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Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in the pathogenesis of inflammatory diseases, and its pro- and anti-inflammatory effects have been reported for different ALOX-isoforms. Human ALOX15B oxygenates arachidonic acid to its 15-hydroperoxy derivative, whereas the corresponding 8-hydroperoxide is formed by mouse Alox15b (Alox8). This functional difference impacts the biosynthetic capacity of the two enzymes for creating pro- and anti-inflammatory eicosanoids. To explore the functional consequences of the humanization of the reaction specificity of mouse Alox15b in vivo, we tested Alox15b knock-in mice that express the arachidonic acid 15-lipoxygenating Tyr603Asp and His604Val double mutant of Alox15b, instead of the arachidonic acid 8-lipoxygenating wildtype enzyme, in two different animal inflammation models. In the dextran sodium sulfate-induced colitis model, female Alox15b-KI mice lost significantly more bodyweight during the acute phase of inflammation and recovered less rapidly during the resolution phase. Although we observed significant differences in the colonic levels of selected pro- and anti-inflammatory eicosanoids during the time-course of inflammation, there were no differences between the two genotypes at any time-point of the disease. In Freund’s complete adjuvant-induced paw edema model, Alox15b-KI mice were less susceptible than outbred wildtype controls, though we did not observe significant differences in pain perception (Hargreaves-test, von Frey-test) when the two genotypes were compared. our data indicate that humanization of the reaction specificity of mouse Alox15b (Alox8) sensitizes mice for dextran sodium sulfate-induced experimental colitis, but partly protects the animals in the complete Freund’s adjuvant-induced paw edema model.

Glutathione peroxidase 4 (Gpx4) and arachidonic acid 15 lipoxygenase (Alox15) are counterplayers in oxidative lipid metabolism and both enzymes have been implicated in spermatogenesis. However, the roles of the two proteins in acrosomal exocytosis have not been explored in detail. Here we characterized Gpx4 distribution in mouse sperm and detected the enzyme not only in the midpiece of the resting sperm but also at the anterior region of the head, where the acrosome is localized. During sperm capacitation, Gpx4 translocated to the post-acrosomal compartment. Sperm from Gpx4+/Sec46Ala mice heterozygously expressing a catalytically silent enzyme displayed an increased expression of phosphotyrosyl proteins, impaired acrosomal exocytosis after in vitro capacitation and were not suitable for in vitro fertilization. Alox15-deficient sperm showed normal acrosome reactions but when crossed into a Gpx4-deficient background spontaneous acrosomal exocytosis was observed during capacitation and these cells were even less suitable for in vitro fertilization. Taken together, our data indicate that heterozygous expression of a catalytically silent Gpx4 variant impairs acrosomal exocytosis and in vitro fertilization. Alox15 deficiency hardly impacted the acrosome reaction but when crossed into the Gpx4-deficient background spontaneous acrosomal exocytosis was induced. The detailed molecular mechanisms for the observed effects may be related to the compromised redox homeostasis.

Lipoxygenases (LOX) are a family lipid oxygenating enzymes that can generate bioactive lipids of clinical relevance from polyunsaturated fatty acids. Most LOXs display a Ca

After 300 million years of evolution, the first land-living mammals reentered the marine environment some 50 million years ago. The driving forces for this dramatic lifestyle change are still a matter of discussion but the struggle for food resources and the opportunity to escape predators probably contributed. Reentering the oceans requires metabolic adaption putting evolutionary pressure on a number of genes. To explore whether eicosanoid signaling has been part of this adaptive response, we first explored whether the genomes of marine mammals involve functional genes encoding for key enzymes of eicosanoid biosynthesis. Cyclooxygenase (COX) and lipoxygenase (ALOX) genes are present in the genome of all marine mammals tested. Interestingly, ALOX12B, which has been implicated in skin development of land-living mammals, is lacking in whales and dolphins and genes encoding for its sister enzyme (ALOXE3) involve premature stop codons and/or frameshifting point mutations, which interrupt the open reading frames. ALOX15 orthologs have been detected in all marine mammals, and the recombinant enzymes exhibit similar catalytic properties as those of land-living species. All marine mammals express arachidonic acid 12-lipoxygenating ALOX15 orthologs, and these data are consistent with the Evolutionary Hypothesis of ALOX15 specificity. These enzymes exhibit membrane oxygenase activity and introduction of big amino acids at the triad positions altered the reaction specificity in favor of arachidonic acid 15-lipoxygenation. Thus, the ALOX15 orthologs of marine mammals follow the Triad concept explaining their catalytic specificity.

Aims: Most mammalian genomes involve several genes encoding for functionally distinct arachidonate lipoxygenase (ALOX isoforms). Proinflammatory leukotrienes are formed via the ALOX5 pathway, but 12/15-lipoxygenating ALOX isoforms have been implicated in the biosynthesis of pro-resolving mediators. In vitro mutagenesis of the triad determinants abolished the leukotriene synthesizing activity of ALOX5, but the biological consequences of these alterations have not been studied. To fill this gap, we created Alox5 knock-in mice, which express the 12/15-lipoxygenating Phe359Trp + Ala424Ile + Asn425Met Alox5 triple mutant and characterized its phenotypic alterations. Results: The mouse Alox5 triple mutant functions as arachidonic acid 15-lipoxygenating enzyme, which also forms 12S-hydroxy and 8S-hydroxy arachidonic acid. In contrast to the wild-type enzyme, the triple mutant effectively oxygenates linoleic acid to 13S-hydroxy linoleic acid (13S-HODE), which functions as activating ligand of the type-2 nuclear receptor peroxisome proliferator-activated receptor gamma (PPARg). Knock-in mice expressing the mutant enzyme are viable, fertile, and develop normally. The mice cannot synthesize proinflammatory leukotrienes but show significantly attenuated plasma levels of lipolytic endocannabinoids. When aging, the animals gained significantly more body weight, which may be related to the fivefold higher levels of 13-HODE in the adipose tissue. Innovation: These data indicate for the first time that in vivo mutagenesis of the triad determinants of mouse Alox5 abolished the biosynthetic capacity of the enzyme for proinflammatory leukotrienes and altered the catalytic properties of the protein favoring the formation of 13-HODE. Conclusion:In vivo triple mutation of the mouse Alox5 gene impacts the body weight homeostasis of aging mice via augmented formation of the activating PPARg ligand 13-HODE.

His596 of human ALOX12 has been suggested to interact with the COO--group of arachidonic acid during ALOX catalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange might contribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 for ALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigated the impact of these mutations on structural, catalytic and allosteric enzyme properties. To shed light on the molecular basis of the observed functional alterations we performed in silico substrate docking studies and molecular dynamics simulations and also explored the impact of Gln596 exchange on the protein structure. The combined theoretical and experimental data suggest that Gln596 may not directly interact with the COO--group of arachidonic acid. In contrast, mutations at Gln596 destabilize the secondary and tertiary structure of ALOX15 orthologs, which may be related to a disturbance of the electrostatic interaction network with other amino acids in the immediate surrounding. Moreover, our MD-simulations suggest that the geometry of the dimer interface depends on the structure of substrate bound inside the substrate-binding pocket and that Gln596Ala exchange impairs the allosteric properties of the enzyme. Taken together, these data indicate the structural and functional importance of Gln596 for ALOX15 catalysis.

For the specificity of ALOX15 orthologs of different mammals the geometry of the amino acids Phe353, Ile418, Met419 and Ile593 (?triad determinants?) is important and mutagenesis of these residues altered the reaction specificity of these enzymes. Here we expressed wildtype human ALOX5 and its F359W/A424I/N425M/A603I mutant in Sf9 insect cells and characterized the catalytic differences of the two enzyme variants. We found that wildtype ALOX5 converted arachidonic acid mainly to 5(S)-HpETE. In contrast, 15(S)- and 8(S)-H(p)ETE were formed by the mutant enzyme. In addition to arachidonic acid, wildtype ALOX5 accepted EPA as substrate but C18 fatty acids were not oxygenated. The quadruple mutant also accepted linoleic acid, alpha- and gamma-linolenic acid as substrate. Structural analysis of the oxygenation products and kinetic studies with stereospecifically labeled 11(S)- and 11(R)-deutero linoleic acid suggested alternative ways of substrate orientation at the active site. In silico docking studies, molecular dynamics simulations and QM/MM calculations confirmed this hypothesis. These data indicate that ?triad determinant? mutagenesis alters the catalytic properties of ALOX5 abolishing its leukotriene synthase activity but improving its biosynthetic capacity for pro-resolving lipoxins. Fil: Ivanov, Igor. Russian Technological University; Fil: Golovanov, Alexey B.. Russian Technological University; Fil: Ferretti, Cristián Alejandro. Universidad Nacional del Litoral. Instituto de Química Aplicada del Litoral. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Química Aplicada del Litoral.; Argentina Fil: Canyelles-Niño, Miquel. Universitat Autònoma de Barcelona; España Fil: Heydeck, Dagmar. Humboldt University Berlin; Fil: Stehling, Sabine. Humboldt University Berlin; Fil: Lluch, José M.. Universitat Autònoma de Barcelona; España Fil: González Lafont, Ángels. Universitat Autònoma de Barcelona; España Fil: Kühn, Hartmut. Humboldt University Berlin

The tree shrew (Tupaia belangeri) is a rat-sized mammal, which is more closely related to humans than mice and rats. However, the use of tree shrew to explore the patho-mechanisms of human inflammatory disorders has been limited since nothing is known about eicosanoid metabolism in this mammalian species. Eicosanoids are important lipid mediators exhibiting pro- and anti-inflammatory activities, which are biosynthesized via lipoxygenase and cyclooxygenase pathways. When we searched the tree shrew genome for the presence of cyclooxygenase and lipoxygenase isoforms we found copies of functional COX1, COX2 and LOX genes. Interestingly, we identified four copies of ALOX15 genes, which encode for four structurally distinct ALOX15 orthologs (tupALOX15a-d). To explore the catalytic properties of these enzymes we expressed tupALOX15a and tupALOX15c as catalytically active proteins and characterized their enzymatic properties. As predicted by the Evolutionary Hypothesis of ALOX15 specificity we found that the two enzymes converted arachidonic acid predominantly to 12S-HETE and they also exhibited membrane oxygenase activities. However, their reaction kinetic properties (KM for arachidonic acid and oxygen, T- and pH-dependence) and their substrate specificities were remarkably different. In contrast to mice and humans, tree shrew ALOX15 isoforms are highly expressed in the brain suggesting a role of these enzymes in cerebral function. The genomic multiplicity and the tissue expression patterns of tree shrew ALOX15 isoforms need to be considered when the results of in vivo inflammation studies obtained in this animal are translated into the human situation.

Background The guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the hnRNP H/F family, which has been implicated in erythropoiesis, regulation of the redox homeostasis, embryonic brain development, mitochondrial function and cellular senescence. The molecular basis for GRSF1-RNA interaction has extensively been studied in the past but for the time being GRSF1 binding proteins have not been identified. Methods To search for GRSF1 binding proteins we first employed the yeast two-hybrid system and screened a cDNA library of human fetal brain for potential GRSF1 binding proteins. Subsequently, we explored the protein-protein-interaction of the recombiant proteins, carried out immunoprecipitation experiments to confirm the interaction of the native proteins in living cells and performed truncation studies to identify the protein-binding motif of GRSF1. Results Using the yeast two-hybrid system we identified the COMM-domain containing protein 1 (COMMD1) as specific GRSF1 binding protein and in vitro truncation studies suggested that COMMD1 interacts with the alanine-rich domain of GRSF1. Co-immunoprecipitation strategies indicated that COMMD1-GRSF1 interaction was RNA independent and also occurred in living cells expressing the two native proteins. Conclusion In mammalian cells the COMM-domain containing protein 1 (COMMD1) specifically interacts with the Ala-rich domain of GRSF1 in an RNA-independent manner. General significance This is the first report describing a specific GRSF1 binding protein.

Arachidonic acid lipoxygenases (ALOXs) are lipid-metabolizing enzymes that have been implicated in cell differentiation, but also in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Most mammalian genomes involve six or seven functional ALOX genes and among the corresponding ALOX-isoforms the ALOX15 orthologs are somewhat unique since they exhibit variable reaction specificity using arachidonic acid as substrate. The Evolutionary Hypothesis of mammalian ALOX15 reaction specificity (Prog. Lipid Res. 72, 55, 2018) suggests that ALOX15 orthologs of primates ranked higher in evolution than gibbons are 15-lipoxygenating enzymes. In contrast, mammals ranking lower than gibbons express dominantly 12-lipoxygenating lipoxygenases and gibbon ALOX15 constitutes a transition enzyme with pronounced dual reaction specificity. Here we predicted the reaction specificity of 95 different prototherian, metatherian and eutherian ALOX15 orthologs on the basis of their primary structures and characterized experimentally the reaction specificity of ten novel metatherian/eutherian enzymes representing different stages of mammalian evolution (gorilla, opossum, cape golden mole, dog, horseshoe bat, hedgehog, Sunda flying lemur, pika, chinchilla, kangaroo rat). We found that 97% of the currently sequenced mammalian ALOX15 including the enzymes of living and extinct hominids follow the Evolutionary Hypothesis. However, the ALOX15 orthologs of rabbits and of the Ord's kangaroo rat violate this mechanistic concept. Taken together, this data confirms the Evolutionary Hypothesis of ALOX15 reaction specificity and puts this concept on a more reliable experimental basis.

Lipoxygenases are lipid peroxidizing enzymes, which frequently occur in higher plants and mammals. These enzymes are also expressed in lower multicellular organisms but here they are not widely distributed. In bacteria, lipoxygenases rarely occur and evaluation of the currently available bacterial genomes suggested that

The Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the heterogeneous nuclear ribonucleoprotein F/H family and has been implicated in RNA processing, RNA transport and translational regulation. Amino acid alignments and homology modeling suggested the existence of three distinct RNA-binding domains and two auxiliary domains. Unfortunately, little is known about the molecular details of GRSF1/RNA interactions. To explore the RNA-binding mechanisms we first expressed full-length human GRSF1 and several truncation mutants, which include the three separated qRRM domains in E. coli, purified the recombinant proteins and quantified their RNA-binding affinity by RNA electrophoretic mobility shift assays. The expression levels varied between 1 and 10mg purified protein per L bacterial liquid culture and for full-length human GRSF1 a binding constant (KD-value) of 0.5μM was determined. In addition, our mechanistic experiments with different truncation mutants allowed the following conclusions: i) Deletion of either of the three RNA-binding domains impaired the RNA-binding affinity suggesting that the simultaneous presence of the three domains is essential for high-affinity RNA-binding. ii) Deletion of the Ala-rich auxiliary domain did hardly affect RNA-binding. Thus, this structural subunit may not be involved in RNA interaction. iii) Deletion of the acidic auxiliary domain improved the RNA-binding suggesting a regulatory role for this structural motif. iv) The isolated RNA-binding domains did not exhibit sizeable RNA-binding affinities. Taken together these data suggest that a cooperative interaction of the three qRRMs is required for high affinity RNA-binding.

The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D–models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling.

Mammalian lipoxygenases (LOX) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Although the reaction specificity of mammalian LOX with n-6 fatty acids (linoleic acid, arachidonic acid) has been explored in detail little information is currently available on the product patterns formed from n-3 polyenoic fatty acids, which are of particular nutritional importance and serve as substrate for the biosynthesis of pro-resolving inflammatory mediators such as resolvins and maresins. Here we expressed the ALOX15 orthologs of eight different mammalian species as well as human ALOX12 and ALOX15B as recombinant his-tag fusion proteins and characterized their reaction specificity with the most abundantly occurring polyunsaturated fatty acids (PUFAs) including 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA). We found that the LOX isoforms tested accept these fatty acids as suitable substrates and oxygenate them with variable positional specificity to the corresponding n-6 and n-9 hydroperoxy derivatives. Surprisingly, human ALOX15 as well as the corresponding orthologs of chimpanzee and orangutan, which oxygenates arachidonic acid mainly to 15S-H(p)ETE, exhibit a pronounced dual reaction specificity with DHA forming similar amounts of 14- and 17-H(p)DHA. Moreover, ALOX15 orthologs prefer DHA and EPA over AA when equimolar concentrations of n-3 and n-6 PUFA were supplied simultaneously. Taken together, these data indicate that the reaction specificity of mammalian LOX isoforms is variable and strongly depends on the chemistry of fatty acid substrates. Most mammalian ALOX15 orthologs exhibit dual positional specificity with highly unsaturated n-3 polyunsaturated fatty acids.

12/15-Lipoxygenases (12/15-LOX) have been implicated in inflammatory and hyperproliferative diseases but the numerous aspects of structural biology of these enzymes are far from clear. Early mutagenesis data and structural modeling of enzyme-substrate complexes suggested that Arg403, which is localized at the entrance of the putative substrate binding pocket, might interact with the fatty acid carboxylic group. On the other hand, side-chain of Arg403 is a part of an ionic network with the residues of α2-helix, which undergoes pronounced conformation changes upon inhibitor binding. To explore the role of Arg403 for catalysis in more detail we exchanged positively charged Arg403 to neutral Leu and quantified structural and functional consequences of the alteration at the site of mutation using fluorometric techniques. We found that a loss of electrostatic interaction between Arg403 and negatively charged amino acid residues of α2-helix has only minor impact on protein folding, but partially destabilized the tertiary structure of the enzyme. We hypothesize that interaction of Arg403 with the substrate's carboxylate might be involved in a complex mechanism triggering conformational changes of the α2-helix, which are required for formation of the catalytically competent dimer r12/15-LOX complex at pre-catalytic stages.

ALOX15 (12/15-lipoxygenase) orthologs have been implicated in maturational degradation of intracellular organelles and in the biosynthesis of antiinflammatory and proresolving eicosanoids. Here we hypothesized that lower mammals (mice, rats, pigs) express 12-lipoxygenating ALOX15 orthologs. In contrast, 15-lipoxygenating isoforms are found in higher primates (orangutans, men), and these results suggest an evolution of ALOX15 specificity. To test this hypothesis we first cloned and characterized ALOX15 orthologs of selected Catarrhini representing different stages of late primate evolution and found that higher primates (men, chimpanzees) express 15-lipoxygenating orthologs. In contrast, lower primates (baboons, rhesus monkeys) express 12-lipoxygenating enzymes. Gibbons, which are flanked in evolution by rhesus monkeys (12-lipoxygenating ALOX15) and orangutans (15-lipoxygenating ALOX15), express an ALOX15 ortholog with pronounced dual specificity. To explore the driving force for this evolutionary alterations, we quantified the lipoxin synthase activity of 12-lipoxygenating (rhesus monkey, mouse, rat, pig, humIle418Ala) and 15-lipoxygenating (man, chimpanzee, orangutan, rabbit, ratLeu353Phe) ALOX15 variants and found that, when normalized to their arachidonic acid oxygenase activities, the lipoxin synthase activities of 15-lipoxygenating ALOX15 variants were more than fivefold higher (P0.01) [corrected]. Comparative molecular dynamics simulations and quantum mechanics/molecular mechanics calculations indicated that, for the 15-lipoxygenating rabbit ALOX15, the energy barrier for C13-hydrogen abstraction (15-lipoxygenation) was 17 kJ/mol lower than for arachidonic acid 12-lipoxygenation. In contrast, for the 12-lipoxygenating Ile418Ala mutant, the energy barrier for 15-lipoxygenation was 10 kJ/mol higher than for 12-lipoxygenation. Taken together, our data suggest an evolution of ALOX15 specificity, which is aimed at optimizing the biosynthetic capacity for antiinflammatory and proresolving lipoxins.

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.

12/15-Lipoxygenases (12/15-LOXs) have been implicated in inflammatory and hyperproliferative diseases but the structural biology of these enzymes is not well developed. Most LOXs constitute single polypeptide chain proteins that fold into a two-domain structure. In the crystal structure the two domains are tightly associated, but small angle X-ray scattering data and dynamic fluorescence studies suggested a high degree of structural flexibility involving movement of the N-terminal domain relative to catalytic subunit. When we inspected the interdomain interface we have found a limited number of side-chain contacts which are involved in interactions of these two structural subunits. One of such contact points involves tyrosine 98 of N-terminal domain. This aromatic amino acid is invariant in vertebrate LOXs regardless of overall sequence identity. To explore in more detail the role of aromatic interactions in interdomain association we have mutated Y98 to various residues and quantified the structural and functional consequences of these alterations. We have found that loss of an aromatic moiety at position 98 impaired the catalytic activity and membrane binding capacity of the mutant enzymes. Although CD and fluorescence emission spectra of wild-type and mutant enzyme species were indistinguishable, the mutation led to enlargement of the molecular shape of the enzyme as detected by analytic gel filtration and this structural alteration was shown to be associated with a loss of protein thermal stability. The possible role of tight interdomain association for the enzyme's structural performance is discussed.

Human lipoxygenases (LOXs) and their metabolites have a great impact on human homeostasis and are of interest for targeted drug design. This goal requires detailed knowledge of their structures and an understanding of structure–function relationship. At the moment, there are two complete crystal structures for mammalian LOX [rabbit 12/15LOX (r-12/15LOX) and human 5LOX (h-5LOX)] and a fragment of human 12LOX. The low-resolution structures in solution for various LOX isoforms have brought about controversial results. Here we explored the behavior of r-12/15LOX in aqueous solution under different conditions (salt and pH) by small-angle X-ray scattering (SAXS) and compared it with human platelet-type 12S-LOX (hp-12LOX) and h-5LOX. Thermodynamic calculations concerning the stability of molecular assemblies, thermal motion analysis [TLSMD (translation, libration, and screw rotation motion detection based on crystallographic temperature factor Bj)], and results of SAXS analyses brought about the following conclusions: (i) in contrast to its crystal structure, r-12/15LOX functions as a monomer that dominates in solution; (ii) it dimerizes at higher protein concentrations in the presence of salt and with increasing degree of motional freedom of the N-terminal PLAT domain, as suggested by the Y98,614 → R double mutant; (iii) in aqueous solutions, hp-12LOX is stable as a dimer, in contrast to h-5LOX and r-12/15LOX, which are monomeric; and (iv) all three mammalian isozymes show a high level of flexibility not only for the PLAT domain but also for other subdomains of the catalytic part in TLS (translation, libration, and screw rotation) analysis and hp-12LOX in SAXS.

Collagen type I inhibits the co-stimulatory activity of DPPIV/CD26, and hence modulates the T cell activation. The enzymatic activity of DPPIV is not necessary for the co-stimulatory activity of this molecule in T cell activation. DPPIV/CD26 plays an important role in the development, maturation, activation and differentiation of T cells as well as on their functions in the immune system. Deficiency of DPPIV/CD26 results in an impaired development and maturation of CD4 lymphocytes and a disturbed response to PWM stimulation. The IL-4 secretion was decreased, and hence the IgG production was reduced and isotype switching to IgE was affected.