Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Seeing double: two different homospermidine oxidases are involved in pyrrolizidine alkaloid biosynthesis in different organs of comfrey (Symphytum officinale).

Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey (Symphytum officinale, Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper-containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, SoCuAO1 is expressed in young leaves, while SoCuAO5 is expressed in roots. CRISPR/Cas9-mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA-free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of SoCuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species-specific, tight spatio-temporal regulation, and independent evolutionary origins in multiple plant lineages.

Insights into polyamine metabolism: homospermidine is double-oxidized in two discrete steps by a single copper-containing amine oxidase in pyrrolizidine alkaloid biosynthesis.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

ω-(5-Phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds as inhibitors of the amine oxidase vascular adhesion protein-1 (VAP-1).

Vascular adhesion protein-1 (VAP-1), also known as plasma amine oxidase or semicarbazide-sensitive amine oxidase, is an enzyme that degrades primary amines to aldehydes with the formation of hydrogen peroxide and ammonia. Among others, it plays a role in inflammatory processes as it can mediate the migration of leukocytes from the blood to the inflamed tissue. We prepared a series of ω-(5-phenyl-2H-tetrazol-2-yl)alkyl-substituted glycine amides and related compounds and tested them for inhibition of purified bovine plasma VAP-1. Compounds with submicromolar activity were obtained. Studies on the mechanism of action revealed that the glycine amides are substrate inhibitors, i.e., they are also converted to an aldehyde derivative. However, the reaction proceeds much more slowly than that of the substrate used in the assay, whose conversion is thus blocked. Examination of the selectivity of the synthesized glycine amides with respect to other amine oxidases showed that they inhibited diamine oxidase, which is structurally related to VAP-1, but only to a much lesser extent. In contrast, the activity of monoamine oxidase A and B was not affected. Selected compounds also inhibited VAP-1 in human plasma. The IC50 values measured were higher than those determined with the bovine enzyme. However, the structure-activity relationships obtained with the glycine amides were similar for both enzymes.

Serum Vascular Adhesion Protein-1 and Endothelial Dysfunction in Hepatic Cirrhosis: Searching for New Prognostic Markers.

Endothelial dysfunction plays a key role in the development of liver cirrhosis. Among the biomarkers of endothelial dysfunction, the soluble form of Vascular Adhesion Protein-1 (sVAP-1) is an unconventional and less known adhesion molecule endowed also with amine oxidase activity. The aim of this study was to explore and correlate the behavior of sVAP-1 with that of the soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) and with the severity of liver cirrhosis. A cross-sectional study was carried out by enrolling 28 controls, 59 cirrhotic patients without hepatocellular carcinoma, and 56 patients with hepatocellular carcinoma (HCC), mainly caused by alcohol abuse. The levels of adhesion molecules and of the pro-inflammatory cytokines (IL-6 and TNF-αα) were determined by immunoassay and the enzymatic activity of sVAP-1 by a fluorometric assay. In non-diabetic patients without HCC, a specific behavior of sVAP-1 was highlighted. Differently from sVCAM-1, sICAM-1, and cytokines, the sVAP-1 level was significantly increased only in the early stage of disease, and then, it decreased in the last stage (866 ± 390 ng/mL vs. 545 ± 316 ng/mL, in Child-Pugh class A vs. C, respectively, p < 0.05). Bivariate analysis correlates sVAP-1 to sVCAM-1, in the absence of HCC (Spearman's rho = 0.403, p < 0.01). Multiple linear regression analysis revealed that sVCAM-1 appears to be a predictor of sVAP-1 (ß coefficient = 0.374, p = 0.021). In conclusion, in non-diabetic and non-HCC cirrhotic patients, sVAP-1 may be a potential prognostic biomarker that, together with sVCAM-1 and pro-inflammatory cytokines, may provide information on the progression of sinusoidal liver endothelium damage.

Strikingly conserved gene expression changes of polyamine regulating enzymes among various forms of acute and chronic kidney injury.

The polyamines spermidine and spermine and their common precursor molecule putrescine are involved in tissue injury and repair. Here, we test the hypothesis that impaired polyamine homeostasis contributes to various kidney pathologies in mice during experimental models of ischemia-reperfusion, transplantation, rhabdomyolysis, cyclosporine treatment, arterial hypertension, diabetes, unilateral ureteral obstruction, high oxalate feeding, and adenine-induced injuries. We found a remarkably similar pattern in most kidney pathologies with reduced expression of enzymes involved in polyamine synthesis together with increased expression of polyamine degrading enzymes. Transcript levels of amine oxidase copper-containing 1 (Aoc1), an enzyme which catalyzes the breakdown of putrescine, were barely detectable by in situ mRNA hybridization in healthy kidneys. Aoc1 was highly expressed upon various experimental kidney injuries resulting in a significant reduction of kidney putrescine content. Kidney levels of spermine were also significantly reduced, whereas spermidine was increased in response to ischemia-reperfusion injury. Increased Aoc1 expression in injured kidneys was mainly accounted for by an Aoc1 isoform that harbors 22 additional amino acids at its N-terminus and shows increased secretion. Mice with germline deletion of Aoc1 and injured kidneys showed no decrease of kidney putrescine content; although they displayed no overt phenotype, they had fewer tubular casts upon ischemia-reperfusion injury. Hyperosmotic stress stimulated AOC1 expression at the transcriptional and post-transcription levels in metanephric explants and kidney cell lines. AOC1 expression was also significantly enhanced after kidney transplantation in humans. These data demonstrate that the kidneys respond to various forms of injury with down-regulation of polyamine synthesis and activation of the polyamine breakdown pathway. Thus, an imbalance in kidney polyamines may contribute to various etiologies of kidney injury.

P19-derived neuronal cells express H1, H2, and H3 histamine receptors: a biopharmaceutical approach to evaluate antihistamine agents.

Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.

Molecular cloning, expression, and functional analysis of copper amine oxidase gene from mulberry (Morus alba L.).

In this study, we investigated a key enzyme encoded by the gene copper amine oxidase (MaCAO), which is involved in the biosynthetic pathway of 1-deoxynojirimycin (DNJ)1, an active ingredient in mulberry leaves. The 1680 bp long MaCAO was successfully cloned (GenBank accession no: MH205733). Subsequently, MaCAO was heterologously expressed using a recombinant plasmid, pET-22b (+)/MaCAO in Escherichia coli BL21 (DE3). A protein with a molecular mass of 62.9 kDa was obtained, whose function was validated through enzymatic reaction. Bioinformatics analysis identified that MaCAO contained the same conserved domain as that of copper amine oxidases ("NYDY"). Furthermore, the tertiary structure of the predicted protein using homology modeling revealed 46% similarity with that of copper amine oxidase (Protein Data Bank ID: 1W2Z). Gas chromatography-mass spectrometry analysis of the enzymatic reaction revealed that MaCAO could catalyze 1,5-pentanediamine to produce 5-aminopentanal. Additionally, levels of mulberry leaf DNJ content were significantly positively correlated with expression levels of MaCAO (P < 0.001). Our results conclude that MaCAO is the key enzyme involved in the biosynthetic pathway of DNJ. The function of MaCAO is validated, providing a foundation for the further analysis of biosynthetic pathways of DNJ in mulberry leaves using tools of synthetic biology.

Recombinant human diamine oxidase prevents hemodynamic effects of continuous histamine infusion in guinea pigs.

OBJECTIVE: To test whether recombinant human diamine oxidase (rhDAO) with a mutated heparin-binding motif (mHBM), which shows an increased alpha-distribution half-life, prevents histamine-induced hemodynamic effects. MATERIAL: Thirty-eight female guinea pigs were either pretreated with rhDOA_mHBM or buffer. TREATMENT AND METHODS: Guinea pigs received a continuous infusion of histamine. Heart rate (HR), body core temperature and mean arterial pressure (MAP) were measured and blood was collected. RESULTS: Continuous intravenous infusion of 8 µg/kg/min histamine increased mean peak plasma histamine levels from 5 (± 0.3 SEM) to 28 ng/mL (± 4.9 SEM) after 30 min but had no effect on oxygen saturation. Guinea pigs pretreated with 4 mg/kg rhDAO_mHBM showed lower mean HR (p = 0.008), histamine plasma concentrations (p = 0.002), and higher body core temperatures at the end of the histamine challenge (p = 0.02) compared to controls. Cessation of histamine infusion led to a rebound increase in MAP, but this hemodynamic instability was prevented by rhDAO_mHBM. Pretreatment with 4 mg/kg rhDAO_mHBM reduced urinary histamine (p = 0.004) and 1-Methylhistamine (p < 0.0001) concentrations compared to controls. CONCLUSIONS: Prophylactic infusion of rhDAO_mHBM prevents hemodynamic effects in a guinea pig model of continuous histamine infusion. These findings might help in the translation from animals to humans and in the selection of the optimal dosing of rhDAO_mHBM during human histamine challenge studies.

Pathogenetic Mechanisms of Mental Disorders: Endogenous Intoxication.

The review describes the syndrome of endogenous intoxication in patients with mental disorders. Oxidative stress, middle-mass endotoxic molecules, impaired functional properties of serum albumin and albumin thiol groups, neurotrophic factors, and enzymes, including monoamine oxidase and semicarbazide-sensitive amine oxidase contribute to the development of endogenous intoxication. Possible pathogenetic mechanisms of the endogenous intoxication development in mental disorders and approaches to its treatment are discussed.

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing.

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.

The Biological Implication of Semicarbazide-Sensitive Amine Oxidase (SSAO) Upregulation in Rat Systemic Inflammatory Response under Simulated Aerospace Environment.

The progress of space science and technology has ushered in a new era for humanity's exploration of outer space. Recent studies have indicated that the aerospace special environment including microgravity and space radiation poses a significant risk to the health of astronauts, which involves multiple pathophysiological effects on the human body as well on tissues and organs. It has been an important research topic to study the molecular mechanism of body damage and further explore countermeasures against the physiological and pathological changes caused by the space environment. In this study, we used the rat model to study the biological effects of the tissue damage and related molecular pathway under either simulated microgravity or heavy ion radiation or combined stimulation. Our study disclosed that ureaplasma-sensitive amino oxidase (SSAO) upregulation is closely related to the systematic inflammatory response (IL-6, TNF-α) in rats under a simulated aerospace environment. In particular, the space environment leads to significant changes in the level of inflammatory genes in heart tissues, thus altering the expression and activity of SSAO and causing inflammatory responses. The detailed molecular mechanisms have been further validated in the genetic engineering cell line model. Overall, this work clearly shows the biological implication of SSAO upregulation in microgravity and radiation-mediated inflammatory response, providing a scientific basis or potential target for further in-depth investigation of the pathological damage and protection strategy under a space environment.

Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of ß-N-Oxalyl-L-α,ß-diaminopropionic Acid (ß-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, ß-N-Oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify ß-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for ß-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though ß-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of ß-ODAP/kg body/day). CDC Amarillo showed 5000-fold less ß-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

The Impact of Semicarbazide Sensitive Amine Oxidase Activity on Rat Aortic Vascular Smooth Muscle Cells.

Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and H2O2. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and H2O2, as well as methylglyoxal and H2O2. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** p < 0.0001), while ßAPN demonstrated inhibitory potential only in benzylamine-treated cells (* p < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** p < 0.0001); the addition of MDL72527 and ßAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.

Genome-Wide Identification, Characterization, and Expression Analysis of the Copper-Containing Amine Oxidase Gene Family in Mangrove Kandelia obovata.

Copper-containing amine oxidases (CuAOs) are known to have significant involvement in the process of polyamine catabolism, as well as serving crucial functions in plant development and response to abiotic stress. A genome-wide investigation of the CuAO protein family was previously carried out in sweet orange (Citrus sinensis) and sweet cherry (Prunus avium L.). Six CuAO (KoCuAO1-KoCuAO6) genes were discovered for the first time in the Kandelia obovata (Ko) genome through a genome-wide analysis conducted to better understand the key roles of the CuAO gene family in Kandelia obovata. This study encompassed an investigation into various aspects of gene analysis, including gene characterization and identification, subcellular localization, chromosomal distributions, phylogenetic tree analysis, gene structure analysis, motif analysis, duplication analysis, cis-regulatory element identification, domain and 3D structural variation analysis, as well as expression profiling in leaves under five different treatments of copper (CuCl2). Phylogenetic analysis suggests that these KoCuAOs, like sweet cherry, may be subdivided into three subgroups. Examining the chromosomal location revealed an unequal distribution of the KoCuAO genes across four out of the 18 chromosomes in Kandelia obovata. Six KoCuAO genes have coding regions with 106 and 159 amino acids and exons with 4 and 12 amino acids. Additionally, we discovered that the 2.5 kb upstream promoter region of the KoCuAOs predicted many cis elements linked to phytohormones and stress responses. According to the expression investigations, CuCl2 treatments caused up- and downregulation of all six genes. In conclusion, our work provides a comprehensive overview of the expression pattern and functional variety of the Kandelia obovata CuAO gene family, which will facilitate future functional characterization of each KoCuAO gene.

LOXL2 in Cancer: A Two-Decade Perspective.

Lysyl Oxidase Like 2 (LOXL2) belongs to the lysyl oxidase (LOX) family, which comprises five lysine tyrosylquinone (LTQ)-dependent copper amine oxidases in humans. In 2003, LOXL2 was first identified as a promoter of tumour progression and, over the course of two decades, numerous studies have firmly established its involvement in multiple cancers. Extensive research with large cohorts of human tumour samples has demonstrated that dysregulated LOXL2 expression is strongly associated with poor prognosis in patients. Moreover, investigations have revealed the association of LOXL2 with various targets affecting diverse aspects of tumour progression. Additionally, the discovery of a complex network of signalling factors acting at the transcriptional, post-transcriptional, and post-translational levels has provided insights into the mechanisms underlying the aberrant expression of LOXL2 in tumours. Furthermore, the development of genetically modified mouse models with silenced or overexpressed LOXL2 has enabled in-depth exploration of its in vivo role in various cancer models. Given the significant role of LOXL2 in numerous cancers, extensive efforts are underway to identify specific inhibitors that could potentially improve patient prognosis. In this review, we aim to provide a comprehensive overview of two decades of research on the role of LOXL2 in cancer.

Enhanced Stability of Vegetal Diamine Oxidase with Trehalose and Sucrose as Cryoprotectants: Mechanistic Insights.

Enteric dysfunctions are common for various histamine-related intestinal disorders. Vegetal diamine oxidase (vDAO), an enzyme able to decompose histamine and thus alleviate histamine-related dysfunctions, was formulated in gastro-resistant tablet forms for oral administration as a food supplement and possible therapeutic agent. A major challenge for the use of proteins in the pharmaceutical field is their poor stability. In this study, vDAO was freeze-dried in the absence or in the presence of sucrose or trehalose as cryoprotectants and then formulated as tablets by direct compression. The stability of the obtained preparations was followed during storage at 4 °C and -20 °C for 18 months. In vitro dissolution tests with the vDAO powders formulated as tablets were performed in simulated gastric and in simulated intestinal fluids. The tablets obtained with the powder of the vDAO lyophilized with sucrose or trehalose cryoprotectants offered better protection for enzyme activity. Furthermore, the release of the vDAO lyophilized with the cryoprotectants was around 80% of the total loaded activity (enzyme units) compared to 20% for the control (vDAO powder prepared without cryoprotectants). This report revealed the potential of sucrose and trehalose as cryoprotectants to protect vDAO from freeze-drying stress and during storage, and also to markedly improve the vDAO release performance of tablets obtained with vDAO powders.

Histamine intolerance.

Histamine intolerance (HIT) is a non-immunological disorder associated with an impaired ability to metabolize ingested histamine. Manifestation of HIT includes gastrointestinal and non-gastrointestinal symptoms. Clinical symptoms of HIT are non-specific and can imitate different diseases such as allergies, food intolerance, mastocytosis and other. The diagnosis of HIT is difficult. There are several candidate tests to detect DAO insufficiency, but their informative value is questionable. Currently, a positive clinical effect of a low-histamine diet is the most important for establishing the diagnosis. Equally in the treatment, a low-histamine diet is the most crucial approach. Other therapeutic options such as DAO supplementation treatment with antihistamines or probiotics are considered as complementary treatments. Our article provides a review on histamine intolerance, focusing on etiology and the diagnostic and treatment possibilities.

Glycosylation site Asn168 is important for slow in vivo clearance of recombinant human diamine oxidase heparin-binding motif mutants.

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants.