5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234]

Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences

CHARACTERIZATION OF THE GLYCOME AND GLYCOPROTEOME IN ALZHEIMER’S DISEASE PATIENTS BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY

Chapter I introduces the importance and challenges of studying glycosylation. The chapter describes the long-term associations between glycosylation and brain development. However, emphasis is placed in the interplay between protein glycosylation and Alzheimer’s Disease. The chapter further discusses the key aspects of employing mass spectrometry techniques for glycomic and glycoproteomic analysis. In Chapter II, a comprehensive map of the N-glycome and glycoproteome of the human brain is presented. The study involved four subjects, two with confirmed Alzheimer Disease neuropathology and the remaining two termed non-cognitive impaired. This map involved 11 functional human brain regions per subject. The research described in Chapter III, resulted from the human brain glycome map findings. This study only focused on three functional brain regions affected differently in the Alzheimer’s Disease pathology. The study design involved higher biological replicates per region and was characterized using a novel nano-liquid chromatography chip-quadrupole-time of flight mass spectrometry (nanoLC-Chip-Q-ToF MS) platform.The motivation behind Chapter IV, involved the increased demand for the development of novel blood-based biomarkers for early detection, prevention, or intervention in Alzheimer’s Disease. This research sought out to determine whether serum glycopeptide analysis holds potential for identifying novel diagnostics and prognostics of Alzheimer’s Disease. This work used an ultra-high-performance liquid-chromatography triple quadrupole mass spectrometry (UHPLC-QqQ-MS) operated in dynamic multiple reaction monitoring (dMRM) mode to monitor glycopeptides alterations in Alzheimer’s Disease patients. Together these chapters highlight the powerful application of mass spectrometry techniques in biological and biomarker research discovery.

Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences

Supplementation with omega‐3 or omega‐6 fatty acids attenuates platelet reactivity in postmenopausal women

Postmenopausal women are at increased risk for a cardiovascular event due to platelet hyperactivity. There is evidence suggesting that ω-3 polyunsaturated fatty acids (PUFAs) and ω-6 PUFAs have cardioprotective effects in these women. However, a mechanistic understanding of how these fatty acids regulate platelet function is unknown. In this study, we supplemented postmenopausal women with fish oil (ω-3 fatty acids) or evening primrose oil (ω-6 fatty acids) and investigated the effects on their platelet activity. The effects of fatty acid supplementation on platelet aggregation, dense granule secretion, and activation of integrin αIIbβ3 at basal levels and in response to agonist were tested in postmenopausal women following a supplementation and washout period. Supplementation with fish oil or primrose oil attenuated the thrombin receptor PAR4-induced platelet aggregation. Supplementation with ω-3 or ω-6 fatty acids decreased platelet dense granule secretion and attenuated basal levels of integrin αIIbβ3 activation. Interestingly, after the washout period following supplementation with primrose oil, platelet aggregation was similarly attenuated. Additionally, for either treatment, the observed protective effects post-supplementation on platelet dense granule secretion and basal levels of integrin activation were sustained after the washout period, suggesting a long-term shift in platelet reactivity due to fatty acid supplementation. These findings begin to elucidate the underlying mechanistic effects of ω-3 and ω-6 fatty acids on platelet reactivity in postmenopausal women. Hence, this study supports the beneficial effects of fish oil or primrose oil supplementation as a therapeutic intervention to reduce the risk of thrombotic events in postmenopausal women. https://clinicaltrials.gov/ct2/show/NCT02629497

Probing the Electrostatic and Steric Requirements for Substrate Binding in Human Platelet-Type 12-Lipoxygenase.

Human platelet ALOX12 (hALOX12 or h12-LOX) has been implicated in a variety of human diseases. The present study investigates the active site of hALOX12 to more thoroughly understand how it positions the substrate and achieves nearly perfect regio- and stereospecificities (i.e., 100 ± 5% of the 12(S)-hydroperoxide product), utilizing site-directed mutagenesis. Specifically, we have determined that Arg402 is not as important in substrate binding as previously seen for hALOX15 but that His596 may play a role in anchoring the carboxy terminal of the arachidonic acid during catalysis. In addition, Phe414 creates a π-stacking interaction with a double bond of arachidonic acid (Δ11), and Ala417/Val418 define the bottom of the cavity. However, the influence of Ala417/Val418 on the profile is markedly less for hALOX12 than that seen in hALOX15. Mutating these two residues to larger amino acids (Ala417Ile/Val418Met) only increased the generation of 15-HpETE by 24 ± 2%, but conversely, smaller residues at these positions converted hALOX15 to almost 100% hALOX12 reactivity [Gan et al. (1996) J. Biol. Chem. 271, 25412-25418]. However, we were able to increase 15-HpETE to 46 ± 3% by restricting the width of the active site with the Ala417Ile/Val418Met/Ser594Thr mutation, indicating both depth and width of the active site are important. Finally, residue Leu407 is shown to play a critical role in positioning the substrate correctly, as seen by the increase of 15-HpETE to 21 ± 1% for the single Leu407Gly mutant. These results outline critical differences between the active site requirements of hALOX12 relative to hALOX15 and explain both their product specificity and inhibitory differences

Transcriptomic and glycomic analyses highlight pathway-specific glycosylation alterations unique to Alzheimer’s disease

Glycosylation has been found to be altered in the brains of individuals with Alzheimer's disease (AD). However, it is unknown which specific glycosylation-related pathways are altered in AD dementia. Using publicly available RNA-seq datasets covering seven brain regions and including 1724 samples, we identified glycosylation-related genes ubiquitously changed in individuals with AD. Several differentially expressed glycosyltransferases found by RNA-seq were confirmed by qPCR in a different set of human medial temporal cortex (MTC) samples (n = 20 AD vs. 20 controls). N-glycan-related changes predicted by expression changes in these glycosyltransferases were confirmed by mass spectrometry (MS)-based N-glycan analysis in the MTC (n = 9 AD vs. 6 controls). About 80% of glycosylation-related genes were differentially expressed in at least one brain region of AD participants (adjusted p-values < 0.05). Upregulation of MGAT1 and B4GALT1 involved in complex N-linked glycan formation and galactosylation, respectively, were reflected by increased concentrations of corresponding N-glycans. Isozyme-specific changes were observed in expression of the polypeptide N-acetylgalactosaminyltransferase (GALNT) family and the alpha-N-acetylgalactosaminide alpha-2,6-sialyltransferase (ST6GALNAC) family of enzymes. Several glycolipid-specific genes (UGT8, PIGM) were upregulated. The critical transcription factors regulating the expression of N-glycosylation and elongation genes were predicted and found to include STAT1 and HSF5. The miRNA predicted to be involved in regulating N-glycosylation and elongation glycosyltransferases were has-miR-1-3p and has-miR-16-5p, respectively. Our findings provide an overview of glycosylation pathways affected by AD and potential regulators of glycosyltransferase expression that deserve further validation and suggest that glycosylation changes occurring in the brains of AD dementia individuals are highly pathway-specific and unique to AD