Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging

Abstract

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation 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 orthologs. 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 arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop 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 humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest.

Arachidonate 15-lipoxygenase type B: Regulation, function, and its role in pathophysiology

“…Recently, these in vitro mutagenesis studies have been confirmed through the generation of Alox15b knock-in mice, in which the reported amino acids of Alox8 were substituted by those from human ALOX15B. As these humanized Alox15b knock-in mice formed only small amounts of 8-HETE, the substitution from Tyr 603 /His 604 in Alox8 towards Asp 602 /Val 603 in Alox15b confirms that these amino acid pairs are responsible for the different regiospecificity of the two enzymes ( Schäfer et al, 2022 ).…”

“…Recent data from Alox15b knock-in mice, humanized by mutation of the identified amino acid pair in Alox8, show that the hematopoietic system including erythrocyte number, hematocrit and hemoglobin is impaired in male mice. Also, this is paralleled by a premature growth arrest ( Schäfer et al, 2022 ). These mice provide a rare insight into the biological function of human ALOX15B and mouse Alox8 in vivo , as there are no commercially available Alox8-deficient mice to date.…”

Knock-in mice expressing a humanized arachidonic acid 15-lipoxygenase (Alox15) carry a partly dysfunctional erythropoietic system

“…The Alox15-KI mice characterized here were created in collaboration with Cyagen Bioscience (Santa Clara, USA). For this purpose, we employed a similar strategy that was used to humanize the reaction specificity of mouse Alox15b [ 23 ] but the protocol was adopted to mouse Alox15. The experimental details are explained in the Methodological Additional file 1 .…”

“…Body weight kinetics of Alox15 -KI mice (either sex) and of wildtype controls ( n = 10) were quantified as described previously [ 23 ]. Statistic evaluation of the experimental raw data was performed using two-way ANOVA.…”

“…To investigate, whether the altered reaction specificity of mouse Alox15 might have altered the pattern of plasma oxylipins we quantified more than 40 different free oxygenated PUFAs in the blood plasma [ 26 ]. The methodological details of sample workup and of the analysis procedure [ 23 , 26 ] are described in the Additional file 1 : Methodological supplement including Table S3, Table S4, Table S5 and Table S6.…”