2: amino acids 323-335;

Fig. 1B), we were able to generate a polyclonal

antibody that recognized both AKR1B15.1 and

AKR1B15.2, but did not cross-react with other

recombinant human AKR proteins (Fig. 6A). This

polyclonal antibody did not cross-react with

AKR1B10 even when loading high amounts

(200 ng) of purified protein (data not shown). In

Western blot analysis of HEK293 transiently

transfected with different plasmids encoding either

AKR1B15.1 or AKR1B15.2, we could clearly

detect the different overexpressed AKR1B15

isoforms. However, the polyclonal antibody also

bound nonspecifically to other proteins of the

HEK293 cells, including those having a similar

Subcellular localization of AKR1B15 isoforms –

To characterize the two AKR1B15 isoforms in

more detail, we determined their subcellular

localization in the HeLa cell line overexpressing

AKR1B15 isoforms using different constructs by

immunocytochemistry. We found N- and C-

terminally tagged AKR1B15.2 (expressed from N-

myc-pcDNA3-AKR1B15.2 and pcDNA4-myc/

HisB-AKR1B15.2, respectively) in the cytosol

(Fig. 7-a, Fig. 7-c). A cytosolic localization was

-9-

Characterization of novel human AKR1B15

observed also for AKR1B15.1, but only when

fused to an N-terminal myc-tag (expressed from

into protein in vivo. Consistent with the low

expression of mRNA, the abundance of the

AKR1B15 protein was low as well; therefore,

fractionation of subcellular components was

necessary to detect the endogenous protein.

Nevertheless, we were able to identify both

endogenous AKR1B15 protein isoforms in the

800xg and 9000xg pellet of homogenates of the

placental-derived BeWo cells, but were unable to

detect endogenous AKR1B15 isoforms in

commercial total protein human tissue lysates and

total lysates of BeWo cells (data not shown). It

appears that additional steps such as enrichment of

subcellular components or immunoprecipitation

will be necessary to characterize the expression of

AKR1B15 in tissues and overcome its low

abundance. Additionally, the detection of the

proteins could be confounded by post-translational

modifications which could reduce the affinity of

the monoclonal antibody to the endogenous

protein. Post-translational prediction algorithms

indicated that both AKR1B15 isoforms could

N-myc-pcDNA3-AKR1B15.1;

terminally tagged AKR1B15.1 (expressed from

pcDNA4-myc/HisB-AKR1B15.1) co-localized

Fig. 7-b).

C-

with mitochondria (Fig. 7-d), indicating that the

N-terminal amino acid sequence, which is

different from that of AKR1B15.2, is important

for the mitochondrial localization of AKR1B15.1.

These results are in accord with theoretical

analysis of AKR1B15 localization using the

iPSORT prediction algorithm. The algorithm

predicted

a

mitochondrial localization of

AKR1B15.1 and a cytosolic localization of

AKR1B15.2 when considering the N-terminal

amino acid leader sequences Met1-Glu30 and

Met1-Leu30, respectively. The mitochondrial

localization of AKR1B15.1 (Fig. 7-f, Fig. 7-h) and

cytosolic localization of AKR1B15.2 (Fig. 7-e,

Fig. 7-g) was verified in HeLa cells transiently

transfected

with

untagged

AKR1B15.1

(pcDNA3.1(+)-AKR1B15.1) or AKR1B15.2

(pcDNA3.1(+)-AKR1B15.2) and stained with

either the polyclonal rabbit-anti-AKR1B15

antibody or the monoclonal rat-anti-AKR1B15

antibody. Thus, AKR1B15.1 is the first AKR

localized to the mitochondria.

undergo

various

modifications

(e.g.,

phosphorylation, SUMOylation, or ubiquitination)

at several sites, including residues of the

monoclonal antibody epitope (data not shown).

These modifications could shift the apparent

molecular of the proteins, destabilize the proteins

or prevent antibody binding and thus interfere with

the detection of endogenous AKR1B15 proteins.

Additional research is required to investigate these

possibilities.

DISCUSSION

In this work we demonstrate, that a novel human

gene, AKR1B15, a member of the AKR super-

family, is expressed in human tissues with the

highest level found in reproductive organs,

adipose tissue and skeletal muscle. In addition, we

found that the AKR1B15 gene undergoes

alternative splicing producing two open reading

frames corresponding to the protein isoforms

AKR1B15.1 and AKR1B15.2. Both mRNA

transcripts are expressed in vivo, although

expression of both variants is limited in abundance

and is not as ubiquitous as that of the highly

homologous AKR1B10. For the most part, the

transcript AKR1B15-201 (referred to as

AKR1B15.1, encoding protein AKR1B15.1) was

more abundant than AKR1B15-001 (referred to as

AKR1B15.2, encoding protein AKR1B15.2) in

tissues expressing both variants.

Importantly, AKR1B15.1 showed enzymatic

activity with sex steroids, both androgens and

estrogens, and 3-keto-acyl-CoA thioesters, such as

acetoacetyl-CoA. In contrast, AKR1B15.2

appeared to be an inactive enzyme, despite the fact

that it possesses all four conserved amino acid

residues of the catalytic tetrad (Asp72, Tyr77,

Lys106, and His139). These findings are

corroborated by the inability of AKR1B15.2 to

bind nicotinamide adenine dinucleotide cofactors,

whereas AKR1B15.1 binds NADPH and NADP⁺

with an affinity in the nanomolar range.

AKR1B15.1 displayed absolute specificity for

phosphorylated dinucleotide cofactors, as neither

binding nor activity has been observed with

NADH or NAD⁺.

Using the monoclonal rat-anti-AKR1B15 (9A5)

antibody, generated in house, we were able to

show that both AKR1B15 transcripts are translated

Currently, it is difficult to distinguish whether the

lack of nucleotide binding by AKR1B15.2 is due

to improper folding or to an intrinsic property of

-10-

Characterization of novel human AKR1B15

this protein. The undetectable enzymatic activity

in both the artificial bacterial and the mammalian

expression system, supports the latter hypothesis,

suggesting that the long N-terminus influences the

protein structure in a way that it prevents

nucleotide and/or substrate access or binding.

While the shorter AKR1B15 isoform, AKR1B15.1

like all other known AKR1B family members is

316 amino acids long, and shares 91 % amino acid

and the fact that estrogens and androgens are

substrates of AKR1B15.1, we measured the

activity of purified AKR1B10 with estrone, 17β-

estradiol, Δ4-androstenedione, as well as

testosterone. Although it is published that

AKR1B10 is inhibited by steroids (49), we found

that AKR1B10 is able to catalyze oxidation or

reduction of those steroids in the nanomolar range,

too. However, in contrast to AKR1B15,

AKR1B10 preferentially catalyzed oxidative

reactions of steroids (data not shown).

sequence

identity

with

AKR1B10,

the

AKR1B15.2 isoform displays greater differences

because the N-terminus of AKR1B15.2 has no

homology with other AKR1Bs and is 28 residues

longer. In the crystal structure of AKR1B proteins

the N-terminus folds into a hairpin of two β-sheets

and creates a bottom of the (β/α)₈barrel (2, 42,

43). The alternative N-terminus in AKR1B15.2

substitutes the first 22 amino acid residues of other

AKR1Bs, which might lead to a disarrangement of

the bottom hairpin and the first β-sheet of the

(β/α)₈barrel. This suggests the intriguing

possibility that the N-terminus of AKR1B15.2

might serve as a modulatory domain, regulating

access to the active site, by changing its

conformation in response to protein modification

such as phosphorylation. Alternatively, the non-

homologous N-terminal loop of AKR1B15.2 may

perform some additional function, analogous to

the N-terminus of AKR6 family members (Kvβ

proteins), which forms a “ball and chain” structure

involved in the regulation of ion flow kinetics of

voltage-gated potassium (Kv) channels (44, 45).

Further investigation is needed to systematically

address these possibilities.

Our studies revealed that AKR1B15.1 is a

predominantly reductive enzyme and that it co-

localizes with mitochondria. The subcellular

localization was surprising as most other human

AKRs are cytosolic enzymes. Although in silico

subcellular localization prediction is hypothetical

and often does not agree with in vivo localization

(50), iPSORT predicted

a

mitochondrial

localization of AKR1B15.1 as well as a cytosolic

localization of AKR1B15.2 and AKR1B10, which

is in agreement with our results. The different

behavior of AKR1B15.1 and AKR1B10

concerning localization can probably be explained

by the different amino acid composition in their N-

termini at positions 22 and 24. AKR1B15.1

possesses an arginine at position 22 and

AKR1B10 features a lysine at this position, both

of which have similar physicochemical properties.

In contrast, the physicochemical properties of the

amino acids at position 24 of AKR1B15.1 (Leu24)

and AKR1B10 (Pro24) are clearly different.

Several previous studies have shown that proline

residues serve as a helix-breaker (51, 52). We

therefore presume that Pro24 may be the amino

acid responsible for different localization of

AKR1B15.1 and AKR1B10. In silico prediction

with iPSORT indicated a mitochondrial location of

the AKR1B10 P24L mutant. This was confirmed

in localization studies with N-terminal sequences

fused to GFP, in which we were able to show that

the substitution P24L in the N-terminus of

AKR1B10 was sufficient to switch the subcellular

localization of the respective GFP reporter

construct from cytosolic to mitochondrial (data not

shown). The results of Western blotting were

consistent with the mitochondrial localization of

AKR1B15.1, as endogenous AKR1B15.1 was

detected in the 800xg and 9000xg pellets of BeWo

cell homogenates. The 800xg pellet contains the

nuclear fraction together with unbroken cells, cell

debris and remains of the supernatant, whereas the

Our measurements of enzymatic parameters show

that AKR1B15.1 possesses K_Mvalues in the low

micromolar range for 17-keto-steroids, which are

similar to the K_Mvalues of other 17β-

hydroxysteroid dehydrogenases (17β-HSDs), e.g.,

HSD17B1, HSD17B5, and HSD17B12 (46–48).

We found that the enzyme is selective towards the

carbonyl group located at the C17 position on the

steroid nucleus. The comparatively low k_catvalues

observed with purified enzyme may result from

the purification procedure, as AKR1B15.1

expressed in E. coli is an insoluble protein which

needs to be reconstituted from inclusion bodies. In

our k_catcalculations, we assumed that all protein

molecules are properly folded, however, it is more

likely that only a fraction of the purified enzyme is

in the right conformation. Due to the high

homology between AKR1B15.1 and AKR1B10

-11-

Characterization of novel human AKR1B15

9000xg pellet represents the mitochondrial

fraction (37). Surprisingly, AKR1B15.2 which

Although it might seem surprising that a single

enzyme reduces such unrelated compounds as

steroids and keto-acyl-CoA derivatives, it appears

that many 17β-HSDs exhibit a wide substrate

spectrum, which includes fatty acid derivatives,

bile acids, and retinoids (55). The 17β-HSDs

belong to two genetic superfamilies: aldo-keto

reductases (AKRs) and short-chain dehydro-

genases (SDRs) (56). To-date at least 14 types of

17β-HSDs have been identified, among which

only type 5 belongs to the AKR superfamily

(AKR1Cs). No activity with 3-keto-acyl-CoAs has

been reported for AKR1C enzymes; however,

17β-HSDs of type 3, 4, 10, and 12, which belong

to the SDR superfamily, possess activity with both

steroids and keto-acyl-CoA conjugates (55).

Therefore, we propose that although structurally

a member of the AKR superfamily, AKR1B15.1

seems

localized

to

the

cytosol

by

immunohistochemistry, was found mainly in the

9000xg pellet rather than the supernatant,

suggesting strong association with subcellular

organelles, possibly lysosomes, which are likely to

be found in the 9000xg pellet (37).

Among the AKRs, only AKR7A2 has been

suggested to be associated with mitochondria in

SH-SY5Y neuroblastoma cells (53). However, its

rat ortholog, AKR7A4, has also been reported

localized to the Golgi apparatus (54), therefore the

subcellular localization of this enzyme is still

unclear. Hence, we conclude that we characterized

the first AKR1 family member co-localizing with

mitochondria; however, it still needs to be clarified

whether AKR1B15.1 is located inside the

mitochondria or strongly associated with the outer

membrane.

functionally

is

a

17β-hydroxysteroid

dehydrogenase. Among the 17β-HSDs only one

enzyme, HSD17B10, is mitochondrial and it

catalyzes the NAD-dependent oxidoreduction of

short-chain 3-keto-acyl-CoAs, along with sex

steroids, as well as bile acid isomerization and

glucocorticoid and gestagen catabolism (57). This

enzyme is also called SCHAD (short-chain

hydroxyl-acyl-CoA dehydrogenase) and it acts

primarily in oxidative direction. Defects in this

enzyme lead to hyper-insulinemic hypoglycemia

(58, 59), abnormal thermogenesis and lower body

weight in mice (60), as well as neural disorders

such as Alzheimer’s and Parkinson’s diseases,

Having established that AKR1B15.1 is localized

to the mitochondria, we investigated whether

mitochondria-specific carbonyls are potential

substrates of the enzyme. We found that a 3-keto-

acyl-CoA compound, acetoacetyl-CoA, can be

reduced by AKR1B15.1 with a K_Mof about

60 µM. We presume that AKR1B15.1 possesses

low oxidizing activity with DL-3-hydroxy-butyryl-

CoA, too, although the conversion could not be

detected by our assays, probably due to limitations

in the sensitivity of NADPH absorption; the

readout of our assay. Longer chain 3-keto-acyl-

CoAs, such as 3-keto-palmitoyl-CoA could also

serve as substrates of AKR1B15.1. However, up to

now we were unable to verify the proposed

conversion due to limitations in substrate amounts

and lack of sensitive and stable detection assays.

Development of a more sensitive assay possibly

based on product detection rather than nucleotide

absorbance is necessary to confirm the reaction.

The free oxo-(di)-carboxylic acids oxaloacetic

acid and 2-oxobutyric acid, as well as the CoA-

thioesters of dicarboxylic acids methylmalonyl-

CoA and succinyl-CoA do not appear to be

substrates of AKR1B15.1. This indicates that only

carbonyl and not carboxyl groups can be reduced

by AKR1B15.1. Moreover, it seems likely that all

substrates need to possess a bulky ring backbone

for their orientation in the substrate binding pocket

of AKR1B15.1.

mental

retardation

and

infantile

neuro-

degeneration (61).

It could be argued that the results from the activity

tests are somehow inconsistent with the

subcellular localization of AKR1B15.1, because

AKR1B15.1 preferably catalyzes reductive

reactions, but the mitochondrial matrix has an

oxidative environment, where among other

reactions, β-oxidation of fatty acids and the very

first steps of the steroid synthesis (from

cholesterol to pregnenolone) take place (62).

However, with the current data, the role of

AKR1B15.1 in mitochondria can only be

hypothesized. Different studies have shown that

steroids and steroid receptors are present in

mitochondria and affect their metabolism (63–65).

One function of AKR1B15.1 may be the activation

of the steroid signaling in mitochondria, as

AKR1B15.1 catalyzes, among other reactions, the

conversion of biologically low active estrone to

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Characterization of novel human AKR1B15

highly active 17β-estradiol, which binds to the

mitochondrial estrogen receptor with high affinity

(65). Like the nuclear genome, the mitochondrial

genome contains hormone-responsive elements,

e.g., the estrogen response element, regulating the

expression of important ribosomal and structural

proteins, as well as mitochondrially encoded

proteins of the oxidative phosphorylation system

(64, 66, 67). In addition, several studies have

shown that 17β-estradiol protects the function of

mitochondria in cells by reducing the amount of

reactive oxygen species and therefore prevents

cells from aging (64). Thus, AKR1B15.1 might

provide the active steroid hormones which are

known to reduce aging in mitochondria and cells.

This hypothesis is supported by a recent

publication by Yashin et al., correlating a SNP in

AKR1B15 with longevity (68). Another function of

AKR1B15.1 might relate to the reduction of

3-keto-acyl-CoAs, such as acetoacetyl-CoA.

Reduction of 3-keto-acyl-CoAs is an important

step in fatty acid synthesis although not expected

in mitochondria, where the reverse process, the

β-oxidation of fatty acids, takes place. However,

several investigators have been able to show that

de novo fatty acid synthesis (FAS) does occur in

mitochondria via the FAS II pathway (69–71) and

that components of the FAS II pathway might

interact with the Complex I of the respiratory

chain (72).

It has been recently reported that a naturally

occurring mutation in AKR1B15 gene (leading to

an S8R mutation in AKR1B15.1, Fig. 1B) is

linked to an infantile mitochondrial disease

characterized by severe depletion of Complex I

activity (73). Interactions between AKR1B15.1

and Complex I would explain why the mutation

was associated with this infantile lethal phenotype

(73). Here, direct protein-protein interactions in

addition to the enzymatic activity of AKR1B15.1

might be of importance.

In conclusion, AKR1B15 is a novel member of the

AKR superfamily with potential roles in steroid

metabolism, regulation of the mitochondrial

function and aging. Given the potential role of the

enzyme in several key metabolic processes, further

research is required to fully characterize its

substrate specificity and mechanism, as well as its

role in normal physiology and the significance of

genetic polymorphisms in the development of

pathological conditions, such as mitochondrial

disease.

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Characterization of novel human AKR1B15

References

1.

2.

3.

Penning, T. M., and Drury, J. E. (2007) Human aldo-keto reductases: Function, gene regulation,

and single nucleotide polymorphisms. Arch. Biochem. Biophys. 464, 241–250

Barski, O. A., Tipparaju, S. M., and Bhatnagar, A. (2008) The aldo-keto reductase superfamily and

its role in drug metabolism and detoxification. Drug Metab. Rev. 40, 553–624

Kondo, K. H., Kai, M. H., Setoguchi, Y., Eggertsen, G., Sjöblom, P., Setoguchi, T., Okuda, K. I.,

and Björkhem, I. (1994) Cloning and expression of cDNA of human delta 4-3-oxosteroid 5 beta-

reductase and substrate specificity of the expressed enzyme. Eur. J. Biochem. 219, 357–363

4.

Fujii, Y., Watanabe, K., Hayashi, H., Urade, Y., Kuramitsu, S., Kagamiyama, H., and Hayaishi, O.

(1990) Purification and characterization of rho-crystallin from Japanese common bullfrog lens. J.

Biol. Chem. 265, 9914–9923

5.

6.

Weng, J., Cao, Y., Moss, N., and Zhou, M. (2006) Modulation of voltage-dependent Shaker family

potassium channels by an aldo-keto reductase. J. Biol. Chem. 281, 15194–15200

Barski, O. A., Papusha, V. Z., Ivanova, M. M., Rudman, D. M., and Finegold, M. J. (2005)

Developmental expression and function of aldehyde reductase in proximal tubules of the kidney.

Am. J. Physiol. Renal Physiol. 289, F200–F207

7.

8.

Penning, T. M. (2011) Human hydroxysteroid dehydrogenases and pre-receptor regulation:

insights into inhibitor design and evaluation. J. Steroid Biochem. Mol. Biol. 125, 46–56

Kabututu, Z., Manin, M., Pointud, J.-C., Maruyama, T., Nagata, N., Lambert, S., Lefrançois-

Martinez, A.-M., Martinez, A., and Urade, Y. (2009) Prostaglandin F2alpha synthase activities of

aldo-keto reductase 1B1, 1B3 and 1B7. J. Biochem. 145, 161–168

9.

Lemonde, H. A., Custard, E. J., Bouquet, J., Duran, M., Overmars, H., Scambler, P. J., and

Clayton, P. T. (2003) Mutations in SRD5B1 (AKR1D1), the gene encoding delta(4)-3-oxosteroid

5beta-reductase, in hepatitis and liver failure in infancy. Gut 52, 1494–1499

10.

11.

12.

Lyon, R. C., Johnston, S. M., Watson, D. G., McGarvie, G., and Ellis, E. M. (2007) Synthesis and

catabolism of gamma-hydroxybutyrate in SH-SY5Y human neuroblastoma cells: role of the aldo-

keto reductase AKR7A2. J. Biol. Chem. 282, 25986–25992

Srivastava, S., Chandra, A., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998)

Identification of cardiac oxidoreductase(s) involved in the metabolism of the lipid peroxidation-

derived aldehyde-4-hydroxynonenal. Biochem. J. 329, 469–475

Baba, S. P., Barski, O. A., Ahmed, Y., O’Toole, T. E., Conklin, D. J., Bhatnagar, A., and

Srivastava, S. (2009) Reductive metabolism of AGE precursors: a metabolic route for preventing

AGE accumulation in cardiovascular tissue. Diabetes 58, 2486–2497

-14-

Characterization of novel human AKR1B15

13.

Guengerich, F. P., Cai, H., McMahon, M., Hayes, J. D., Sutter, T. R., Groopman, J. D., Deng, Z.,

and Harris, T. M. (2001) Reduction of aflatoxin B1 dialdehyde by rat and human aldo-keto

reductases. Chem. Res. Toxicol. 14, 727–737

14.

15.

Maser, E. (2004) Significance of reductases in the detoxification of the tobacco-specific carcinogen

NNK. Trends Pharmacol. Sci. 25, 235–237

Liu, S.-Q., Jin, H., Zacarias, A., Srivastava, S., and Bhatnagar, A. (2001) Binding of pyridine

coenzymes to the beta-subunit of the voltage sensitive potassium channels. Chem. Biol. Interact.

130-132, 955–962

16.

Malup, T. K., Kobzev, V. F., Zhdanova, L. G., Slobodianiuk, Si., and Sviridov, S. M. (2000)

[Analysis of nucleotide sequences in a region of S100b protein gene in AKR/J, DBA/dJ and

BALB/cLac mice]. Mol. Biol. (Mosk). 34, 366–367

17.

18.

Ramana, K. V. (2011) Aldose reductase: new insights for an old enzyme. Biomol. Concepts 2,

103–114

Vikramadithyan, R. K., Hu, Y., Noh, H.-L., Liang, C.-P., Hallam, K., Tall, A. R., Ramasamy, R.,

and Goldberg, I. J. (2005) Human aldose reductase expression accelerates diabetic atherosclerosis

in transgenic mice. J. Clin. Invest. 115, 2434–2443

19.

20.

Gabbay, K. H. (2004) Aldose reductase inhibition in the treatment of diabetic neuropathy: where

are we in 2004? Curr. Diab. Rep. 4, 405–408

Dvornik, E., Simard-Duquesne, N., Krami, M., Sestanj, K., Gabbay, K. H., Kinoshita, J. H.,

Varma, S. D., and Merola, L. O. (1973) Polyol accumulation in galactosemic and diabetic rats:

control by an aldose reductase inhibitor. Science 182, 1146–1148

21.

22.

Kinoshita, J. H. (1990) A thirty year journey in the polyol pathway. Exp. Eye Res. 50, 567–573

Bril, V., and Buchanan, R. A. (2006) Long-term effects of ranirestat (AS-3201) on peripheral nerve

function in patients with diabetic sensorimotor polyneuropathy. Diabetes Care 29, 68–72

23.

Alexiou, P., Pegklidou, K., Chatzopoulou, M., Nicolaou, I., and Demopoulos, V. J. (2009) Aldose

reductase enzyme and its implication to major health problems of the 21(st) century. Curr. Med.

Chem. 16, 734–752

24.

25.

Srivastava, S., Spite, M., Trent, J. O., West, M. B., Ahmed, Y., and Bhatnagar, A. (2004) Aldose

reductase-catalyzed reduction of aldehyde phospholipids. J. Biol. Chem. 279, 53395–53406

Vander Jagt, D. L., Robinson, B., Taylor, K. K., and Hunsaker, L. A. (1992) Reduction of trioses

by NADPH-dependent aldo-keto reductases. Aldose reductase, methylglyoxal, and diabetic

complications. J. Biol. Chem. 267, 4364–4369

26.

Srivastava, S., Vladykovskaya, E., Barski, O. A., Spite, M., Kaiserova, K., Petrash, J. M., Chung,

S. S., Hunt, G., Dawn, B., and Bhatnagar, A. (2009) Aldose reductase protects against early

atherosclerotic lesion formation in apolipoprotein E-null mice. Circ. Res. 105, 793–802

-15-

Characterization of novel human AKR1B15

27.

28.

Shinmura, K., Bolli, R., Liu, S.-Q., Tang, X.-L., Kodani, E., Xuan, Y., Srivastava, S., and

Bhatnagar, A. (2002) Aldose reductase is an obligatory mediator of the late phase of ischemic

preconditioning. Circ. Res. 91, 240–246

Ramana, K. V, Chandra, D., Srivastava, S., Bhatnagar, A., Aggarwal, B. B., and Srivastava, S. K.

(2002) Aldose reductase mediates mitogenic signaling in vascular smooth muscle cells. J. Biol.

Chem. 277, 32063–32070

29.

30.

31.

Cao, D., Tat Fan, S., and Chung, S. S. M. (1998) Identification and characterization of a novel

human aldose reductase-like gene. J. Biol. Chem. 273, 11429–11435

Hyndman, D. J., and Flynn, T. G. (1998) Sequence and expression levels in human tissues of a new

member of the aldo-keto reductase family. Biochim. Biophys. Acta 1399, 198–202

Gallego, O., Belyaeva, O. V, Porté, S., Ruiz, F. X., Stetsenko, A. V, Shabrova, E. V, Kostereva, N.

V, Farrés, J., Parés, X., and Kedishvili, N. Y. (2006) Comparative functional analysis of human

medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases

with retinoids. Biochem. J. 399, 101–109

32.

33.

Yoshitake, H., Takahashi, M., Ishikawa, H., Nojima, M., Iwanari, H., Watanabe, A., Aburatani, H.,

Yoshida, K., Ishi, K., Takamori, K., Ogawa, H., Hamakubo, T., Kodama, T., and Araki, Y. (2007)

Aldo-keto reductase family 1, member B10 in uterine carcinomas: a potential risk factor of

recurrence after surgical therapy in cervical cancer. Int. J. Gynecol. Cancer 17, 1300–1306

Martin, H.-J., Breyer-Pfaff, U., Wsol, V., Venz, S., Block, S., and Maser, E. (2006) Purification

and characterization of AKR1B10 from human liver: role in carbonyl reduction of xenobiotics.

Drug Metab. Dispos. 34, 464–470

34.

35.

36.

Zhong, L., Shen, H., Huang, C., Jing, H., and Cao, D. (2011) AKR1B10 induces cell resistance to

daunorubicin and idarubicin by reducing C13 ketonic group. Toxicol. Appl. Pharmacol. 255, 40–47

Salabei, J. K., Li, X.-P., Petrash, J. M., Bhatnagar, A., and Barski, O. A. (2011) Functional

expression of novel human and murine AKR1B genes. Chem. Biol. Interact. 191, 177–184

Wabitsch, M., Brenner, R. E., Melzner, I., Braun, M., Möller, P., Heinze, E., Debatin, K. M., and

Hauner, H. (2001) Characterization of a human preadipocyte cell strain with high capacity for

adipose differentiation. Int. J. Obes. Relat. Metab. Disord. 25, 8–15

37.

38.

39.

Schmitt, S., Saathoff, F., Meissner, L., Schropp, E.-M., Lichtmannegger, J., Schulz, S., Eberhagen,

C., Borchard, S., Aichler, M., Adamski, J., Plesnila, N., Rothenfusser, S., Kroemer, G., and

Zischka, H. (2013) A semi-automated method for isolating functionally intact mitochondria from

cultured cells and tissue biopsies. Anal. Biochem. 443, 66–74

Mindnich, R., Haller, F., Halbach, F., Moeller, G., Hrabé de Angelis, M., and Adamski, J. (2005)

Androgen metabolism via 17beta-hydroxysteroid dehydrogenase type 3 in mammalian and non-

mammalian vertebrates: comparison of the human and the zebrafish enzyme. J. Mol. Endocrinol.

35, 305–316

Barski, O. A., Gabbay, K. H., Grimshaw, C. E., and Bohren, K. M. (1995) Mechanism of human

aldehyde reductase: characterization of the active site pocket. Biochemistry 34, 11264–11275

-16-

Characterization of novel human AKR1B15

40.

41.

42.

Bannai, H., Tamada, Y., Maruyama, O., Nakai, K., and Miyano, S. (2002) Extensive feature

detection of N-terminal protein sorting signals. Bioinformatics 18, 298–305

Barski, O. A., Mindnich, R., and Penning, T. M. (2013) Alternative splicing in the aldo-keto

reductase superfamily: implications for protein nomenclature. Chem. Biol. Interact. 202, 153–158

Wilson, D. K., Bohren, K. M., Gabbay, K. H., and Quiocho, F. A. (1992) An unlikely sugar

substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in

diabetic complications. Science 257, 81–84

43.

44.

Gallego, O., Ruiz, F. X., Ardèvol, A., Domínguez, M., Alvarez, R., de Lera, A. R., Rovira, C.,

Farrés, J., Fita, I., and Parés, X. (2007) Structural basis for the high all-trans-retinaldehyde

reductase activity of the tumor marker AKR1B10. Proc. Natl. Acad. Sci. 104, 20764–20769

McCormack, K., McCormack, T., Tanouye, M., Rudy, B., and Stühmer, W. (1995) Alternative

splicing of the human Shaker K+ channel beta 1 gene and functional expression of the beta 2 gene

product. FEBS Lett. 370, 32–36

45.

46.

Pongs, O., and Schwarz, J. R. (2010) Ancillary subunits associated with voltage-dependent K+

channels. 90, 755–796

Puranen, T. J., Poutanen, M. H., Peltoketo, H. E., Vihko, P. T., and Vihko, R. K. (1994) Site-

directed mutagenesis of the putative active site of human 17 beta-hydroxysteroid dehydrogenase

type 1. Biochem. J. 304, 289–293

47.

48.

49.

50.

51.

52.

Byrns, M. C., Jin, Y., and Penning, T. M. (2011) Inhibitors of type 5 17β-hydroxysteroid

dehydrogenase (AKR1C3): overview and structural insights. J. Steroid Biochem. Mol. Biol. 125,

95–104

Luu-The, V., Tremblay, P., and Labrie, F. (2006) Characterization of type 12 17beta-

hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydroxysteroid dehydrogenase

responsible for estradiol formation in women. Mol. Endocrinol. 20, 437–443

Endo, S., Matsunaga, T., Mamiya, H., Ohta, C., Soda, M., Kitade, Y., Tajima, K., Zhao, H.-T., El-

Kabbani, O., and Hara, A. (2009) Kinetic studies of AKR1B10, human aldose reductase-like

protein: endogenous substrates and inhibition by steroids. Arch. Biochem. Biophys. 487, 1–9

Keller, B., Meier, M., and Adamski, J. (2009) Comparison of predicted and experimental

subcellular localization of two putative rat steroid dehydrogenases from the short-chain

dehydrogenase/reductase protein superfamily. Mol. Cell. Endocrinol. 301, 43–46

Alías, M., Ayuso-Tejedor, S., Fernández-Recio, J., Cativiela, C., and Sancho, J. (2010) Helix

propensities of conformationally restricted amino acids. Non-natural substitutes for helix breaking

proline and helix forming alanine. Org. Biomol. Chem. 8, 788–792

Lam, C.-W., Yuen, Y.-P., Cheng, W.-F., Chan, Y.-W., and Tong, S.-F. (2006) Missense mutation

Leu72Pro located on the carboxyl terminal amphipathic helix of apolipoprotein C-II causes

familial chylomicronemia syndrome. Clin. Chim. Acta. 364, 256–259

-17-

Characterization of novel human AKR1B15

53.

54.

Keenan, C., Ghaffar, S., Grant, A. W., Hinshelwood, A., Li, D., McGarvie, G., and Ellis, E. M.

(2006) in Enzymology and Molecular Biology of Carbonyl Metabolism. Vol. 12. West Lafayette

(Weiner, H., Plapp, B., Lindahl, R., and Maser, E., eds.) pp. 388–395, Purdue University Press

Kelly, V. P., Sherratt, P. J., Crouch, D. H., and Hayes, J. D. (2002) Novel homodimeric and

heterodimeric rat gamma-hydroxybutyrate synthases that associate with the Golgi apparatus define

a distinct subclass of aldo-keto reductase 7 family proteins. Biochem. J. 366, 847–861

55.

56.

Mindnich, R., Möller, G., and Adamski, J. (2004) The role of 17 beta-hydroxysteroid

dehydrogenases. Mol. Cell. Endocrinol. 218, 7–20

Peltoketo, H., Luu-The, V., Simard, J., and Adamski, J. (1999) 17beta-hydroxysteroid

dehydrogenase (HSD)/17-ketosteroid reductase (KSR) family; nomenclature and main

characteristics of the 17HSD/KSR enzymes. J. Mol. Endocrinol. 23, 1–11

57.

58.

Shafqat, N., Marschall, H.-U., Filling, C., Nordling, E., Wu, X.-Q., Björk, L., Thyberg, J.,

Mårtensson, E., Salim, S., Jörnvall, H., and Oppermann, U. (2003) Expanded substrate screenings

of human and Drosophila type 10 17beta-hydroxysteroid dehydrogenases (HSDs) reveal multiple

specificities in bile acid and steroid hormone metabolism: characterization of multifunctional

3alpha/7alpha/7beta/17beta/20beta/21-H. Biochem. J. 376, 49–60

Clayton, P. T., Eaton, S., Aynsley-Green, A., Edginton, M., Hussain, K., Krywawych, S., Datta,

V., Malingré, H. E. M., Berger, R., and van den Berg, I. E. T. (2001) Hyperinsulinism in short-

chain L-3-hydroxyacyl-CoA dehydrogenase deficiency reveals the importance of beta-oxidation in

insulin secretion. J. Clin. Invest. 108, 457–465

59.

60.

Heslegrave, A. J., and Hussain, K. (2013) Novel insights into fatty acid oxidation, amino acid

metabolism, and insulin secretion from studying patients with loss of function mutations in 3-

hydroxyacyl-CoA dehydrogenase. J. Clin. Endocrinol. Metab. 98, 496–501

Schulz, N., Himmelbauer, H., Rath, M., van Weeghel, M., Houten, S., Kulik, W., Suhre, K.,

Scherneck, S., Vogel, H., Kluge, R., Wiedmer, P., Joost, H.-G., and Schürmann, A. (2011) Role of

medium- and short-chain L-3-hydroxyacyl-CoA dehydrogenase in the regulation of body weight

and thermogenesis. Endocrinology 152, 4641–4651

61.

62.

63.

Yang, S.-Y., He, X.-Y., and Schulz, H. (2005) 3-Hydroxyacyl-CoA dehydrogenase and short chain

3-hydroxyacyl-CoA dehydrogenase in human health and disease. FEBS J. 272, 4874–4883

Miller, W. L., and Auchus, R. J. (2011) The molecular biology, biochemistry, and physiology of

human steroidogenesis and its disorders. Endocr. Rev. 32, 81–151

Yang, S.-H., Liu, R., Perez, E. J., Wen, Y., Stevens, S. M., Valencia, T., Brun-Zinkernagel, A.-M.,

Prokai, L., Will, Y., Dykens, J., Koulen, P., and Simpkins, J. W. (2004) Mitochondrial localization

of estrogen receptor beta. Proc. Natl. Acad. Sci. U. S. A. 101, 4130–4135

64.

65.

Vasconsuelo, A., Milanesi, L., and Boland, R. (2013) Actions of 17β-estradiol and testosterone in

the mitochondria and their implications in aging. Ageing Res. Rev. 12, 907–917

Pedram, A., Razandi, M., Wallace, D. C., and Levin, E. R. (2006) Functional estrogen receptors in

the mitochondria of breast cancer cells. Mol. Biol. Cell 17, 2125–2137

-18-

Characterization of novel human AKR1B15

66.

67.

Demonacos, C., Djordjevic-Markovic, R., Tsawdaroglou, N., and Sekeris, C. E. (1995) The

mitochondrion as a primary site of action of glucocorticoids: the interaction of the glucocorticoid

receptor with mitochondrial DNA sequences showing partial similarity to the nuclear

glucocorticoid responsive elements. J. Steroid Biochem. Mol. Biol. 55, 43–55

Demonacos, C. V, Karayanni, N., Hatzoglou, E., Tsiriyiotis, C., Spandidos, D. A., and Sekeris, C.

E. (1996) Mitochondrial genes as sites of primary action of steroid hormones. Steroids 61, 226–

232

68.

69.

70.

Yashin, A. I., Wu, D., Arbeev, K. G., and Ukraintseva, S. V (2010) Joint influence of small-effect

genetic variants on human longevity. Aging (Albany. NY). 2, 612–620

Witkowski, A., Joshi, A. K., and Smith, S. (2007) Coupling of the de novo fatty acid biosynthesis

and lipoylation pathways in mammalian mitochondria. J. Biol. Chem. 282, 14178–14185

Hiltunen, J. K., Schonauer, M. S., Autio, K. J., Mittelmeier, T. M., Kastaniotis, A. J., and

Dieckmann, C. L. (2009) Mitochondrial fatty acid synthesis type II: more than just fatty acids. J.

Biol. Chem. 284, 9011–9015

71.

Parl, A., Mitchell, S. L., Clay, H. B., Reiss, S., Li, Z., and Murdock, D. G. (2013) The

mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-

mitochondrial cross talk through the PPAR system of transcriptional activation. Biochem. Biophys.

Res. Commun. 441, 418–424

72.

73.

Schulte, U. (2001) Biogenesis of respiratory complex I. J. Bioenerg. Biomembr. 33, 205–212

Calvo, S. E., Compton, A. G., Hershman, S. G., Lim, S. C., Lieber, D. S., Tucker, E. J., Laskowski,

A., Garone, C., Liu, S., Jaffe, D. B., Christodoulou, J., Fletcher, J. M., Bruno, D. L., Goldblatt, J.,

DiMauro, S., Thorburn, D. R., and Mootha, V. K. (2012) Molecular diagnosis of infantile

mitochondrial disease with targeted next-generation sequencing. Sci. Transl. Med. 4, 118ra10

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Characterization of novel human AKR1B15

Acknowledgments - The authors are grateful to Anna Rast, Aurelia Weber and Xiao-Ping Li for their

excellent technical assistance as well as Dr. Janina Tokarz for reading the manuscript. We also thank Dr.

Margarita Ivanova for initial immunocytochemistry work and helpful discussions as well as Sabine

Schmitt for her expertise in the enrichment of mitochondria.

FOOTNOTES

*This work was supported in part by National Institutes of Health Grants HL55477, HL59378, and

GM103492.

¹To whom correspondence may be addressed:

Oleg A. Barski, NIGMS, NIH, 45 Center Dr. Rm. 2AS55C, Bethesda, MD 20892, USA. Tel: (301)-496-

1511; Fax: (301)-480-2802; E-mail: oleg.barski@nih.gov (present address).

Disclaimer: This article was prepared while Oleg A. Barski was employed at the University of Louisville.

The opinions expressed in this article are the author's own and do not reflect the view of the National

Institutes of Health, the Department of Health and Human Services, or the United States government.

or

Jerzy Adamski , Helmholtz Zentrum Muenchen, German Research Center for Environmental Health,

Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstr. 1, 85764 Neuherberg,

Germany. Tel.: 049-89-3187-3155; Fax: 049-89-3187-3225; E-mail: Adamski@helmholtz-muenchen.de

²The abbreviations used are:

A-diol, 3α,17β-androstandiol; A-dione, androstanedione; AKR, aldo-keto reductase; AN, androsterone;

D4-AE, Δ4-androstenedione; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; E1, estrone;

E2, 17β-estradiol; ER, endoplasmic reticulum; FAS, fatty acid synthesis; HSD, hydroxysteroid

dehydrogenase; KP_i, potassium phosphate buffer; NaP_i, sodium phosphate buffer; RT, reverse

transcriptase; SD, standard deviation; SE, standard error; T, testosterone.

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Characterization of novel human AKR1B15

FIGURE LEGENDS

FIGURE 1: Comparison of AKR1B10 and AKR1B15 on gene, transcript, and protein level.

(A) Schematic illustrations of intron-exon structures of AKR1B10 and AKR1B15. Alternative use of exons

in the 5’-region of AKR1B15 generates two splice variants, referred to as AKR1B15.1 (Ensembl

transcript: AKR1B15-201) and AKR1B15.2 (Ensembl transcript: AKR1B15-001), which are translated into

the protein isoforms AKR1B15.1 and AKR1B15.2, respectively. Black straight lines depict introns, while

exons are shown as numbered rectangles. Translated exons of AKR1B10 are colored in green, alternatively

spliced exons in the 5’-region of AKR1B15 are colored orange (for AKR1B15.1) or yellow (for

AKR1B15.2) followed by orange-yellow-striped common exons. UTR’s are shown in white. Arrows

depict annealing sites for transcript specific primer pairs: AKR10-15.1-fwd (a), AKR15.2-fwd (b),

AKR15-rev (c), AKR10-rev (d). (B) Alignment of the protein sequences of AKR1B10, AKR1B15.1, and

AKR1B15.2. Alternative splicing of the 5’- region of AKR1B15 leads to a totally different and longer N-

terminus of AKR1B15.2 compared with that of AKR1B15.1. AKR1B15.1 and AKR1B10 possess high

homology in their N-termini. Amino acids of the catalytic tetrad are highlighted in bold, the serine at

position 8 of AKR1B15.1, which is mutated in a phenotype with a mitochondrial disease, is colored in

blue, and the proline at position 24 of AKR1B10, which is responsible for its cytosolic localization, is

colored in green. The recognition region for the monoclonal rat-anti-AKR1B15 antibody is highlighted by

a green box; the C-terminal recognition region for the polyclonal rabbit-anti-AKR1B15 antibody is

highlighted by a red box.

FIGURE 2: Expression of AKR1B10, AKR1B15.1, and AKR1B15.2 in tissues and cell lines.

(A) Semi-quantitative end-point RT-PCR with cDNA from tissues and cell lines shows different

expression patterns for AKR1B15 and AKR1B10. GAPDH as well as reactions without reverse

transcriptase (no RT) or cDNA (H₂O) serve as controls. (B) Quantitative real-time PCR with cDNA of

selected tissues validate differences in expression levels of AKR1B15.1, AKR1B15.2, and AKR1B10.

Expression levels of AKR1B15.1 (black bars), AKR1B15.2 (gray bars), and AKR1B10 (open bars)

transcripts are shown on a logarithmic scale and are normalized to the expression of AKR1B15.1 in

placenta after correction of the C_Tvalues by the average of the three housekeeping genes: GAPDH,

HPRT, and 18S RNA; error bars represent standard deviations from two independent cDNA sets.

FIGURE 3: Expression and purification of AKR1B10, AKR1B15.1, and AKR1B15.2 in E. coli BL21

(DE3). Protein bands in Coomassie stained SDS-polyacrylamide gels are shown.

(A) Lysates of IPTG-induced (I) but not uninduced (U) cell pellets show clear bands of AKR1B10,

AKR1B15.1, and AKR1B15.2, respectively. (B) After centrifugation of induced cell lysates both

AKR1B15 isoforms show up as insoluble proteins in the pellet fraction (P), while AKR1B10 is soluble

and therefore present in the supernatant (SN). (C) Purification of AKR1B15.1 and AKR1B15.2 leads to

sufficiently pure protein, containing only minute amounts of degradation or truncation products.

FIGURE 4: Binding of dinucleotide cofactors to AKR1B15.1.

(A) NADPH (open circles) -, or NADP⁺(filled circles) - dependent decline in the fluorescence of

AKR1B15.1 measured in 20 mM KP_ibuffer pH 7.4 using an excitation wavelength of 280 nm and

emission wavelength of 340 nm. Data are shown as discrete points and curves are best fits of equation [1]

to the data. Changes in fluorescence were normalized to the value of the initial fluorescence of the protein;

data are presented as mean ± SD of two independent measurements. (B) Parameters gained from cofactor

titration studies. K_dand maximum quenching (ΔF_max) were calculated from the fluorescence curves by

fitting equation [1] to the data.

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Characterization of novel human AKR1B15

FIGURE 5: AKR1B15.1 catalyzes redox reactions with steroids.

(A) AKR1B15.1 exhibits strong preference for phosphorylated cofactor by catalyzing redox reactions only

in the presence of NADP(H) but not NAD(H). Activity tests were carried out using 10⁶HEK293 cells

either non-transfected (HEK293) or transiently transfected with pIRES-hrGFP-1α-AKR1B15.1

3

(HEK293+AKR1B15.1), 15 nM H-labeled estrone or 10 nM H-labeled 17β-estradiol, and 350 µM

cofactor in reaction buffer. Bars represent the mean of steroid conversion in % after 60 min (for estrone)

and 120 min (for 17β-estradiol) incubation; error bars show standard deviations of three replicates. (B)

AKR1B15.1 possesses activity on the C17 β-position of the steroid nucleus (C17) but not on the C3

position (C3). Activity tests were carried out using 10⁶HEK293 cells transiently transfected with pIRES-

3

hrGFP-1α-AKR1B15.1, 10 to 40 nM H-labeled steroids, and 350 µM NADPH (+ NADPH) or NADP⁺

(+ NADP⁺) cofactor in reaction buffer. Bars represent conversion in %. (C) Comparison of reaction

velocities with different steroids. Activity tests were carried out using 90 nM purified AKR1B15.1, 20 nM

(corresponding to 10 pmol per reaction) ³H-labeled steroids, and 300-325 µM cofactors in reaction buffer.

Results of reductive reactions using NADPH are represented by open symbols, those of oxidative

reactions using NADP⁺by filled symbols. Data represent mean ± SD (n=3).

FIGURE 6: Generation of specific polyclonal and monoclonal anti-AKR1B15 antibodies and detection of

endogenous AKR1B15 isoforms.

(A) Different His-tagged human AKRs (AKR6A3, AKR1B1, AKR1B10, AKR1B15.1, AKR1B15.2, and

AKR1A1) were expressed in E. coli BL21 (DE3) and analyzed by Western blot using polyclonal rabbit-

anti-AKR1B15, monoclonal rat-anti-AKR1B15 (9A5) or polyclonal rabbit-anti-His-tag (Cell Signaling)

antibodies as primary antibodies and HRP-conjugated goat-anti-rabbit (Life Technologies) and mouse-

anti-rat-IgG2A (in-house production) secondary antibodies. Non-transformed E. coli BL21 (DE3) served

as negative control. The anti-His-tag staining showed that all proteins were expressed (lower panel). (B)

Untagged AKR1B15.1 and AKR1B15.2 were overexpressed in HEK293 cells and analyzed by Western

blot using polyclonal rabbit-anti-AKR1B15 and monoclonal rat-anti-AKR1B15 (9A5) antibodies as

primary antibodies and goat-anti-rabbit (Life Technologies) and mouse-anti-rat-IgG2A (in-house

production) antibodies as HRP-conjugated secondary antibodies, respectively. Non-transfected HEK293

cells served as negative control. While the polyclonal antibody is fairly unspecific, as it recognizes several

proteins, the monoclonal antibody shows high specificity to both AKR1B15 isoforms with no cross-

reactivity. (C) Fractions (800xg and 9000xg pellets) of the enrichment of mitochondria from BeWo cells

were analyzed for the presence of endogenous AKR1B15 isoforms by Western blot using monoclonal rat-

anti-AKR1B15 (9A5) antibody as primary and IR-dye conjugated goat-anti-rat-AlexaFluor 790 (Dianova)

antibody as secondary. A mixture of extracts from HEK293 cells overexpressing untagged AKR1B15.1 or

AKR1B15.2 served as a positive control and non-transfected HEK293 cells as well as a secondary

antibody only hybridization (left panel) served as negative controls. Endogenous AKR1B15.1 is marked

by an asterisk and AKR1B15.2 by an arrowhead.

FIGURE 7: Subcellular distribution of the two AKR1B15 isoforms.

HeLa cells were transiently transfected with either N-myc-pcDNA3-AKR1B15.2 (a), N-myc-pcDNA3-

AKR1B15.1 (b), pcDNA4-myc/His B-AKR1B15.2 (c), pcDNA4-myc/His B-AKR1B15.1 (d),

pcDNA3.1(+)-AKR1B15.2 (e, g), or pcDNA3.1(+)-AKR1B15.1 (f, h) in order to overexpress N-

terminally myc-tagged (a, b), C-terminally myc-tagged (c, d), or untagged (e-h) protein. Nuclei were

stained using Hoechst 33342 dye (A); mitochondria were stained using Mito-Tracker Orange CMTM-Ros

(B); myc-tagged AKR1B15.1 or AKR1B15.2 were stained using mouse-anti-myc / goat-anti-mouse-

AlexaFluor 488 antibodies and untagged AKR1B15.1 was stained using polyclonal rabbit-anti-

AKR1B15 / goat-anti-rabbit-AlexaFluor 488 antibodies or monoclonal rat-anti-AKR1B15 / goat-anti-rat-

AlexaFluor 488 antibodies (C). The individual staining as well as the overlays (D) demonstrate that

AKR1B15.2 is a cytoplasmic protein, while C-terminally myc-tagged AKR1B15.1 as well as untagged

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Characterization of novel human AKR1B15

AKR1B15.1 co-localize with mitochondria. Nuclei are shown in blue, mitochondria in red, AKR1B15 in

green, and co-localization in yellow.

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Characterization of novel human AKR1B15

TABLE 1:

Primer names and sequences used for cloning and semi-quantitative RT-PCR (RT) or qPCR (Q).

Restriction sites are underlined and coding sequences are shown in capital letters.

primers for cloning into pET28a(+):

AKR1B15.1-NdeI_fwd:

AKR1B15.2-NdeI_fwd:

AKR1B15-XhoI_rev (1)

5’-cccttccatATGGCCACGTTTGTGGAGCT-3’

5’-tcaagctacatATGGTCTTACAAATGGAA-3’

5’-agatccctcgagTCAATATTCTGCATCGAA-3’

primers for cloning into pcDNA3.1(+)

AKR1B15.1-NotI_fwd (1):

AKR1B15.2-NotI_fwd (1):

AKR1B15-XhoI_rev (1):

5’-aagaagcggccgcaccATGGCCACGTTTGTGGAGCT-3’

5’-aagaagcggccgcaccATGGTCTTACAAATGGA-3’

5’-agatccctcgagTCAATATTCTGCATCGAA-3’

primers for cloning into N-myc-pcDNA3

AKR1B15.1-NotI_fwd (2):

AKR1B15.2-NotI_fwd (2):

5’-ttttgcggccgcaaGCCACGTTTGTGGAGCTC-3’

5’-ttttgcggccgcaaGTCTTACAAATGGAACCCCA-3’

5’-tttctcgagTCAATATTCTGCATCGAAGGGAAAGT-3’

AKR1B15-XhoI_rev (2):

primers for cloning into pcDNA4-myc/HisB

AKR1B15.1-HindIII_fwd:

AKR1B15.2-HindIII_fwd:

AKR1B15-NotI_rev:

5’-ttttaagcttATGGCCACGTTTGTGGAGCTC-3’

5’-ttttaagcttATGGTCTTACAAATGGAACCCCA-3’

5’-ttttgcggccgctATATTCTGCATCGAAGGGAAAGT-3’

primers for cloning into pIRES-hrGFP-1α

pET28-AKR1B15.1-NotI_fwd:

5’-aagaagcggccgcaccATGGGCAGCAGCCAT-3’

5’-aaatggcggccgcaccatgggcagcagccatcatcatcatcatcacagcagcggcctggtgc-

-cggcggcagccatATGGTCTTACAAATGGAACCCCAAGTGAACTCA-3’

5’-agatccctcgagTCAATATTCTGCATCGAA-3’

AKR1B15.2-NotI-His_fwd:

AKR1B15-XhoI_rev (1):

primers for RT-PCR (RT) and qPCR (Q)

AKR10-15.1_fwd:

AKR15.2_fwd:

AKR10_rev:

5’-CCACGTTTGTGGAGCTCAGT-3’

5’-CCCTTTGACTGGCCTAAAGA-3’

5’-AACGTTGCTTTTCCACCGATGGC-3’

5’-AACGTTCCTTTTCCACTGATCAT-3’

5’-AGTCAACGGATTTGGTCGTA-3’

(RT, Q)

(RT)

(Q)

AKR15_rev:

GAPDH_fwd (1):

GAPDH_rev (1):

GAPDH_fwd (2):

GAPDH_rev (2):

HPRT1:

5’-ATGACAAGCTTCCCGTTCT-3’

5’-GGTGGTCTCCTCTGACTTCAACA-3’

5’-GTTGCTGTAGCCAAATTCGTTGT-3’

RT²qPCR Primer Assay for Human HPRT1 (SABiosciences)

18S-RNA:

RT²qPCR Primer Assay for Human 18SrRNA (SABiosciences) (Q)

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Characterization of novel human AKR1B15

TABLE 2: Kinetic parameters of AKR1B15.1.

Kinetic parameters of AKR1B15.1 were determined with purified enzyme and cofactor NADPH (300 µM)

for reductive reactions or NADP⁺(325 µM) for oxidative reactions. K_Mand k_catvalues were calculated by

Michaelis-Menten fit (SigmaPlot) of initial reaction velocities measured with increasing concentrations of

either unlabeled steroids, spiked with 10 pmol ³H-labeled steroids, or unlabeled acetoacetyl-CoA.

K_M, Michaelis constant; k_cat, turnover number; k_cat/ K_M, catalytic efficiency. Values are mean ± SE (n=3).

K_M

[µM]

k_cat

k_cat/ K_M

substrate

[min^-1]

[µM^-1min^-1]

androsterone

∆4-androstenedione

estrone

2.8 ± 0.2

1.9 ± 0.2

2.5 ± 0.4

63.4 ± 7.4

19.2 ± 2.3

7.1 ± 1.5

9.1 ± 1.2

1.7 ± 0.1

1.1 ± 0.1

1.0 ± 0.1

0.5 ± 0.1

3.0 ± 0.3

0.6 ± 0.1

0.5 ± 0.1

0.61

0.60

0.38

0.01

0.16

0.09

0.06

acetoacetyl-CoA

3α,17β-androstandiol

testosterone

17β-estradiol

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Characterization of novel human AKR1B15

Figure 1:

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Characterization of novel human AKR1B15

Figure 2:

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Characterization of novel human AKR1B15

Figure 3:

-28-

Characterization of novel human AKR1B15

Figure 4:

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Characterization of novel human AKR1B15

Figure 5:

-30-

Article Doi

Aldo-keto Reductase 1B15 (AKR1B15): A mitochondrial human aldo-keto reductase with activity toward steroids and 3-keto-acyl-CoA conjugates

DOI: 10.1074/jbc.M114.610121

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.M114.610121

Full text of DOI:10.1074/jbc.M114.610121

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