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Mathematisch-Naturwissenschaftliche Fakultät
Marjann Schäfer | Kumar Reddy Kakularam | Florian Reisch | Michael Rothe |
Sabine Stehling | Dagmar Heydeck | Gerhard Paul Püschel | Hartmut Kuhn
Male Knock-in Mice Expressing an
Arachidonic Acid Lipoxygenase 15B
(Alox15B) with Humanized Reaction
Specificity Are Prematurely Growth Arrested
When Aging
Suggested citation referring to the original publication:
Biomedicines 10 (2022), Art. 1379 pp. 1 - 22
DOI https://doi.org/10.3390/biomedicines10061379
ISSN 2227-9059
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Citation: Schäfer, M.; Kakularam,
K.R.; Reisch, F.; Rothe, M.; Stehling,
S.; Heydeck, D.; Püschel, G.P.; Kuhn,
H. Male Knock-in Mice Expressing an
Arachidonic Acid Lipoxygenase 15B
(Alox15B) with Humanized Reaction
Specificity Are Prematurely Growth
Arrested When Aging. Biomedicines
2022,10, 1379. https://doi.org/
10.3390/biomedicines10061379
Academic Editors: Robert Gurke and
Marco Sisignano
Received: 12 May 2022
Accepted: 3 June 2022
Published: 10 June 2022
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biomedicines
Article
Male Knock-in Mice Expressing an Arachidonic Acid
Lipoxygenase 15B (Alox15B) with Humanized Reaction
Specificity Are Prematurely Growth Arrested When Aging
Marjann Schäfer 1,2, Kumar R. Kakularam 1, Florian Reisch 1,2, Michael Rothe 3, Sabine Stehling 1,
Dagmar Heydeck 1, Gerhard P. Püschel 2,*,† and Hartmut Kuhn 1 ,*,†
1Department of Biochemistry, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität
Berlin and Humboldt Universität zu Berlin, Charitéplatz 1, 10117 Berlin, Germany;
marjann.schaefer@googlemail.com (M.S.); kumar.kakularam@charite.de (K.R.K.);
florian.reisch@charite.de (F.R.); sabine.stehling@charite.de (S.S.); dagmar.heydeck@charite.de (D.H.)
2Institute for Nutritional Sciences, University Potsdam, Arthur-Scheunert-Allee 114-116,
14558 Potsdam, Germany
3Lipidomix GmbH, Robert-Rössle-Straße 10, 13125 Berlin, Germany; michael.rothe@lipidomix.de
*Correspondence: gpuesche@uni-potsdam.de (G.P.P.); hartmut.kuehn@charite.de (H.K.)
† These authors contributed equally to this work.
Abstract:
Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differ-
entiation and in the pathogenesis of inflammation. The mouse genome involves seven functional
Alox genes and the encoded enzymes share a high degree of amino acid conservation with their
human orthologs. There are, however, functional differences between mouse and human ALOX or-
thologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative
(
15S-HpETE
), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis
for this functional difference has been explored and
in vitro
mutagenesis humanized the reaction
specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the
reaction specificity of mouse Alox15b
in vivo
, we created Alox15b knock-in mice expressing the arachi-
donic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating
wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and de-
velop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain
significantly less body weight than outbred wildtype controls, but this effect was not observed for
female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we
determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited
significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here
again, these differences were not observed in female individuals. These data suggest that human-
ization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic
system in males, which is paralleled by a premature growth arrest.
Keywords: eicosanoids; lipid peroxidation; oxidative stress; polyenoic fatty acids; erythropoiesis
1. Introduction
Arachidonic acid lipoxygenases (ALOX isoforms) form a family of oxygen-metabolizing
enzymes that convert polyunsaturated fatty acids into corresponding hydroperoxy deriva-
tives [
1
–
4
]. The mouse genome involves seven functional Alox genes and most of them are
localized in a joint Alox gene cluster on chromosome 11 [
5
]. In the human genome an orthol-
ogous gene exists for each mouse Alox isoform and most of these genes are clustered on the
short arm of chromosome 17 [
6
]. When arachidonic acid (AA) is used as the substrate, mouse
Alox isoforms exhibit different reaction specificities and AA 12-lipoxygenating (Alox15 [7]
Alox12 [
8
], Alox12b [
9
], Aloxe12 [
10
], Aloxe3 [
11
]), AA 5-lipoxygenating (Alox5, [
12
]) and
Biomedicines 2022,10, 1379. https://doi.org/10.3390/biomedicines10061379 https://www.mdpi.com/journal/biomedicines
Biomedicines 2022,10, 1379 2 of 22
AA 8-lipoxygenating (Alox15b, [
13
]) enzymes have been reported. Despite the high de-
gree of amino acid conservation (>80%), several human and mouse ALOX orthologs show
remarkable functional differences. For instance, under physiological conditions, human
ALOXE3 does not exhibit a fatty acid oxygenase activity [
14
], but under hyperoxic condi-
tions the formation of dioxygenation products has been reported [
15
]. On the other hand,
under normoxic conditions, mouse Aloxe3 was identified as AA 12-lipoxygenating en-
zyme [
11
]. Mouse Alox15 catalyzes AA 12-lipoxygenation [
7
], but the human ortholog
produces 15-H(p)ETE as the dominant AA oxygenation product [
16
]. The structural basis
for the distinct reaction specificities of mouse and human ALOX15 orthologs has been
explored and critical amino acid residues have been identified [
17
–
19
]. Human ALOX15B
converts AA almost exclusively to 15-HETE [
20
], but mouse Alox15b, which is called Alox8
according to the AA-based ALOX nomenclature, catalyzes AA 8-lipoxygenation [
13
,
21
].
Here again, the structural basis for the different reaction specificities of human and mouse
ALOX15b orthologs has been explored and sequence determinants have been identified [
22
].
When Tyr603 and His604 of recombinant mouse Alox15b were mutated to the amino acids,
which are present at these positions in human ALOX15B (Asp and Val, respectively), the
corresponding double mutant catalyzed almost exclusively the formation of 15S-HETE [
22
].
When an inverse mutagenesis strategy was applied for human ALOX15B, the reaction
specificity of this enzyme was shifted in favor of AA 8S-lipoxygenation [
22
]. These data
suggested that Tyr603 and His604 might function as sequence determinants for the reaction
specificity of these enzymes [22].
In addition to different reaction specificities, mouse and human ALOX15B orthologs
show different tissue-specific expression profiles. Northern blot analyses indicated that
human ALOX15B is mainly expressed in lung and prostate but no ALOX15B transcripts
were detected in commercial mRNA preparations of spleen, thymus, testis, ovary, small
intestine, colon, mixed leukocytes, heart, brain, placenta, liver, skeleton muscle, kidney and
pancreas [
20
]. Mouse Alox15b mRNA was found at high concentrations in brain. At lower
levels this mRNA was also present in heart, but it was not detected in spleen, lung, liver,
skeletal muscle, kidney and testis [
21
]. In normal mouse skin, Alox15b mRNA was also
detected in small amounts. However, after treatment with phorbol myristic acid (PMA) a
strong stimulation of Alox15b expression was observed [
13
,
21
]. According to these data,
PMA-treated mouse skin was the richest source of Alox15b.
To find out whether Tyr603Asp+His604Val exchange also modifies the reaction speci-
ficity of mouse Alox15b
in vivo
and to explore the functional consequences of this human-
ization of the reaction specificity of the enzyme, we created knock-in mice (Alox15b-KI
mice), which express the AA 15-lipoxygenating Tyr603Asp + His604Val double mutant
of mouse Alox15b instead of the AA 8-lipoxygenating wildtype enzyme. These mice are
viable, breed normally and female individuals display similar body weight kinetics as
outbred wildtype controls. In contrast, male Alox15b-KI mice gain significantly less body
weight than wildtype controls when aging and this effect was paralleled by compromised
erythropoiesis. Taken together, these data suggest that
in vivo
humanization of the reaction
specificity of mouse Alox15b attenuated the functionality of the erythropoietic system in
males, which is paralleled by a gender-specific premature growth arrest.
2. Materials and Methods
Chemicals—The chemicals used for this study were obtained from the following
sources: Arachidonic acid (AA) and authentic HPLC standards of HETE-isomers (15S-
HETE, 15S/R-HETE, 12S/R-HETE, 12S-HETE, 8R/S-HETE, 8S-HETE, 5S/R-HETE, 5S-HETE)
from Cayman Chem. (distributed by Biomol GmbH, Hamburg, Germany); acetic acid from
Carl Roth GmbH (Karlsruhe, Germany); sodium borohydride from Life Technologies, Inc.
(Eggenstein, Germany); isopropyl-
β
-thiogalactopyranoside (IPTG) from Carl Roth GmbH
(Karlsruhe, Germany); calcium ionophore A23187 and butylhydroxy toluene (BHT) from
Merck (Darmstadt, Germany); restriction enzymes from ThermoFisher (Schwerte, Ger-
many); the E. coli strain Rosetta2 DE3 pLysS from Novagen (Merck-Millipore, Darmstadt,
Biomedicines 2022,10, 1379 3 of 22
Germany). Oligonucleotide synthesis was performed at BioTez Berlin Buch GmbH (Berlin,
Germany). Nucleic acid sequencing was carried out at Eurofins MWG Operon (Ebersberg,
Germany). HPLC grade methanol, acetonitrile, n-hexane, 2-propanol and water were from
Fisher Scientific (Waltham, MA, USA). Phorbol 12-myristate 13-acetate (PMA) was pur-
chased from PeproTech (Hamburg, Germany). The origins of other chemicals employed in
this study are specified in the description of the methods for which they have been used.
For oxylipidomic measurements, the following chemicals were used: Deuterated standards
(LTB4-d4, 20-HETE-d6, 15-HETE-d8, 13-HODE-d4, 14,15-DHET-d11, 9,10-DiHOME-d4,
12,13-EpOME-d4, 8,9-EET-d11, PGE2-d4; 10 ng/mL each) from Cayman Chem. (Ann Arbor,
MI, USA); acetonitrile, methanol and acetic acid from Merck (Darmstadt, Germany); ethyl
acetate, n-hexane, NaOH, Na
2
HPO4, KH
2
PO4 from Fisher Scientific (Schwerte, Germany)
Bacterial expression of mouse Alox15b variants—Wildtype and mutant mouse Alox15b
were expressed in E. coli as N-terminal his-tag fusion proteins. For this purpose, the coding
region of the mouse Alox15b cDNA was cloned into the bacterial expression plasmid pET28b.
Recombinant expression and preparation of the enzymes were performed as described
before [23,24] and crude bacterial cell lysate supernatants were used as enzyme source.
Site-directed mutagenesis of mouse Alox15b—To humanize the reaction specificity of
mouse Alox15b, site-directed mutagenesis was carried out using the PfuUltra II Hotstart
PCR Master Mix kit (Agilent Technologies Germany GmbH & Co. KG, Waldbronn, Ger-
many) as described before [
23
]. To create the Tyr603Asp+His604Val double mutant, the
following mutation primers were synthesized. Upstream primer: 5
0
-GTT AAT TCG TCA
AGT GAT GTC ATC ATT GCT CTC TGG-3
0
; downstream primer: 3
0
-CCA GAG AGC AAT
GAT GAC ATC ACT TGA CGA ATT AAC-50.
In vitro
activity assays of recombinant mouse Alox15b variants—To assay the catalytic
activities of the recombinant enzymes, variable amounts of cell lysate supernatants were
added to 0.5 mL of PBS containing arachidonic acid (AA) as substrate at a final concentration
of 100
µ
M. Incubation and analysis of the reaction products were performed with RP-HPLC
as described before [
23
]. To resolve the hydroxy fatty acid enantiomers, combined normal
phase/chiral phase HPLC (NP/CP-HPLC) was carried out. For this purpose, the conjugated
dienes formed were purified with RP-HPLC and further analyzed by combined normal
phase/chiral phase HPLC. For these analyses, a Chiralpak AD-H column (
4.6 ×250 mm
,
5
µ
m particle size, Daicel, Osaka, Japan) was connected with a Nucleosil pre-column
(
4.6 ×30 mm
, 5
µ
m particle size, Macherey-Nagel, Düren, Germany) and the analytes were
eluted isocratically using a solvent system consisting of n-hexane/methanol/ethanol/acetic
acid (96/3/1/0.1, by vol.) at a flow rate of 1 mL/min.
Creation of Alox15b-KI mice—The Alox15b-KI mice characterized in this study were
created in collaboration with Cyagen Bioscience (Santa Clara, CA, USA). To modify the
Alox15b gene (GenBank accession number: NM_009661.4; Ensemble: ENSMUSG00000020891)
that is located on mouse chromosome 11, we introduced two consecutive point mutations
(
Tyr603Asp + His604Val
) into the wildtype mouse Alox15b gene using the Crispr/Cas9 muta-
genesis strategy. The mouse Alox15b gene involves fourteen exons and its ATG start codon is lo-
cated in exon 1, but the TAA stop codon is in exon 14. The target amino acids Tyr603 and His604
are both located in exon 13. For
in vivo
mutagenesis, the following gRNA targeting vectors and
the donor oligonucleotide (with targeting sequence, flanked by 130 bp homologous sequences
on both sides) were designed: (i) gRNA1 (matches forward strand of the gene): GTT ATC ACA
TCA TTG CTC TCT GG (https://www.vectorbuilder.com/vector/VB171128-1087vxv.html,
accessed on 10 May 2022); (ii) gRNA2 (matches reverse strand of the gene): AAC TTG ACG
AAT TAA CTG CTG GG (https://www.vectorbuilder.com/vector/VB171128-1088vvs.html,
accessed on 10 May 2022); (iii) donor oligonucleotide sequence: ACT ACT TCC AAA GGC
CAG GCC CGG CCT GGA TTT CAT AGC CAG CTG CCA CAT TAA TTC GTC AAG TGA
CGT CAT CAT TGC TCT CTG GCT GCT AAG CGC AGA ACC TGG GAC CAA GTA AGT
AAG GAG CTG GGA. The mutation sites Tyr603Asp and His604Val (TAT CAC to GAC GTC)
were introduced into exon 13 of the Alox15b gene by homology-directed repair. Binding and
recutting of the sequence after homology-directed repair was prevented by introduction of a
Biomedicines 2022,10, 1379 4 of 22
silent mutation (CTC to CTG) (Figure 1). gRNA generated by
in vitro
transcription and the
donor oligonucleotide as well as the Cas9 mRNA were co-injected into fertilized eggs for the
production of knock-in mice. The pups were genotyped with PCR followed by nucleotide
sequence analysis of the amplification product. To reduce the possibility that our mutagenesis
strategy introduced major off-target alterations into the mouse genome, we carried out off-
target analyses. For this purpose, the mouse genome was screened in silico for the targeting
sequence GTT ATC ACA TCA TTG CTC TCT GG and five potential off-target sites were
identified. To exclude off-target alterations in these regions, genomic PCR was carried out
and sequencing of the PCR products did not reveal any additional mutations (Table S1).
Figure 1. Crispr/Cas9 strategy for creating Alox15b-KI mice.
To create Alox15b-KI mice, we employed
the Crispr/Cas9 strategy to introduce the Tyr603Asp + His604Val double mutant into the wildtype
Alox15b gene. The resulting knock-in mice express the AA 15-lipoxygenating Tyr603Asp + His604Val
double mutant instead of the AA 8-lipoxygenating wildtype enzyme. For this purpose, the donor
oligonucleotide (TAT-CAC to GAC-GTC) was introduced into exon 13 by homology-directed repair
mechanisms. In addition, a silent mutation (CTC to CTG) was introduced.
Genotyping—The target region of the mouse Alox15b gene locus was amplified with
PCR (annealing temperature 60
◦
C) with specific primers (mouse Alox15b forward: 5
0
-CGG
GAA GCC CTG GTC CAG TAT ATC-3
0
; mouse Alox15b reverse: 5
0
-AGC CTC ACC CTG
CCT CTA CTC TAA GT-3
0
) and the 656 bp PCR product was sequenced using the forward
primer 50-ATA TTC ACC TGC TCA GCC AAG CAT G-30to confirm in vivo mutagenesis.
Ex vivo activity assays using PMA-treated mouse skin—To explore whether our
in vivo
mutagenesis strategy altered the reaction specificity of mouse Alox15b, we carried out ex
vivo activity assays. For this purpose, we used PMA-treated mouse skin as the enzyme
source. To obtain the skin we sacrificed three wildtype mice and three Alox15b-KI animals,
removed the tails and incubated them for 2 h in PBS containing 5
µ
M PMA. Afterwards, we
prepared the epidermis and extracted total RNA from the proximal 3 cm. The remaining
tissue was cut into small pieces and homogenized in 1 mL of PBS using a Fast-Prep-24
sample preparation system (MP Biomedicals, Irvine, USA). Cell debris was spun down
and 0.6 mL of the homogenate supernatant was mixed with 0.4 mL of PBS containing
100
µ
M AA as Alox substrate. After a 30 min incubation period at room temperature, the
reaction products were reduced by the addition of solid sodium borohydride, the sample
was acidified by the addition of 35
µ
L acetic acid and the formed eicosanoids were extracted
Biomedicines 2022,10, 1379 5 of 22
twice with 1 mL of ethylacetate. The extracts were combined, the solvent was evaporated
under vacuum and the remaining lipids were reconstituted in 250
µ
L of acetonitrile. After
vortexing for 2 min, 250
µ
L of water and 5
µ
L of acetic acid were added, the sample was
centrifuged to remove insoluble material and 300
µ
L of the supernatant was injected for the
RP-HPLC analysis. As for the
in vitro
activity assays (see above), a Shimadzu instrument
(LC20 AD) equipped with a diode array detector (SPD M20A) was used and the hydroxy
fatty acids were separated on a Nucleodur C18 Gravity column (Macherey-Nagel, Düren,
Germany; 250
×
4 mm, 5
µ
m particle size) coupled with a guard column (8
×
4 mm, 5
µ
m
particle size). A solvent system consisting of acetonitrile:water:acetic acid (70:30:0.1, by
vol.) was employed at a flow rate of 1 mL/min and analytes were eluted isocratically
at 25
◦
C. To resolve the hydroxy fatty acid enantiomers, combined normal phase/chiral
phase HPLC was carried out. For this purpose, the conjugated dienes formed were purified
with RP-HPLC and further analyzed by combined normal phase/chiral phase HPLC.
For these analyses, a Chiralpak AD-H column (4.6
×
250 mm, 5
µ
m particle size, Daicel,
Osaka, Japan) was connected with a Nucleosil pre-column (4.6
×
30 mm, 5
µ
m particle
size, Macherey-Nagel, Düren, Germany) and the analytes were eluted isocratically using a
solvent system consisting of n-hexane:methanol:ethanol:acetic acid (96:3:1:0.1, by vol.) at a
flow rate of 1 mL/min.
Determination of the basic hematological parameters—Basic blood parameters (Hb, HK,
erythrocyte count, leucocyte count, MCV, MHC, MCHC) of the two genotypes in three
different age groups (young mice, 10–20 weeks; middle-aged mice, 30–40 weeks; old mice,
70–80 weeks) of either sex were determined. These analyses were performed at the Institut
für Veterinärmedizinische Diagnostik GmbH (Berlin, Germany).
Quantification of body weight kinetics—Male and female Alox15b-KI mice and outbred
wildtype control animals (n= 10 in each experimental group) were housed in separate
cages (5 mice/cage) with water and standard chow diet ad libitum. The body weights
were taken once a week over the time period indicated. The body weight kinetics were
visualized using the GraphPad Prism version 8.2.0 for Windows (GraphPad Software, San
Diego, CA, USA,) and statistic evaluation was carried out using the Wilcoxon test.
Osmotic resistance of erythrocytes—To assess the resistance of the red blood cells for
osmotic stress, we followed the experimental protocol described before [
25
]. For this
purpose, 2
µ
L of blood was diluted in 200
µ
L phosphate buffer (pH 7.4) containing NaCl
at different concentrations (0–0.85%) and the cells were incubated for 30 min at room
temperature. The samples were centrifuged at 200
×
gfor 10 min and the supernatant
containing the free hemoglobin, which was released during hemolysis, was quantified
measuring the absorbance at 540 nm. The absorbance values were expressed as percentage
of complete hemolysis, which was achieved when the cells were incubated in the absence
of NaCl. This absorbance was defined as 100% hemolysis.
Oxylipidomics—To explore whether humanization of the reaction specificity of mouse
Alox15b may impact the pattern of free plasma oxylipins, we quantified the amounts
of free oxygenated PUFAs in the blood plasma. These data do not include the esterified
derivatives. For this purpose, EDTA blood was drawn from sacrificed mice by heart
puncture and the blood plasma was prepared by centrifugation. Then, 10
µ
L of blood
plasma was mixed with 450
µ
L of water and 10
µ
L of a mixture of internal standards
(LTB4-d4, 20-HETE-d6, 15-HETE-d8, 13-HODE-d4, 14,15-DHET-d11, 9,10-DiHOME-d4,
12,13-EpOME-d4, 8,9-EET-d11, PGE2-d4; 10 ng/mL each) and 5
µ
L butylhydroxytoluene
(BHT) were added. Plasma proteins were precipitated by the addition of 100
µ
L of a
1:4 mixture (by vol.) of glycerol:water and 500
µ
L acetonitrile. The pH was adjusted to 6.0
by the addition of 2 mL phosphate buffer (0.15 M), the precipitated proteins were removed
by centrifugation and the clear supernatant was used for solid phase lipid extraction
on a 200 mg Agilent Bond-Elute-Certify II cartridge (Agilent Technologies, Santa Clara,
CA, USA). Before sample application, the cartridge was conditioned with 3 mL methanol
and 3 mL phosphate buffer (0.15 M, pH 6.0). After the sample was applied, the column
was washed with 3 mL of a 1:1 mixture (by vol.) of methanol:water and the oxygenated
Biomedicines 2022,10, 1379 6 of 22
fatty acids were eluted with a 74:25:1 mixture (by vol.) of ethyl acetate:n-hexane:acetic
acid. The solvents were evaporated in a stream of nitrogen and the remaining lipids
were reconstituted in 100
µ
L of a 6:4 mixture (by vol.) of methanol:water and used for
LC-MS/MS analysis.
LC-MS/MS was carried out on an Agilent 1290/II LC-MS system consisting of a
binary pump, an autosampler and a column oven (Agilent Technologies, Waldbronn,
Germany). As stationary phase, we employed an Agilent Zorbax Eclipse C18 UPLC column
(
150 ×2.1 mm
, 1.8
µ
m particle size). The temperature was set at 30
◦
C. As mobile phase, we
used a solvent gradient that was mixed out of two stock solutions. Stock A: Water containing
0.05% acetic acid. Stock B: 1:1 mixture (by vol.) of methanol:acetonitrile. The solvent
gradient employed for our analyses is specified in Table S2. To avoid chromatographic
artefacts, the injection system was rinsed after each injection with a 5:4:1 mixture (by vol.)
of methanol:water:isopropanol. The HPLC system was connected with a triple quadrupole
MS system (Agilent 6495 System, Agilent Technologies, Santa Clara, CA, USA). Negative
electrospray ionization was carried out and the ionization parameters are given in Table S3.
The mass spectrometer was run in dynamic MRM-mode and each metabolite was detected
simultaneously by two independent mass transitions (Table S3). Experimental raw data
were evaluated with the Agilent Mass-Hunter software package, version B10.0. For all
metabolites analyzed in this study, individual calibration curves were set up (Table S4) and
the lower detection limits were determined (Table S5).
Ex vivo Alox5 activity assay—EDTA blood was removed from sacrificed Alox15b-KI
mice and outbred wildtype controls (n= 5 for each genotype). A 200
µ
L amount of the
whole blood was incubated for 15 min at 37
◦
C in the absence or presence of 5
µ
M calcium
ionophore A23187, which activates the Alox5 pathway. After the incubation period, the
blood cells were removed by centrifugation and the plasma was quickly shock frozen in
liquid nitrogen. Oxylipins were extracted and the LTB4 concentrations were quantified
with LC-MS (see “Oxylipidomics” in the Material and Methods section).
Statistics—Statistic evaluation of the activity data and quantification of the patterns
of AA oxygenation products was carried out with the two-sided Student’s t-test using the
Microsoft Excel software package (Excel 2016) or the unpaired t-test using the GraphPad
prism program. Numeric p-values <0.05 were considered statistically significant. Fertility
data and blood parameters were analyzed using the Mann–Whitney U-test and body weight
kinetics and osmotic resistance using the Wilcoxon test performed with the GraphPad Prism
software package, version 8.2.0 for Windows (GraphPad Software, San Diego, CA, USA).
3. Results
Tyr603Asp + His604Val exchange of mouse Alox15b humanized the reaction specificity of the
recombinant enzyme—To confirm that the reaction specificity of mouse Alox15b catalyzed AA
oxygenation is humanized by Tyr603Asp + His604Val exchange, we first expressed wildtype
mouse Alox15b and its Tyr603Asp + His604Val double mutant as recombinant N-terminal
his-tag fusion proteins in E. coli and quantified the pattern of conjugated dienes formed
during a 10 min incubation period of the recombinant enzymes with 100
µ
M arachidonic
acid. As indicated in Figure 2A, conjugated dienes (inset to Figure 2A) co-migrating with
authentic standards of 12-HETE and 8-HETE were formed by the recombinant wildtype
enzyme. Smaller amounts of 15S-HETE were also detected. It should be stressed at this
point that in our standard HPLC system 12-HETE and 8-HETE were not well resolved and
thus, we analyzed the reaction product of the wildtype enzyme with an LC-MS method,
in which the two critical HETE-isomers are well separated (inset to Figure 2B). Here,
we identified the major AA oxygenation product as 8-HETE. Taken together, our HPLC
and LC-MS analyses of the reaction products of recombinant wildtype mouse Alox15b
indicated that 8-HETE is the major conjugated diene formed by this enzyme. Moreover,
in the wildtype enzyme incubation, we observed small amounts of 5-HETE (Figure 2A).
Similar amounts of 5-HETE were also detected in no-enzyme control incubation (data not
Biomedicines 2022,10, 1379 7 of 22
shown) and thus this compound must be classified as an AA auto-oxidation product that
was already present in the substrate stock solution.
Figure 2. Reaction specificity of wildtype and mutant recombinant mouse Alox15b.
Wildtype and
mutant (Tyr603Asp + His604Val) Alox15b were expressed as N-terminal his-tag fusion proteins as
described in Section 2and aliquots of the bacterial lysis supernatants were used as enzyme source.
After a 10 min incubation period with exogenous AA (100
µ
M), the reaction products were analyzed
with RP-HPLC, as reported in Section 2. (
A
) Representative partial RP-HPLC chromatogram of
the AA oxygenation products formed by wildtype mouse Alox15b. Retention time of authentic
standards are indicated by the arrows above the traces. Inset: UV-spectrum of the conjugated dienes
eluting with a retention time of 8.5 min. (
B
) Representative partial RP-HPLC chromatogram of the
AA oxygenation products formed by the Tyr603Asp + His604Val double mutant of mouse Alox15b.
Inset: LC-MS analyses of the AA oxygenation products formed by wildtype and mutant Alox15b.
(
C
) Statistical evaluation of the major AA oxygenation products formed by wildtype and mutant
mouse Alox15b (n= 4).
In contrast, 15S-HETE was the major conjugated diene formed from AA by the
Tyr603Asp + His604Val double mutant (Figure 2B). To obtain independent evidence for the
chemical structure of the major AA oxygenation product formed by the mutant enzyme,
the conjugated dienes formed were also analyzed with LC-MS. Here, we confirmed that
15-HETE was the major AA oxygenation product formed by the mutant enzyme (inset in
Figure 2B). Statistic evaluation of the RP-HPLC raw data is given in Figure 2C. In summary,
our analytical data confirmed [
22
] that wildtype mouse Alox15b oxygenates AA domi-
Biomedicines 2022,10, 1379 8 of 22
nantly to 8-HETE, whereas the Tyr603Asp + His604Val exchange humanized the reaction
specificity of this enzyme.
Corresponding
in vivo
mutagenesis of the Alox15b gene humanized the reaction specificity
of the enzyme—To explore whether the reaction specificity of mouse Alox15b can also be
humanized when an identical mutagenesis strategy was employed
in vivo
, we created
knock-in mice expressing the Alox15b Tyr603Asp + His604Val double mutant instead of the
wildtype enzyme using the Crispr/Cas9 strategy. For this purpose, a gRNA targeting vector
as well as donor oligonucleotides were designed, which involved the targeting sequence
flanked by 130 bp homologous sequences on both sides. The Tyr603Asp+His604Val (TAT-
CAC to GAC-GTC) mutation sites were introduced into exon 13 by homology-directed
repair mechanisms. In addition, a silent mutation (CTC to CTG) was introduced upstream
of the targeting sequence to prevent the binding and re-cutting of the sequence after
homology-directed repair (Figure 1). The Cas9 mRNA, the gRNA generated by
in vitro
transcription and the donor oligonucleotide were co-injected into fertilized eggs for the
production of knock-in mice. The resulting pups were genotyped with PCR followed by
sequence analysis of the amplification products. Heterozygous founder animals were
mated, homozygous wildtype controls as well as homozygous knock-in mice were se-
lected and colonies of Alox15b knock-in mice as well as outbred wildtype control animals
were established.
Mouse Alox15b is expressed at high levels in PMA-treated skin [
21
]. To re-explore
the tissue-specific expression pattern of mouse Alox15b, we first extracted total RNA from
different tissues of wildtype mice (no PMA treatment) and quantified with qRT-PCR the
expression levels of Alox15b mRNA. Using our qRT-PCR system we detected the highest
expression levels in skin and lung but we also observed low-level expression of the enzyme
in other tissues such as liver, kidney and bone marrow (Figure 3A). Since previous literature
reports indicated that expression of Alox15b is strongly augmented in skin by treatment
with PMA [
13
,
21
], we decided to use PMA-treated skin as the enzyme source for ex vivo
activity assays. To prepare PMA-treated skin, we sacrificed Alox15b-KI mice and outbred
wildtype controls, removed the tails and incubated them for 2 h at room temperature in PBS
containing 5
µ
M PMA. After washing in PBS, the epidermis was prepared, total RNA was
extracted from the proximal part (3 cm) of the tail epidermis and qRT-PCR was carried out
to quantify the steady-state concentrations of the mRNAs encoding for the seven mouse
Alox isoforms. From Figure 3B, it can be seen that in wildtype mice Alox12b, Alox15b and
Aloxe3 are expressed at higher levels when compared with Alox12 and Alox12e. A similar
expression pattern was observed in Alox15b-KI mice, but here elevated mRNA levels were
observed for Alox12, Aloxe12 and Alox15b when wildtype animals were compared with the
Alox15-KI mice. For Alox15 and Alox5, we did not detect significant mRNA concentrations
in PMA-treated tail skin (data not shown). The most interesting result for the present
study was that expression of Alox15b in Alox15b-KI mice was higher (about 3-fold) than
in wildtype controls. Similar differences were also observed for Alox12 and Aloxe12, but
the molecular bases for these findings have not been explored. For Alox15b, the increased
expression levels in the Alox15b-KI mice might be interpreted as a compensatory response
of the skin towards the lacking 8-HETE formation. Although a direct repressive effect of
8-HETE on the expression of the Alox15b gene has not been reported, it might be part of a
feedback-control mechanism.
Biomedicines 2022,10, 1379 9 of 22
Figure 3. Expression of Alox isoforms in different mouse tissues.
(
A
) Expression of Alox15b in
different mouse tissues. Total RNA was extracted from different mouse tissues and Alox15b mRNA
was quantified with qRT-PCR as described in Section 2. Two independent measurements (n= 2) were
carried out for each RNA extract. (
B
) Expression of Alox isoforms in PMA-treated mouse skin. Total
RNA was extracted from PMA-treated tail epidermis of Alox15b-KI mice (red columns) and outbred
wildtype controls (blue columns). Expression of the mRNA of different Alox isoforms was quantified
by qRT-PCR. Two independent measurements (n= 2) were carried out for each RNA extract. We also
attempted to quantify expression of Alox15 and Alox5 but did not get specific PCR signals. Thus,
these two Alox isoforms are not expressed on PMA-treated epidermis.
To explore whether our Alox15b-KI mice express an Alox15b with humanized reaction
specificity, we next performed ex vivo activity assays. For this purpose, the epidermis of
the PMA-treated mouse tails was homogenized in PBS (n= 3 for each genotype). After
centrifugation (10,000
×
g), 0.6 mL of the homogenate supernatant was taken and 0.4 mL of
PBS containing 100 µM of AA was added. After 30 min at room temperature, the reaction
products were reduced, extracted and analyzed with RP-HPLC. From Figure 4A, it can be
seen that conjugated dienes co-eluting with authentic standards of 12- and 8-HETE were
formed by PMA-treated epidermis of wildtype mice. In addition, small amounts of 5-HETE
were also detected, but combined NP/CP-HPLC indicated a racemic mixture, which was
already present in the substrate solution. In contrast, formation of 15-HETE was hardly
detected. Identical analyses of the reaction products formed by PMA-treated epidermis
of Alox15b-KI mice also revealed the dominant formation of 12- and/or 8-HETE. Both
compounds were not well resolved under these chromatographic conditions (Figure 4A).
Importantly, in these activity assays we observed a clear 15-HETE peak (Figure 4B). When
we compared the shape of two 8/12-HETE peaks formed by PMA-treated epidermis
of wildtype and Alox15-KI mice, it became evident that the peak formed by wildtype
epidermis was broader and not as sharp as that formed by Alox15b-KI epidermis. These
data suggest that the wildtype peak might represent a mixture of two or more constituents,
and eventually a mixture of 12- and 8-HETE. To explore the chemical structure of the
reaction products in more detail, we prepared the conjugated dienes with RP-HPLC and
further analyzed the products by combined normal-phase/chiral-phase HPLC (NP/CP-
Biomedicines 2022,10, 1379 10 of 22
HPLC). From Figure 4C, it can be seen that the 8-HETE/12-HETE peak formed by wildtype
epidermis consisted of a 3:1 mixture of 12S- and 8S-HETE, but we did not detect any
15S-HETE. In contrast, the major conjugated dienes formed by Alox15b-KI epidermis
were identified as 12S-HETE and 15S-HETE (Figure 4D). Here, no 8S-HETE was detected.
Quantification of the product patterns and statistical evaluation of the data are shown in
Figure 4E. Taken together, the results of our ex vivo activity assays indicate that the major
AA oxygenation product formed by PMA-treated epidermis was 12S-HETE. The metabolic
origin of this product was not explored in this study. It might well be that Alox12, which
is expressed in mouse skin, might be responsible for the formation of this product and
experiments with Alox12 knockout mice would help to shed light on this open question.
The 8S-HETE formation by wildtype epidermis is likely related to the expression of Alox15b.
The lack of 15S-HETE formation by wildtype epidermis but the detection of this compound
in the product mixture formed by Alox15b-KI epidermis is consistent with our hypothesis
that our genetic manipulation of the Alox15b gene humanized the reaction specificity of
this enzyme
in vivo
. This functional ex vivo activity data is consistent with the results of
our genotyping strategy (insets in Figure 4C,D).
Figure 4. Ex vivo Alox activity assays using PMA-treated tail epidermis as source of mouse Alox15b.
Expression of Alox15b was induced in mouse tail epidermis by treatment with PMA and ex vivo
activity assays were carried out using aliquots of a homogenate supernatant of mouse tail epidermis
(see Section 2). (
A
) RP-HPLC analysis of the product pattern formed from AA when PMA-treated
tail epidermis of wildtype mice was used as enzyme source. (
B
) RP-HPLC analysis of the product
pattern formed from AA when PMA-treated tail epidermis of Alox15b-KI mice was used as enzyme
source. (
C
) The conjugated dienes formed by PMA-treated wildtype epidermis were prepared with
RP-HPLC (panel A) and further analyzed by combined normal phase/chiral phase HPLC (see Section 2).
Inset: Representative genotyping chromatogram. (
D
) The conjugated dienes formed by PMA-treated
Alox15b KI epidermis were prepared with RP-HPLC (panel B) and further analyzed by combined normal
phase/chiral phase HPLC (see Section 2). * This front shoulder peak did not show a conjugated diene
chromophore. Inset: representative genotyping chromatograms. (
E
) Statistical evaluation of the major
AA oxygenation products. For this evaluation, the experimental raw data of NP/CP-HPLC were used.
Biomedicines 2022,10, 1379 11 of 22
Reproduction characteristics of Alox15b-KI mice—Alox15b-KI mice are viable and repro-
duce normally. When we compared these animals with outbred wildtype controls, we did
not observe significant differences between the two genotypes, when litter size (pups per
litter), frequency of pregnancy (litters per female
×
month), number of pups per month
(pups per female and month), number of premature casualties before weaning and the
gender ratio of the newborns were compared (Figure 5). After birth, the newborns of
both genotypes developed normally and there was no obvious evidence for post-partal
developmental defects of the Alox15b-KI mice. In humans, ALOX15B is expressed at high
levels in the skin and the enzyme was originally cloned from human hair roots [
20
]. As in
humans, in mice, this enzyme is also expressed in skin [
13
,
21
] and thus humanization of
its reaction specificity might have affected fur development and maintenance. However,
when we evaluated fur development, we did not observe major differences between the
two genotypes.
Figure 5. Comparison of fertility parameters of Alox15b-KI mice and outbred wildtype controls.
For Alox15b-KI mice, 14 breeding pairs and for outcrossed wildtype controls, 18 breeding pairs (one
male + 2 females) were mated and the different fertility parameters (x-axis) were quantified over a
breeding period of 56 (Alox15b-KI mice) and 65 (wildtype controls) breeding months, respectively. ns,
no significant difference.
Body weight kinetics of Alox15b-KI mice—When we compared the kinetics of the absolute
body weights of male and female Alox15b-KI mice with those of outbred wildtype controls
starting 10 weeks after birth, we did not observe a significant difference (Wilcoxon test,
p= 0.0607
) between the two genotypes when female individuals were followed (Figure 6A).
In fact, the growth curves were almost identical. Similar growth kinetics were also observed
for male individuals during the first 24 weeks of post-partal development (Figure 6B) and
statistic evaluation of the absolute body weights did not reveal significant differences
between the two genotypes (Wilcoxon test, p= 0.6633) during this developmental period.
However, at later time points the curves deviated from each other (Figure 6B) and highly
significant differences (Wilcoxon test, p< 0.0001) were observed between the two genotypes.
From these data, one may conclude that male Alox15b-KI gained significantly less body
weight than outbred wildtype controls when aging and thus they experienced a premature
growth arrest.
Biomedicines 2022,10, 1379 12 of 22
Figure 6. Comparison of the body weight kinetics of Alox15b-KI mice and outbred wildtype controls.
For each genotype, 10 individuals of either sex were housed in separate cages (5 mice/cage) with water
and food (standard chow diet) ad libitum. Absolute body weights were quantified once a week, covering
the developmental period between 10 and 64 weeks. (A) Female individuals, (B) male individuals.
Hematological parameters of Alox15b-KI mice—In principle, there are multiple reasons for
the observed premature growth arrest and a slightly compromised hematopoietic system
might be one of them. To test the functionality of the hematopoietic systems of Alox15b-KI
mice and outbred wildtype controls, we first compared the basic blood parameters (Hb,
HK, erythrocyte count, leukocyte count, MCV, MHC, MCHC) of the two genotypes in three
different age groups (young mice, 10–20 weeks; middle-aged mice, 30–40 weeks; old mice,
70–80 weeks) of either sex. Although all hematological parameters were in the normal
range of the determination method used here, we found that for aged Alox15b-KI mice the
major red blood cell parameters (erythrocyte count, hematocrit, hemoglobin) were signifi-
cantly lower than the corresponding parameters of outbred wildtype controls (Figure 7).
For young and middle-aged males, such differences were not observed. Interestingly, we
did not find such differences for female individuals, irrespective of their age. Thus, as for
the body weight kinetics, we observed gender-specific differences between Alox15b-KI
mice and outbred wildtype controls but it remains to be explored whether the dysfunc-
tionality of the erythropoietic system is responsible for the developmental retardation of
male Alox15b-KI mice. It should be stressed at this point that for most other hematolog-
ical parameters (reticulocyte count, MCV, MHC, MCHC) we did not observe significant
differences between the two genotypes (Figures S1 and S2). Only for middle-aged female
individuals the leukocyte counts of Alox15b-KI mice were significantly elevated (Figure S2),
but the functional relevance of this difference has not been explored. In summary, when
compared with outbred wildtype controls, aged male Alox15b-KI mice suffer from a mild
normochromic normocytic anemia. Such a type of anemia frequently develops in patients
with a primary defect in the erythropoietic system [
26
]. If this is the case for Alox15b-KI
Biomedicines 2022,10, 1379 13 of 22
mice, the
in vivo
life span of peripheral red blood cells should be reduced, which frequently
induces reticulocytosis and splenomegaly. To explore whether Alox15b-KI mice suffer from
splenomegaly, we compared the spleen weights of middle-aged (30–40 weeks) Alox15-KI
mice and outbred wildtype controls but did not detect significant differences between
the two genotypes (Table S6). Moreover, we did not observe reticulocytosis in aged male
Alox15b-KI mice (Figure S1).
Figure 7. Comparison of basic erythrocyte parameters of Alox15b-KI mice and outbred wildtype
controls of either sex.
Alox15b-KI mice and outbred wildtype controls of either sex were classified in
three age categories (young mice, 10–20 weeks; middle-aged mice, 30–40 weeks; old mice 70–80 weeks,
n
≥
5 for each age group). After sacrificing the animals by cervical dislocation under anesthesia,
EDTA blood was removed by heart puncture. The basic hematological parameters were determined
by the Institut für Veterinärmedizinische Diagnostik GmbH (Berlin, Germany). Relevant erythrocyte
parameters are shown in this image. The complete sets of hematological parameters are given in
Figures S1 and S2. * indicates significant differences (p< 0.05). ns, no significant difference.
To obtain independent evidence for this hypothesis, we compared the sensitivity of
red blood cells towards ex vivo osmotic challenge. When erythrocytes are incubated in
hypo-osmotic solutions, they take up water, swell and finally hemolyse. If one determines
the degree of hemolysis at different salt concentrations, the osmotic resistance of the cells
can be quantified and an impaired osmotic resistance may be considered as an indicator for
defective erythrocyte membrane functionality [
27
]. We quantified the osmotic resistance
of wildtype and Alox15b-KI erythrocytes in the three different age categories (young,
Biomedicines 2022,10, 1379 14 of 22
10–20 weeks
; middle-aged, 30–40 weeks; old, 70–80 weeks) of female and male individuals.
From Figure 8A, it can be seen that erythrocytes of young female mice exhibit a higher
osmotic resistance than cells prepared from middle-aged and aged individuals. At a given
NaCl concentration, the extent of ex vivo hemolysis of erythrocytes prepared from young
animals was significantly lower than that determined for cells prepared from middle-aged
and aged individuals. Between the two latter age categories, we did not observe differences
in the degree of hemolysis. Most importantly, for female mice, we did not detect significant
differences in the osmotic resistance between erythrocytes of Alox15b-KI mice and wildtype
controls in either of the three age categories. However, when similar experiments were
carried out with male individuals, the situation was somewhat different (Figure 8B). Here
again, we found that erythrocytes prepared from young individuals were less susceptible
to osmotic challenge than cells of older mice and we did not observe significant differences
between Alox15-KI mice and outbred wildtype controls for young and middle-aged male
individuals. However, erythrocytes of aged Alox15b-KI mice were more resistant against
osmotic challenge than the red cells of outbred wildtype control animals. On the first view,
this finding is inconsistent with our conclusion that the erythropoietic system of Alox15b-KI
mice is compromised since, following this hypothesis, a reduced osmotic resistance would
be expected for Alox15b-KI erythrocytes. However, endogenous compensation reactions
may have occurred and we refer to this point in more detail in the “Discussion” section.
Figure 8.
Comparison of the susceptibility of erythrocytes prepared from Alox15b-KI mice and from
outbred wildtype controls for osmotic challenge. Alox15b-KI mice and outbred wildtype controls
of either sex were classified in three age categories (young mice, 10–20 weeks; middle-aged mice,
30–40 weeks; old mice, 70–80 weeks, n
≥
5 for each age-group with 2–3 replicates/individual). After
sacrificing the animals by cervical dislocation under anesthesia, EDTA blood was removed and the
susceptibility of the red blood cells for osmotic challenge was quantified as described in Section 2.
The degree of hemolysis was calculated and these data are plotted over the NaCl concentration.
Under strongly hypo-osmotic conditions (water, no NaCl), all erythrocytes (100%) hemolyzed. Under
iso-osmotic conditions (0.85% NaCl), more than 80% of the red blood cells survived the hemolysis
period. (A) Female mice, (B) male mice. ns, no significant difference.
Biomedicines 2022,10, 1379 15 of 22
Oxylipidomics—To explore whether our genetic manipulation of the Alox15b gene
impacted the patterns of the plasma oxylipins, we quantified the plasma levels of more
than 40 different oxylipins (Tables S4 and S5). Twelve of them, including a number of
maresins, resolvins and neuroprotectins, were below the detection limits of our analytical
systems (Table S5), but for 32 oxygenated fatty acid derivatives, we obtained reliable
analytic data (Figures S3, S4 and S6–S9). For these experiments, blood plasma of five
middle-aged male individuals of each genotype were explored and the most relevant
findings are briefly discussed below.
When we summed up the different free oxylipins detected in the blood plasma of both
Alox15b-KI mice and outbred wildtype controls, we found significantly more oxygenated
PUFA derivatives in Alox15-KI mice (Figure 9A). This difference was mainly due to the
significantly higher plasma concentrations of 12-HETE (Figure S3), 12-HETrE (Figure S7)
and 13-HODE (Figure S8) in Alox15b-KI mice (compared with wildtype controls). These
three compounds are the dominant free oxylipins in the plasma of both genotypes. Their
metabolic origin remains a matter of discussion. Although our data suggest a role of Alox15b
in their biosynthesis (humanization of the reaction specificity of Alox15b elevated the relative
abundance of these metabolites), the mechanistic details have not yet been defined.
Mouse Alox15b converts AA mainly to 8S-HETE, but the dominant AA oxygenation
product of the humanized enzyme was 15-HETE (Figure 2). If our genetic manipulation of
the Alox15b gene is mirrored on the level of the oxygenated plasma lipids, one would expect
to see elevated plasma concentrations of 15-HETE but reduced plasma levels of 8-HETE
(Alox15-KI mice vs. wildtype controls). Unfortunately, such differences were not observed
(Figure 9B). Similarly, we did not detect the expected changes in the plasma levels of the EPA
oxygenation products (Figure 9C) since 8-HEPE was not reduced in Alox15b-KI mice and 15-
HEPE was not augmented. Wildtype mouse Alox15b converts DHA mainly to 10-HDHA,
but the humanized enzyme forms 17-HDHA as a major DHA oxygenation product (Figure
S5). If these changes were mirrored on the level of the oxygenated plasma lipids, one would
expect to see lower 10-HDHA levels but higher 17-HDHA levels in Alox15b-KI mice than
in wildtype controls. However, we did not observe the expected differences (Figure 9D).
When we finally analyzed the major oxygenation products of 8,11,14-eicosatetraenoic acid,
we measured significantly higher 15-HeTrE levels in Alox15b-KI mice (Figure 9E) and
this finding was predicted as a functional consequence of our genetic manipulation. On
the other hand, we did not observe a concomitant decrease in the 8-HeTrE plasma levels
(Figure 9E). Here again, we measured higher 8-HeTrE concentrations. Since for linoleic
acid and alpha-linolenic acid, the oxygenation products formed by recombinant wildtype
mouse Alox15b and its Tyr603Asp+His604Val double mutant have not been determined, it
was impossible to predict the alterations in the plasma oxylipidomes (Figure S8).
Among the di- and tri-hydroxylated PUFA derivatives, we only detected 5S,12S-
DiHETE (Figure S3) and 10R,17S-DiHDHA (NPx, Figure S9) as plasma oxylipins. However,
there were no significant differences in the plasma concentration of these metabolites
between the two genotypes. Other more complex oxylipins, such as 8S,15S-diHETE, RvE1,
RvD1, RvD1(17R), RvD2, RvD3, RvD4(17epi), RvD5, Mar-1, Mar-2, Mar(7epi) and NPD1,
were below the detection limits of our analytical method and thus we cannot comment on
whether mouse Alox15b or its Tyr603Asp+His604Val double mutant might be involved in
the biosynthesis of these metabolites.
Taken together, our lipidomic data suggest that humanization of the reaction specificity
of mouse Alox15b induces subtle alterations in the blood plasma concentrations of several
oxylipins. However, it remains to be explored how exactly the observed lipidomic changes
may be related to our subtle genetic manipulation.
Biomedicines 2022,10, 1379 16 of 22
Figure 9. Quantification of selected oxylipins in the blood plasma of Alox15b-KI mice and out-
bred wildtype controls.
Male Alox15b-KI mice and outbred wildtype controls (n= 5 for each geno-
type) were sacrificed, EDTA blood was removed, plasma lipids were extracted and the free plasma
oxylipidomes were quantified with LC-MS (for methodological details see Section 2). Quantifications
of selected metabolites are given. (
A
) Sum of all quantified oxylipins (OH-PUFAs). (
B
) Quantifica-
tion of the most relevant arachidonic acid (AA) metabolites, (
C
) Quantification of the most rele-
vant 5,8,11,14,17-eicosapentaenoic acid (EPA) metabolites. (
D
) Quantification of the most relevant
4,7,10,13,16,19-docosahexaenoic acid (DHA) metabolites. (
E
) Quantification of the most relevant 8,11,14-
eicostrienoic acid metabolites. * indicates significant difference at p< 0.05, ** indicates significant
differences at p< 0.01. ns, no significant difference.
Biomedicines 2022,10, 1379 17 of 22
Humanization of Alox15b specificity does not affect the Alox5 pathway—Since Alox5 and
Alox15b are co-expressed in myeloid cells, it might be possible that the two enzymes might
directly or indirectly interact with each other. To explore whether humanization of the
reaction specificity of Alox15b may impact the Alox5 pathway in peripheral blood cells,
we carried out ex vivo Alox5 activity assays. For this purpose, we stimulated whole blood
with calcium ionophore A23187 and quantified the formation of leukotriene B (LTB4) with
LC-MS. From Figure 10 it can be seen that wildtype blood cells form large amounts of
LTB4 when stimulated with A23187. In contrast, no LTB4 was formed in the absence of
this stimulus. When similar incubations were carried out with whole blood prepared
from Alox15b-KI mice, similar amounts of LTB4 were detected. Statistically, there was
no significant difference between the two genotypes. Taken together, these data indicate
that humanization of the reaction specificity of mouse Alox15b hardly impacts the Alox5
pathway of peripheral blood cells.
Figure 10. Ex vivo Alox5 activity assays using whole blood of Alox15b-KI mice and outbred
wildtype controls as experimental system
. Male Alox15b-KI mice and outbred wildtype controls
(n= 5 for each genotype) were sacrificed, heparinized blood was collected and the Alox5 pathway
was stimulated by incubating the blood with 5
µ
M of calcium ionophore A23178. Cells were spun
down and the blood plasma was shock frozen in liquid nitrogen. For analysis, the oxylipins were
extracted and the free plasma LTB
4
levels were quantified with LC-MS (for methodological details
see Section 2). ** indicate significant differences (p< 0.01). ns, no significant difference.
4. Discussion
In vivo
mutagenesis humanizes the reaction specificity of mouse Alox15b—Mouse Alox15b
oxidizes arachidonic acid mainly into 8S-H(p)ETE [
13
,
21
]. In contrast, the human ortholog
constitutes an AA 15-lipoxygenating enzyme [
20
]. The structural basis for this functional
difference has been explored in detail and
in vitro
mutagenesis studies on recombinant
mouse Alox15b (Tyr603Asp + His604Val double mutant) humanized the reaction specificity
of this enzyme [22]. To explore whether a similar mutagenesis strategy will humanize the
reaction specificity
in vivo
, we created knock-in mice carrying a minimally mutated version
of the Alox15b gene, which encodes for the Tyr603Asp + His604Val double mutant. For this
purpose, we employed the Crispr/Cas9 technology and established a colony of homozy-
gous Alox15b-KI animals expressing the Tyr603Asp + His604Val Alox15b double mutant
instead of the wildtype enzyme as well as a colony of outbred wildtype control animals. To
explore whether this minor genomic manipulation (exchange of five nucleotides) indeed in-
duced humanization of the reaction specificity, we carried out ex vivo activity assays using
phorbol ester treated mouse skin as an enzyme source. For these experiments, we incubated
homogenates of PMA-treated tail epidermis of three individuals of each genotype with
exogenous AA and analyzed the oxygenated AA derivatives by different types of HPLC.
Biomedicines 2022,10, 1379 18 of 22
When wildtype epidermis was taken through this experimental protocol, 12S-HETE was
detected as the dominant AA oxygenation product (Figure 4A). The metabolic source of this
compound has not been explored, but arachidonic acid 12-lipoxygenating enzymes, such as
Alox15, Alox12 and Aloxe12, might contribute. In addition, smaller amounts of 8S-HETE
were detected (Figure 4C) and this metabolite most probably originated from PMA-induced
epidermal Alox15b. When we used PMA-treated mouse skin of Alox15b-KI mice as the
enzyme source, the major arachidonic acid oxygenation product was also 12S-HETE. In
addition, we observed significant amounts of 15S-HETE (Figure 4B,D), which was not
present when wildtype skin was used. 8S-HETE was hardly formed in these incubations
(Figure 4A,C). Taken together, these data confirm the predicted functional consequences of
our genetic manipulation and the results suggested that our
in vivo
mutagenesis strategy
humanized the reaction specificity of mouse Alox15b.
Alox15b-KI mice may help to explore the biological function of Alox15b—The biological
role of ALOX15B in humans and mice has not completely been clarified. Since the human
enzyme is expressed at high levels in hair follicles, it has been implicated in hair growth [
20
].
When human peripheral monocytes were differentiated
in vitro
to macrophages, expression
of ALOX15B was strongly augmented [
28
]. In fact, Western blot analysis indicated that
interleukin-4 (IL4), bacterial lipopolysaccharide and hypoxia increased the expression of
ALOX15B but not of ALOX12. Since IL4 drives the differentiation of naive monocytes
to alternatively activated macrophages (M2 macrophages) and since M2 macrophages
have been implicated in inflammatory resolution [
29
,
30
], ALOX15B may play a role in
this process. A similar expression regulation has previously been reported for human
ALOX15 [
31
,
32
], but the regulatory mechanisms appear to be different for the two human
ALOX isoforms. ALOX15B and ALOX15 [
33
,
34
] have been implicated in the biosynthesis of
specialized pro-resolving lipid mediators (SPMs) and this catalytic activity may be related
to the pro-resolving activities of M2-macrophages. However, because of the different
reaction specificities of the two ALOX15 orthologs [
13
,
20
,
21
], the biosynthetic mechanisms
and the SPM profiles in mice and humans should be different. ALOX15B has also been
implicated in the pathogenesis of hyperproliferative diseases [
35
]. In fact, in prostate cancer,
expression of the ALOX15B gene is silenced and mechanistic studies suggested a function
of the ALOX15B gene as a tumor suppressor gene [
36
–
38
]. During development of ovarian
cancer, expression of ALOX15B is strongly augmented and high levels of ALOX15B mRNA
have also been detected in metastatic tissue [
39
]. Although the mechanistic details for this
expression regulation have not been explored, quantification of ALOX15 mRNA in the
blood has been suggested as a marker for ovarian cancer [
39
]. Since mouse Alox15b exhibits
a different reaction specificity than its human ortholog [
13
,
20
,
21
], it remains unclear whether
mouse Alox15b fulfils similar functions in mouse models of prostate and/or ovarian cancer.
For such studies, the Alox15b-KI mice expressing an enzyme with humanized reaction
specificity might be useful as a mechanistic tool.
Male Alox15b knockout mice show impaired recovery from influenza infection when
compared with wildtype littermates [
40
]. In fact, six-month-old Alox15b
-/-
mice displayed a
significantly prolonged state of illness, as indicated by altered body temperatures, impaired
locomotor activities and delayed body weight recovery [
40
]. Moreover, the steady-state con-
centrations of the pro-inflammatory cytokine interleukin 6 in the lungs were significantly
elevated during the phase of inflammatory resolution [
40
]. Interestingly, such differences
were not observed when 3-month-old individuals were taken through the experimental
protocol. These data suggest that Alox15b deficiency compromised the immune response
towards influenza virus infection in aged Alox15b-deficient mice. Obviously, this immuno-
logical defect was well compensated in 3-month-old individuals. Unfortunately, no female
mice were used for these experiments so that a possible gender specificity could not be
explored. In our study, we observed similar age-dependent alterations. The curves of
body weight kinetics of male Alox15b-KI mice were superimposable with those of outbred
wildtype controls (Figure 6B) up to an age of 24 weeks. Afterwards, the curves of the body
weight kinetics deviated from each other and Alox15b-KI mice gained significantly less body
Biomedicines 2022,10, 1379 19 of 22
weight than outbred wildtype controls. Interestingly, this difference was gender-specific
for male individuals since the curves of the body weight kinetics of females remained
superimposable up to an age of 64 weeks. The molecular basis for this gender-specific
premature growth arrest has not been explored in detail but it might be related to our
observation that the erythropoietic system of male Alox15-KI individuals is compromised
in aged male Alox15b-KI mice (Figure 7).
Humanization of the reaction specificity of mouse Alox15b induces malfunction of the ery-
thropoietic system in aged male Alox15b-KI mice—Although Alox15b
-/-
mice develop normally,
their immune response towards influenza virus infection is compromised [
40
]. During the
initial phase of this response, the innate immune system is activated at the site of infection
and natural killer (NK) cells enter this site to eradicate virus-infected cells [
41
]. In addition,
neutrophils and monocytes are recruited to the site of infection, helping to clear the tissue
from infected cells [
42
]. The observation that Alox15b
-/-
mice need more time to recover
from an experimental infection with the influenza virus [
40
] suggests that Alox15b may
play a role for the normal functionality of the immune system. Unfortunately, the precise
function of the enzyme has not been explored and hematopoietic parameters (leukocyte
count, differential blood count) have not been reported. We found that the leukocyte counts
of Alox15b-KI mice and of outbred wildtype controls of either sex were not significantly
different (Figures S1 and S2). However, aged male Alox15b-KI mice expressing a mutant
Alox15b isoform with humanized reaction specificity carry a compromised erythropoietic
system (Figure 7). Interestingly, this erythropoietic defect was not observed in female
individuals. For the time being, the molecular basis for the defective erythropoiesis in
aged male Alox15b-KI mice remains unclear but work is in progress to shed light on this
interesting aspect.
When we quantified the osmotic stability of wildtype erythrocytes and compared it
with that of red blood cells prepared from Alox15b-KI mice, we observed an improved
osmotic resistance of Alox15b-KI erythrocytes (Figure 8). On first view, this observation
is inconsistent with our conclusion that the erythropoietic system of Alox15b-KI mice is
compromised. Following this hypothesis, a reduced osmotic resistance would be expected
for Alox15b-KI erythrocytes. However, it might be possible that the improved osmotic
resistance of Alox15b-KI erythrocytes may be the result of an adaptive response of the
erythropoietic system to balance the reduced capacity of the bone marrow to produce red
blood cells. Osmotic-resistant erythrocytes are likely to live longer and this may compensate
the attenuated erythropoietic capacity. It would be of interest to explore experimentally
whether Alox15b-KI erythrocytes really show a prolonged in vivo life span.
Gender-specific effects of humanization of the reaction specificity of mouse Alox15b—As
indicated in Figures 6and 7, humanization of the reaction specificity of mouse Alox15b
induced a premature gender-specific growth retardation in male Alox15b-KI mice (Figure 6)
and a gender-specific dysfunction of the erythropoietic system (Figures 7and 8) in aged
individuals. Although the molecular mechanisms for the gender-specific character of these
effects remain to be explored, our observation that female individuals do not show these
effects suggests that both the premature growth retardation and the dysfunctionality of the
erythropoietic system may not be the consequence of differences in the genetic background
of the Alox15b-KI mice and wildtype animals. If background problems that can never
be completely excluded were responsible for the observed defects, similar phenotypic
alterations would also be expected for female individuals.
In humans, leukotriene-related diseases such as bronchial asthma and allergic rhinitis
show an obvious gender bias. In fact, adult female individuals are more frequently affected
than males [
43
]. Ex vivo formation of leukotrienes by female blood was significantly higher
than that by male blood and the addition of 5-dehydrotestosteron to female blood reduced
the ex vivo leukotriene biosynthetic capacity of female blood to male levels. These data
indicated that androgens may regulate the catalytic activity of Alox5 and the underlying
mechanisms have been reported [
44
]. Moreover, female individuals are more susceptible
than males for anti-leukotriene therapy [
45
] and taken together these data indicate gender-
Biomedicines 2022,10, 1379 20 of 22
specific differences in ALOX5 functionality. However, for the time being, gender-specific
effects have not been reported for other human ALOX paralogs such as ALOX15, ALOX15B
and ALOX12. Although the molecular basis for the gender-specific premature growth
arrest of male Alox15b-KI mice has not been clarified, this is the first report suggesting that
gender-specific effects may also be related to Alox15b.
Supplementary Materials:
The following spporting information can be downloaded at https://www.
mdpi.com/xxx/s1. Figure S1. Basic hematological parameters of male Alox15-KI mice and outbred
wildtype control animals of different age categories. Figure S2. Basic hematological parameters of female
Alox15-KI mice and outbred wildtype control animals of different age categories. Figure S3. Quantifi-
cation of the major free oxygenated arachidonic acid metabolites in the blood plasma of Alox15b-KI
mice and outbred wildtype controls. Figure S4. Quantification of the major free oxygenated 5,8,11,14,17-
eicosapentaenoic acid metabolites in the blood plasma of Alox15b-KI mice and outbred wildtype
controls. Figure S5. RP-HPLC analysis of the major conjugated dienes formed from 4,7,10,13,16,19-
docosahexaenoic acid (DHA) by recombinant mouse Alox15b and its Tyr603Asp+His604Val double
mutant exhibiting a humanized reaction specificity with arachidonic acid as substrate. Figure S6. Quan-
tification of the major free oxygenated 4,7,10,13,16,19-docosahexaenoic acid metabolites in the blood
plasma of Alox15b-KI mice and outbred wildtype controls. Figure S7. Quantification of the major
free oxygenated metabolites of 8,11,14-eicosatrienoic acid in the blood plasma of Alox15b-KI mice and
outbred wildtype controls. Figure S8. Quantification of the major free oxygenated linoleic acid (HODE-
isomers) and al-pha-linolenic acid metabolites (HOTrE-isomers) in the blood plasma of Alox15b-KI mice
and outbred wildtype controls. Figure S9. Quantification of free NPx in the blood plasma of Alox15b-KI
mice and outbred wildtype controls. Table S1. Off-target analysis of our
in vivo
mutagenesis strategy.
Table S2. Mobile phase gradient used for LC-separation of the plasma oxylipins. Table S3. Ionization
conditions used in our LC-MS based analysis of the blood plasma oxylipins. Table S4. Characteristic
mass transitions, collision energies and retention times of the different oylipins. Table S5. Metabolites
quantified in the frame of our plasma lipidome analysis. Table S6. Spleen weights of male and fe-male
middle-aged Alox15b-KI mice and outbred wildtype controls.
Author Contributions:
Conceptualization, H.K., D.H., K.R.K.; methodology, M.S., K.R.K., F.R., M.R.,
S.S., D.H., H.K.; validation, H.K., D.H., investigation, M.S., K.R.K., F.R., M.R., S.S., D.H., H.K. data
curation, M.S., K.R.K., F.R., M.R., S.S., D.H., H.K.; writing—original draft preparation, H.K.; writing—
review and editing, M.S., K.R.K., F.R., M.R., S.S., D.H., G.P.P., H.K.; visualization, K.R.K., H.K., D.H.;
supervision, H.K., D.H., G.P.P.; project administration, H.K., D.H.; funding acquisition, H.K., D.H. All
authors have read and agreed to the published version of the manuscript.
Funding:
This work was supported by grants from the Deutsche Forschungsgemeinschaft—DFG
(KU961/13-1, KU961/14-1 to H.K., HE8295/1-1 to D.H.).
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki and approved by the Institutional Review Board of the State Animal Care
Committee (Landesamt für Gesundheit und Soziales, Berlin, Germany) and the following permission
number was given: T0437/08.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The original experimental raw data can be obtained from the authors
upon request.
Acknowledgments:
The work of F.R. was financed in part by research fellowship of the Sonnefeld
Foundation. The costs of open access publication were covered by the University of Potsdam and for
this funds of the German Research Foundation (project number 491466077) were provided.
Conflicts of Interest:
The authors declare that they do not have any conflict of interest with the
content of this article.
Biomedicines 2022,10, 1379 21 of 22
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