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UGT1A10 is a high activity and important extrahepatic enzyme – why has its role in intestinal glucuronidation been frequently underestimated? Johanna Troberg, Erkka Järvinen, Guang-Bo Ge, Ling Yang, and Moshe Finel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00852 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 7, 2016

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Molecular Pharmaceutics

Title page

UGT1A10 is a high activity and important extrahepatic enzyme – why has its role in intestinal glucuronidation been frequently underestimated?

Johanna Troberg, Erkka Järvinen, Guang-Bo Ge, Ling Yang and Moshe Finel*

Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Finland (J.T., E.J., M.F.) and Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China (G-B.G., Y.L.).

*Dr. Moshe Finel, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Finland. P.O. Box 56 (Viikinkaari 5), 00014 University of Helsinki, Finland. Telephone: +358 504480748, Fax: +358 9 191 59556, E-mail: [email protected].

1 Environment ACS Paragon Plus


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Abstract The aim of this work was to highlight a considerable and broad problem in UGT1A10 activity assessment that lead to underestimation of its role in intestinal glucuronidation of drugs and other xenobiotics. The reason appeared to be poor activity of the commercial UGT1A10 that is used by many laboratories and here we have tested it by comparison with our recombinant His-tagged UGT1A10 (marked as UGT1A10-H), both expressed in insect cells. The glucuronidation rates of morphine, estradiol, estrone, SN-38, diclofenac, 4-methylumbelliferon,

7-amino-4-methylcoumarin,

N-(3-carboxy

propyl)-4-hydroxy-1,8-

naphthalimide and bavachinin was assayed. The results revealed that the activity of commercial UGT1A10 was low, very low, and in the cases of morphine, estrone, 7-methyl-4aminocoumarin and bavachinin it was below detection limit. On the other hand, under the same conditions, UGT1A10-H exhibited high glucuronidation rates toward all these compounds. Moreover, using estradiol, morphine and estrone, in the presence and absence of suitable inhibitors, nilotinib or atractylenolide I, it was demonstrated that UGT1A10-H, but not the commercial UGT1A10, provides a good tool to study the role of native UGT1A10 in the human intestine. The results also suggest that much of the data in the literature on UGT1A10 activity may have to be re-evaluated.

Keywords UDP-Glucuronosyltransferase, recombinant UGT1A10, intestine microsomes, intestinal glucuronidation, estradiol, estrone, morphine, SN-38, AMC, NCHN, bavachinin

Abbreviations 4-MU, 7-hydroxy-4-methylcoumarin, also called 4-methylumbelliferone; AMC, 7-amino-4methylcoumarin; DMSO, dimethylsulfoxide; HIM, human intestinal microsomes; HLM,


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human liver microsomes; NCHN, N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide; qRTPCR, quantitative real time-PCR; SN-38, 7-ethyl-10-hydroxycamptothecin; UDPGA, UDP-αD-glucuronic

acid; UGT, UDP-glucuronosyltransferase.

Introduction

human UGT. A similar situation is present

UDP-glucuronosyltransferases (UGTs) are

with inhibitors, perhaps with the exception

membrane enzymes of the endoplasmic

of the UGT1A4 inhibitor hecogenin.3 The

reticulum that play important roles in

availability

conjugation and detoxification of various

individual UGTs is also scarce and only a

xenobiotics

UGTs

monoclonal antibody against UGT1A9 was

catalyze glucuronic acid transfer from

reported.4 Thus, it is difficult to validate

UDP-glucuronic acid (UDPGA) to small

the variable, even contradicting, tissue

compounds that carry a nucleophilic

expression levels of the UGTs that were

group(s), particularly hydroxyl, different

published over the years, except that it

amines or carboxylic group.1,2

became clear that several UGTs are not

and

endobiotics.

of

specific

antibodies

to

expressed in the liver. There are 19 different human UGTs in subfamilies 1A, 2A and 2B that use

The small intestine is the first barrier to

UDPGA as their cofactor. Each UGT can

many drugs and drug-like compounds, but

commonly conjugate several different

it has been unclear which of the UGTs are

substrates and there is a rather broad, even

expressed

if partial, overlap in substrate specificity

significant levels. The highly cited review

among the human UGTs. Consequently,

on UGTs5 listed the extrahepatic UGT1A8

there are very few compounds that are

as a small intestine enzyme with high

fully specific substrate for only a single

expression level. The general acceptance

in


the

small

intestine

to


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of that remained deep in people’s mind and

that only four UGTs, namely UGT1A1,

even received support from the first

UGT1A10, UGT2B7 and UGT2B17, are

proteomics results on intestinal UGTs

expressed in the human small intestine to

expression.6

significant levels (at least in the majority of Caucasians11).

However, contrary to that older but still common

view

expression

about of

intestinal

UGT1A8,

Individual UGTs should be expressed in a

two

suitable recombinant system in order to

time-PCR

characterize their substrate specificity.

(qRT-PCR) studies found just low, or very

Some laboratories use mammalian cell

low, mRNA levels of UGT1A8 in the

lines, often HEK293 cells, while others,

small intestine and at least ten fold higher,

among which the commercial suppliers of

if not significantly more, of UGT1A10.7,8

recombinant UGTs employ insect cells.

Moreover, the laboratory that published the

We

first proteomics study on UGTs, published

baculovirus-infected insect cells, with the

another one the following year in which

small difference that our enzymes mostly

the

intestinal

carry a short C-terminal peptide ending

microsomes were very different from their

with 6 His residues (His-tag).12,13 We

earlier one and UGT1A8 was not among

cloned

the detected enzymes.9 Another proteomics

UGT1A10 that carry such C-terminal tags

study was published shortly afterwards by

in our Helsinki laboratory and both

a different laboratory, presented further

recombinant

details on UGTs expression in the human

activity toward test phenolic compounds.14

liver and intestine.10 The results in both

In a study on the glucuronidation of 17α-

latter proteomics studies, as well as in the

estradiol and 17β-estradiol, in which only

two qRT-PCR studies, strongly suggest

our

extensive

level

the

quantitative

results

for

real

human

are

also

and

expressed

enzymes

His-tagged


expressing

UGTs

UGT1A8

displayed

enzymes

were

in

and

good

used,

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UGT1A10 (in the current paper the same

terminal fusion peptide, ending with a His-

enzyme is termed UGT1A10-H) exhibited

tag, and the commercial counterpart, as

very high glucuronidation rates toward the

well as examine which of them better

3-OH of estradiol, by far the highest rates

represents the native human UGT1A10 in

of the other tested UGTs, including

the small intestine microsomes.

UGT1A1.15 These results were in line with the high activity rates that were previously

Materials and Methods

reported for the human UGT1A10, when it

Materials

was expressed in mammalian cell lines in different

laboratories

(e.g.

refs.

16-19

).

Nevertheless, the results that UGT1A10-H catalyzes estradiol glucuronidation at high rates also raised questions regarding the activity of the commercial UGT1A10, since its estradiol glucuronidation rate was reported to be low in another study.

7-ethyl-10-hydroxycamptothecin (SN-38) was purchased from Shanghai Boylechem Co., Ltd (Shanghai, China), nilotinib from Alfa

Aesar

(Shanghai,

China)

and

bavachinin was from Chengdu Pufei De Biotech Co., Ltd. (Chengdu, Sichuan, China). N-(3-carboxy propyl)-4-hydroxy-

20

1,8-naphthalimide

(NCHN)

Additional evidence that something may

glucuronide

synthesized

be wrong with the activity of commercial

Chinese Academy of Sciences (Dalian,

UGT1A10 came from our relative activity

China) as previously described.22 17-β-

factor

magnolol

estradiol and its 3-glucuronide, estrone, 7-

that

amino-4-methylcoumarin

study

glucuronidation.21

on Following

we

were

and

its

in

the

(AMC),

started to pay more attention to this topic

atractylenolide I, 4-methylumbelliferone

and, eventually, decided to carry out a

(4-MU) and its glucuronide, morphine-6-

side-by-side

glucuronide,

comparison

between

our

recombinant, UGT1A10-H, that carry a C-

diclofenac,

UDP-α-D-

glucuronic acid (UDPGA, ammonium



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salt),

sodium

monobasic

Lakes, NJ). Our recombinant UGTs were

hydrogen

expressed in insect cells, as previously

phosphate were from Sigma-Aldrich (St.

described.12,14,23 The preparation is not

Louis, MO). Estrone 3-glucuronide was

identical to human tissue microsomes and

bought

we define it as membranes enriched with

dehydrate

phosphate

Page 6 of 29

and

from

disodium

TRC

(Toronto,

ON).

Morphine dihydrochloride was purchased

recombinant

from

membranes.

Yliopiston

Apteekki

(Helsinki,

UGT,

or

UGT-enriched

Finland). Magnesium chloride hexahydrate Glucuronidation

assays

and

HPLC

and perchloric acid were obtained from methods Merck (Darmstadt, Germany), formic acid from Riedel-deHaën (Seelze, Germany)

Substrates

were

and alamethicin was purchased from A.G.

methanol

Scientific (San Diego, CA). Acetic acid,

evaporated from the assay tubes prior to

dimethylsulfoxide (DMSO), acetonitrile

the addition of reaction mixture, whereas

and methanol were all HPLC grade.

the levels of DMSO were decreased upon

or

dissolved

DMSO.

either

Methanol

in was

dilution to the final assay concentration. Commercial UGTs UGT1A8, UGT1A10, SN-38 was pretreated in DMSO:0.05N UGT2B7,

UGT1A1,

UGT1A9

and

NaOH (50:50) as described previously.24

UGT2B15 (“Supersomes”) were purchased The reaction mixture contained 50 mM from Corning Life Sciences (Corning, NY) phosphate buffer (pH 7.4, with the and from BD Biosciences (Franklin Lakes, exception of pH 6.0 for the diclofenac NJ) the former retailer of “Supersomes”.

assay, see Zhang et al.25), 10 mM MgCl2

Pooled human liver microsomes (HLM, lot and 2-20 µg insect cell membranes no.

18888)

and

human

intestine enriched with recombinant UGT from our

microsomes (HIM, lot no. 3189756) were Helsinki laboratory, commercial UGT bought from BD Biosciences (Franklin


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(“Supersomes”),

cell

10%. In estradiol kinetic assays the

membranes or 5-40 µg HLM or HIM

substrate consumption was kept below

(Table S1, see Supporting Information).

25%, with the exception of the lowest

The

first

substrate concentration point (0.5 µM)

(in the

where estradiol consumption was 44%

reactions

control

insect

mixtures

were

preincubated on ice for 15 min

cases of human microsomes they also included 50 µg alamethicin per mg microsomal protein26), then moved to 37 °C for 5 min before starting reactions by the addition of UDPGA to a final concentration of 5 mM. Reaction tubes were incubated at 37 °C for a set time, between 5 and 60 min. The reactions were terminated by the addition of either methanol, 5% acetic acid in methanol or with 4 M perchloric acid (see Table S1 in

with our UGT1A10-H. Glucuronides and parent compounds were analyzed using an Agilent 1100, equipped with

diode

array

and

fluorescence

detectors (Agilent Technologies, Palo Alto, CA). The column, Poroshell 120 EC-C18 (4.6

x

100

mm,

2.7

µm;

Agilent

Technologies, Palo Alto, CA) was set at temperature 40 °C. The HPLC methods are described

in

Table

S2

(Supporting

Information) and the injection volumes (5

the Supporting Information for detailed –80 µl) were adjusted based on the incubation

times

and

termination sensitivity of the detection in each case. To

methods).

Terminated

samples

were estimate

the

amount

of

formed

centrifuged at 16 000 x g for 10 min and glucuronides

when

no

authentic

the supernatants were subjected to HPLC glucuronide was available for making a analysis.

Enzyme

concentrations

and standard curve, it was made using the

incubation times were selected from the parent compound, based on the assumption linear range of product formation, while that parent compound and its glucuronide keeping the substrate consumption below have closely similar UV absorbance



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Page 8 of 29

maxima27. All the assays were done in

Biotechnology,

triplicates. In addition, two control assays

protein amounts, 10 µg, of the commercial

were carried out, one without UDPGA and

and our UGT1A10 were loaded onto 4-

one without substrate. Screening assays of

15% gradient SDS-PAGE, together with

different UGTs also included a control of

protein standard markers (Kaleidoscope,

insect cells infected with baculovirus does

BioRad Laboratories, Hercules, CA). The

not express any recombinant UGT.

resolved

Rockford,

proteins

were

IL).

Equal

subsequently

immunostained using anti-UGT1A mouse

Data analysis

monoclonal antibody sc-271268 (1:1000) The mean velocities and standard errors of

from Santa Cruz Biotech (Santa Cruz, CA)

triplicate glucuronidation samples of the

as a first antibody and their HRP-

estrogens kinetics were analyzed using

conjugated goat anti-mouse IgG sc-2005

GraphPad Prism version 5.04 for Windows

(1:2500) as a secondary antibody (Santa

(GraphPad Software Inc., San Diego, CA).

Cruz, CA).

Protein concentration determination and Western blot Protein concentrations were determined using the BCA protein assay (Pierce


Page 9 of 29

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Morphine

examine which of the two preparations is

SN-38

more suitable for studying UGT1A10 substrates glucuronidation in the small intestine (see Figure 1 for the structure of 4-MU

AMC

the

compounds

used

as

UGT1A10

substrates in this study and the site of conjugation in them). The two preparations NCHN

Diclofenac were

first

compared

for

the

glucuronidation of morphine (at the 3-OH), SN-38 (the active metabolite of irinotecan) and diclofenac (at pH 6.0), as well as 4Estradiol

Estrone MU, an extensively employed substrate for most UGTs. The commercial UGT2B7 and UGT1A1 were added as references to the

Bavachinin

morphine and SN-38 assays, respectively, and UGT1A8 was added to the 4-MU Figure 1. Structures of the 9 compounds used as glucuronidation substrates in this study. The arrows

assay. The results of these comparisons are shown in Figure 2 and all its four parts

indicate the glucuronidation site by UGT1A10 in

reveal

each compound.

large

differences

in

glucuronidation rates between UGT1A10-

Results

H The commercial recombinant UGT1A10 is commonly used, but we suspected that there is a problem with its activity. Therefore, we set out to test it against the UGT1A10-H

very

we

are

preparing

and

the

commercial

UGT1A10.

Moreover, under these assay conditions and HPLC analysis (Tables S1 and S2 of the Supporting Information) no morphine glucuronidation activity by the commercial

and



UGT1A10 was detected and its diclofenac

UGT1A10 raise the question which of

glucuronidation activity was barely above

them better represents the native intestinal

detection limit (Figure 2).

UGT1A10? In order to explore if our UGT1A10-H provides a good model for

A. SN-38

B. Diclofenac

Rate (nmol/min/mg)

0.06

studying the activity of native UGT1A10

3

in the small intestine, we took advantage of 0.04

2

0.02

1

new inhibitors that became available in recent

years.

Morphine-3-glucuronide

formation rate by HLM and HIM, in the presence and absence of the rather specific

0.20

1A 1A 10 10 -H

1A 1 1A 1 1A 0 10 -H

0.00

Rate (nmol/min/mg)

C. Morphine

0.15

0.10

D. 4-MU

UGT2B7 inhibitor, with respect to UGTs of subfamily 1A, atractylenolide I28, is

9

presented in Figure 3. While UGT2B7 is

5

expressed both in HLM and in HIM,

1 0.5

UGT1A10 is only expressed in HIM10. Therefore, a significantly higher inhibition

0.05

of morphine-3-glucuronide formation by 1A 8 1A 1 1A 0 10 -H

0.00 2B 7 1A 1 1A 0 10 -H

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Page 10 of 29

Figure 2. Glucuronidation rates of SN-38 (A, substrate concentration 5 µM), Diclofenac (B, 100

atractylenolide I is expected in HLM than in HIM, as indeed was confirmed by the results (Figure 3).

µM), Morphine (C, 100 µM) and 4-MU (D, 50 µM) by the commercial UGT1A10 and UGT1A10-H. Commercial UGT1A1, UGT2B7 and UGT1A8 were added as references to assays A, C and D, respectively. See Methods for further details.

The very large rate differences between the two different preparations of recombinant


Page 11 of 29

Morphine ± atractylenolide I 0.25

absence and presence of nilotinib, a rather specific inhibitor of UGT1A1.30 The

0.20

results

clearly

demonstrate

that

the

0.10

is much higher than in HLM. Furthermore,

0.05

in

0.00

glucuronide formation in HIM is almost IM H

1A 10 -

H LM

estradiol-3-glucuronide formation in HIM

H

0.15

2B 7

Rate (nmol/min/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Morphine glucuronidation rates by

UGT2B7, UGT1A10-H, HLM and HIM, in the absence (gray columns) and presence (dark columns) of atractylenolide I. The morphine and

contrary

insensitive

to

to

HLM,

inhibition

estradiol-3-

by

nilotinib

(Figure 4A). Similarly to HIM, this activity in UGT1A10-H is very high and nearly insensitive

to

nilotinib,

whereas

the

atractylenolide I concentrations were 100 µM and

estradiol glucuronidation activity of the

50 µM, respectively.

commercial UGT1A10 is much lower than Estradiol 3-glucuronide formation is often

the activity of UGT1A1 (uninhibited), and

considered to be a typical activity of

commercial recombinant UGT1A8 that is

UGT1A1. In HIM, however, the situation

also included as an additional reference

may be very different since both UGT1A1

(Figure 4A).

and UGT1A10 are expressed in this tissue and, according to our previous results, UGT1A10 catalyzes this reaction at much higher rate than UGT1A1.15,

29

In Figure

4A the comparison between the two UGT1A10 preparations is combined with examination of estradiol glucuronidation rates (at the 3-OH) in HLM and HIM. Moreover, each sample was analyzed in 11 Environment ACS Paragon Plus


considerable rates by the recombinant

A. Estradiol ± nilotinib Rate (nmol/min/mg)

16 13 10 7 4 1

(extrahepatic) UGT1A10-H of the Helsinki laboratory, even if only traces of activity were detected when it was first assayed

0.50

using the commercial UGT1A10.22 In

0.25

analogy with the estradiol glucuronidation 8

1

1A

1A

10 1A

10 -H 1A

H

HL M

IM

0.00

experiment, we have tested the effects of nilotinib,

B. NCNH ± nilotinib

6 Rate (nmol/mg/min)

the

UGT1A1

inhibitor,

on

NCHN glucuronidation in HLM and HIM, 4

as well as its effects on recombinant UGT1A1

2

and

UGT1A10

catalyzing

NCHN glucuronidation (Figure 4B). The 0 H 1A 10 -

H IM

H LM

1A 10

difference in the inhibitor’s effect is not as 1A 1

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Page 12 of 29

strong in the case of NCHN as it was with

Figure 4. A. Analysis of estradiol-3-glucuronide formation in the absence (gray columns) and presence (dark columns) of nilotinib, by UGT1A1,

estradiol

and

it

inhibits

NCHN

glucuronidation by UGT1A10 to a higher

the commercial UGT1A10, our UGT1A10-H, HLM and HIM. The estradiol and nilotinib concentrations

degree when the substrate was estradiol

were 50 µM and 5 µM, respectively. B. Similar

(Figure 4A). In addition, the contribution

analysis as in part A, except that NCHN and nilotinib concentrations were 350 µM and 10 µM, respectively.

of UGT1A1 to NCHN glucuronidation in HIM is most probably larger than in the case of estradiol, as could be inferred from

Figure

4B

presents

results

for

the

comparing the activities of recombinant

fluorescent compound NCHN that we

UGT1A1 and UGT1A10-H toward these

recently reported to be a UGT1A1-specific

substrates

substrate in HLM.22 Later, however, we

Nevertheless, the difference in NCHN

noticed that it could be glucuronidated at 12 Environment ACS Paragon Plus

(Figures

4A

and

4B).

glucuronidation

rate

between

the

commercial UGT1A10 to UGT1A10-H is very

large,

similarly

to

the

other

A. HLM

0.5

no inhibitor Nilotinib

0.4 0.3 0.2

experiments described above and as in the case of morphine (Figure 3), the sensitivity

0.1 0.0 0

inhibitor is significantly lower than the sensitivity of the corresponding activity in HLM.

Taken

together,

the

Rate (nmol/min/mg)

of NCHN glucuronidation by HIM to the

10

20

30

40

50

20

30

40

50

40

50

B. HIM

3

2

1

results 0 0

presented in Figures 2 and 4 show how poor the activity of the commercial recombinant UGT1A10 is, while the results in Figures 3 and 4 demonstrate that

10

C. UGT1A10-H

25 Rate (nmol/min/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Rate (nmol/min/mg)

Page 13 of 29

20 15 10

the native UGT1A10 in HIM has high

5 0 0

10

activity.

20 30 [estradiol], µM

Figure 5. Kinetic analyses of estradiol-3The high estradiol glucuronidation activity

glucuronide formation by HLM (A), HIM (B) and UGT1A10-H (C), in the absence (closed circles)

in HIM and UGT1A10-H was employed to

and presence (open squares) of 5 µM nilotinib.

compare the kinetics of our recombinant

It is clear from these results that there are

enzyme to that of the native enzyme.

nearly identical kinetic curve (Figure 5)

Kinetics analyses of HLM, HIM and

and Km value (Table 1) for our UGT1A10-

1A10-H were carried out, in the presence

H and the native enzyme in HIM, but not

and absence of nilotinib. The results are

the hepatic UGTs UGT1A1 and UGT1A3

shown in Figure 5 and the kinetic constants

that

are listed in Table 1.

formation in HLM (Figure 5).

catalyze


estradiol-3-glucuronide


Table 1. Kinetic constants and enzyme kinetic model for estradiol glucuronidation (in the absence and presence of the inhibitor nilotinib) and for estrone glucuronidation UGT

Km ± S.E.

Vmax ± S.E.

Model,

enzyme

(95% CI)

(95% CI)

r2

N-glucuronidation activity toward some heterocyclic

amines,

but

not

the

commercial UGT1A10 (in that case it was a commercial UGT1A10 from a different

Estradiol, no inhibitor HLM

HIM

1A10-H

0.58 ± 0.04

MM

(14.8–30.3)

(0.49–0.67)

0.97

study).19 We have also encountered high

2.9 ± 0.4

2.4 ± 0.1

MM

N-glucuronidation activity of our insect

(2.0–3.8)

(2.2–2.6)

0.94

2.8 ± 0.4

21.2 ± 0.7

MM

(1.9–3.6)

(19.8–22.7)

0.94

Estradiol, 5 µM Nilotinib HLM

supplier than the one used in the present

22.6 ± 3.7

cells

expressed

UGT1A10-H

when

examining the glucuronidation of AMC, a structural analogue of 4-MU (Figure 1).

30.6 ± 7.3

0.19 ± 0.02

MM

(15.5–45.7)

(0.15–0.24)

0.94

2.8 ± 0.3

2.2 ± 0.1

MM

(2.2–3.4)

(2.1–2.3)

0.97

3.5 ± 0.4

18.0 ± 0.5

MM

(2.6–4.3)

(17.0–19.1)

0.96

2.5

2.0

HIM

1A10-H

Rate (nmol/min/mg)

1.5

1.0

0.5

0.0 1A1-H 1A3-H 1A4-H 1A5-H 1A6-H 1A7-H 1A8-H 1A9-H 1A10-H 2A1-H 2A2-H 2A3-H 2B4-H 2B7-H 2B10-H 2B11-H 2B15 2B17-H 2B28-H 1A10 1A8 1A9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 29

Estrone HIM

1A10-H

13.1 ± 2.5

2.85 ± 0.21

MM

Figure 6. AMC glucuronidation by our different

(7.8–18.4)

(2.41–3.29)

0.92

C-terminally tagged UGTs (indicated by -H), as

8.1 ± 1.1

16.9 ± 0.7

MM

well as the commercial UGT1A8, UGT1A9 and

(5.8–10.4)

(15.4–18.4)

0.94

UGT1A10. The substrate concentration was 200 µM.

The glucuronidation of AMC by 19 Most human UGTs, with the exception of UGT1A4 and UGT2B10, mainly catalyze O-glucuronidation reactions. Nevertheless, it was previously shown that UGT1A10, expressed in HEK293 cells exhibited high

recombinant UGT enzymes was tested and the

results

revealed

that

while

the

glucuronidation rate of the commercial UGT1A10 was below detection limit, the rate of our UGT1A10 was very high, by


Page 15 of 29

far the highest among the human UGTs

Estrone

20

A Rate (nmol/min/mg)

(Figure 6). In a recent study we found that estrone is a rather specific substrate for UGT1A10 and suggested that estrone glucuronidation

recombinant Here

we

used

for

testing

UGT1A10 have

different 31

preparations. compared

the

glucuronidation rates of UGT1A10-H, the

2.5

B.HIM

2.0 1.5 1.0 0.5

0

10

20

30

40

30

40

C. UGT1A10-H

15

also of commercial UGT1A8 since in a

catalyze estrone glucuronidation at much

5

0.0

commercial UGT1A10, HIM, HLM, and

previous study20 UGT1A8 was reported to

10

1A 8 1A 1A 10 10 -H H LM H IM

be

Rate (nmol/min/mg)

could

15

0

Rate (nmol/min/mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10

5

higher rate than UGT1A10. In addition, we 0

have analyzed the enzyme kinetics of estrone glucuronidation by HIM and

0

10

20 [estrone], µM

Figure 7. Estrone glucuronidation rates (20 µM substrate) by selected samples (A) and estrone

UGT1A10-H (Figure 7). The results demonstrate that, under the used assay

glucuronidation

kinetics

by

HIM

(B)

and

UGT1A10-H (C).

conditions, good activity was only found in HIM and in UGT1A10-H (Figure 7).

The estrone kinetic analysis (Figure 7), like the estradiol kinetic analysis (Figure

The higher estrone glucuronidation rates of 5), indicates that a similar high activity UGT1A10-H, in comparison to HIM UGT1A10

is

functioning

in

both

(Figure 7), are similar to the difference UGT1A10-H and HIM (see Table 1 for between the two samples in estradiol kinetic constants), except that there is glucuronidation (Figure 4A). 15 Environment ACS Paragon Plus


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 29

probably significantly more of it, per mg

C-terminal

total

UGT1A10 carry. In any case, it is clear

protein,

in

the

UGT1A10-H

preparation.

fusion

peptide

that

our

that the high activity of UGT1A10-H is not

To examine whether or not the differences in activity between UGT1A10-H and the commercial UGT1A10 are due to much higher expression level of the enzyme in our preparation, the two samples were

simply due to higher expression level per mg total protein. Equally, the poor activity of the commercial UGT1A10 is not due to failure to express the protein, but to express it as an active enzyme.

analyzed by western blotting. Monoclonal antibodies against UGT1As (sc-271268) were used for this test and the results reveal that the commercial UGT1A10 is expressed at much higher levels per total protein (Figure 8). The

large

overexpression

of

the

commercial UGT1A10 is probably one of

Figure 8. Immunoblot analysis of the commercial UGT1A10 (Supersomes) and UGT1A10-H. Equal amounts of protein, 10 µg, were loaded onto each

the reasons for its slightly faster migration

lane. See Methods for further details.

in the gel. The other is the lack of the short reveal

remarkable

glucuronidation

rates

differences between

in them,

Discussion namely

very

low

activity

by

the

In this study, two human recombinant

commercial UGT1A10 in comparison to

UGT1A10 enzymes were examined for the

UGT1A10-H

glucuronidation of 9 different drugs and

Furthermore, glucuronide formation by the

related compounds (Figure 1). The results

commercial UGT1A10 often remained


(Figures

2,

4,

6,

7).

Page 17 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


below detection limit, at least under these

glucuronidation of the test compound at

assay conditions, whereas the activity of

significant rates, namely morphine by

UGT1A10-H toward the same substrates

UGT2B7 or estradiol and NCHN by

was high (Figures 2, 4, 6, 7).

UGT1A1 (see below).

The main issue, however, is not which

Human microsomes pools were used and,

recombinant UGT1A10 has higher activity

according to the test substrate, inhibitors

but which of them better represents the

that are rather specific for other intestinal

activity of the native enzyme in the small

UGTs that catalyze the same activity, were

intestine.

particularly

tested for their effect. These inhibitors

important since there is now a good

were either nilotinib for UGT1A1, or

agreement

and

atractylenolide I for UGT2B7 (note that

proteomics studies that only 4 different

the specificity of atractylenolide I was only

UGTs,

reported

This

topic

between

namely

is

qRT-PCR

UGT1A1,

UGT1A10,

with

respect

to

UGTs

of

UGT2B7 and UGT2B17, are expressed to

subfamily 1A28). The results of the

significant levels in the human small

experiments that included microsomes

intestine.7-10 With this in mind, we have

(Figures 3, 4A, 4B, 7) show that the

tested the activity of intestinal UGT1A10

commercial UGT1A10 does not represent

using morphine (at the 3-OH), estradiol

the

(also at the 3-OH), NCHN and estrone as

UGT1A10. Morphine glucuronidation is

substrates for both HLM and HIM (Figures

an appropriate example since the rates

3-7), alongside the glucuronidation of the

exhibited by the commercial UGT1A10

tested compound by the two recombinant

were below detection limit in the method

UGT1A10 preparations. Attention was

used here. Even if a much a more sensitive

also paid to other highly expressed

method will be used, along with higher

intestinal

protein

UGTs

that

catalyze

the

activity

of

the

concentrations


native

and

intestinal

longer


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 29

incubation times, in an effort to yield

would be faced trying to explain the

detectable glucuronide, it will not be

NCHN and the estrone glucuronidation

enough. The challenge is not being able to

results on the basis of the results obtained

detect morphine glucuronide, but to get

with

high morphine glucuronidation activity, as

UGT1A10 (Figures 4B and 7). It may be

expected

was

added that in the case of estrone (Figure 7)

expressed in HEK293 cells16. UGT1A10-H

no other UGT was tested here since we

exhibited the expected high morphine

recently carried out a comprehensive study

glucuronidation activity, in this case when

on estrone glucuronidation and, with the

expressed in insect cells with a C-terminal

exception of UGT1A10 and low activity of

fusion tag. Moreover, using atractylenolide

UGT1A3, none of the other UGTs

I we have shown that in HIM there is

exhibited significant glucuronidation rate

another UGT, in addition to UGT2B7, that

of this estrogen.31 As with morphine and

is

morphine

SN-38, the high estrone glucuronidation

glucuronidation at significant rates since

activity of UGT1A10-H is in agreement

the morphine glucuronidation activity of

with

HIM is less sensitive to the inhibitor than

UGT1A10

the corresponding activity in HLM (Figure

mammalian cell line, in that case COS-1

3).

cells.18

The high rate of estradiol glucuronidation

The estrone glucuronidation rate could be

in HIM that, unlike in HLM, was largely

used as a “UGT1A10 activity marker”.

insensitive to nilotinib (Figure 4A) would

According to this suggestion and using

also be nearly impossible to explain having

conditions as in Figure 7A, the current

only

recombinant

batch of UGT1A10-H should have been

UGT1A10 at hand. Similar difficulties

diluted about 7-8 fold with control

able

the

from

to

UGT1A10

catalyze

commercial

that

the

commercial

previously


that

UGT1A8

reported was

and

results

for

expressed

in

Page 19 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


membranes in order to have similar

It is likely that UGT1A10 glucuronidation

glucuronidation rates as the HIM pool.

activity in HIM was often assigned to UGT1A8, due to much higher activity by

The commercial enzyme is not inactive. It the commercial UGT1A8. A good example was

previously

reported

to

catalyze for this is bavachinin, a major bioactive

glucuronidation of several compounds, such as curcuminoids32 and curcumin analogs33, but no comparison to the

component of the Chinese traditional medicine Fructus Psoraleae. Among all the tested commercial UGTs, bavachinin

activity of the native intestinal UGT1A10 was recently found to be glucuronidated toward these compounds was presented. In mainly by UGT1A8 and UGT1A1, with the present study we have shown that only low activity by UGT1A3, but none by commercial UGT1A10 is able to catalyze UGT1A10.34 Hence, bavachinin could the glucuronidation of 4-MU, SN-38 and have been a good substrate to test the estradiol (Figures 2 and 4). However, the expression of a functional UGT1A8 in the rates were a small fraction of the rates small

intestine,

following

UGT1A1

exhibited by UGT1A10-H. inhibition by nilotinib (Figure 4). The HIM and HLM experiments (Figures However,

upon

testing

bavachinin

3-7), in combination with the comparisons glucuronidation by UGT1A10-H, it turned between UGT1A10-H to the commercial out

that

it

catalyzes

bavachinin

UGT1A10 (Figures 2,4-7), lead to the glucuronidation more than twice as fast as conclusion

that

there

is

an

active the commercial UGT1A8 (Figure 9).

UGT1A10 in the human small intestine and that the commercial enzyme is not suitable for identifying many substrates of the native intestinal UGT1A10.



It is tempting to ask why one preparation

Bavachinin 3 Rate (nmol/min/mg)

of recombinant UGT1A10 is so much more active than another, even if both were

2

expressed in the same system. We do not have the answer to this, although it is

1

obvious that one difference between them is the presence of a C-terminal fusion tag

0

in UGT1A10-H (for details of the fusion

1A 8 1A 1A 10 10 -H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

tag see Kurkela et al.12). Nevertheless, the same C-terminal fusion peptide is present

Figure 9. Bavachinin glucuronidation rates by the commercial UGT1A8 and UGT1A10, as well as

in most other recombinant UGTs from our laboratory and the activity of commercial

by UGT1A10-H. The substrate concentration was

UGTs, per mg membrane protein, is 10µM.

usually higher than in our preparations These results could explain the high

(e.g. Figure 6, the AMC glucuronidation

bavachinin glucuronidation activity that

rates of 1A9-H and 1A9, even if this may

was observed in HIM, even if the

simply be due to higher expression level).

expression level of UGT1A8 is low

In addition, UGT1A10 that was expressed

(although exceptions are possible). More

in mammalian cells without any C-

importantly, the bavachinin result makes

terminal tag was highly active16-19 and the

one wonder how many other UGT1A10

activity of UGT1A10-H was similar to

substrates were overlooked during the last

these preparations. Hence, it might be

10-15 years, since researchers have started

concluded that, presently, the problem is

to count on the commercial UGT1A10 for

limited to the expression of UGT1A10 in

detection.

insect cells without a C-terminal fusion


Page 20 of 29

Page 21 of 29

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


peptide. It is clearly not in the transcription

stimulated by lowering the reaction pH to

and translation of a full-length protein

6.025. Another interesting activity of

translation (Figure 8), but in a subsequent

UGT1A10 is its N-glucuronidation of

step or steps that lead to correct folding of

certain substrates at high rates (Figure 6

an active enzyme. More studies and better

and Dellinger et al.19). In addition, the use

understanding of insects cell biology might

of estrone glucuronidation as a marker and

help if one aims at solving the reason(s) for

possible ruler for UGT1A10 activity was

the difference in activity between the two

suggested.

preparation.

Alternatively,

an

active These findings, in combination with the

UGT1A10 for in vitro research could be new understanding of the limited number expressed

in

mammalian

cells

and, of UGTs that are expressed in the human

perhaps, in some other systems. small intestine to significant levels, mean In conclusion, we have demonstrated here

that UGT1A10 is an important intestinal

that the recombinant UGT1A10-H, that

enzyme, probably much more important

carry a C-terminal fusion tag, catalyzes

that

multiple different activities and provide a

Researchers, those that test the metabolism

good tool to study the important roles of

of new compounds as well as those that try

the intestinal UGT1A10. Among the

to build models based on previously

activities are the previously reported, but

published activity results, should rethink

somewhat forgotten are the conjugations of

how much they could count on the

the 3-OH of morphine16, SN-3817 and the

commercial UGT1A10 and results that

3-OH of estradiol15 at high rates. In

could be obtained using it, results that were

addition, it is interesting to note the acyl

obtained using a poor activity UGT1A10

glucuronidation of diclofenac, an activity

in the past. It seems that quite much

that, in the case of UGT1A10 is strongly

wrong, largely underestimating, data about

has


been

appreciated

earlier.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the real activity of the human intestinal

Page 22 of 29

Acknowledgment

UGT1A10 is present in the literature. We would like to thank Johanna Mosorin Checking which enzyme source was used for skillful technical assistance, and to to obtain the information may be the first acknowledge the financial support of the step in analyzing it. Sigrid Juselius Foundation (grants no. 47033421 and 4704583), the Academy of Finland (grant no.12600101) and the National Science Foundation of China (grants

no.

81473181

&

81273590).

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