Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells

Öhman, Johan; Jakobsson, Emma; Källström, Ulla; Elmblad, Annette; Ansari, Akbar Ali; Kalderén, Christina; Robertson, Elinor; Danielsson, Eva; Gustavsson, Anna‐Lena; Varadi, Andrea; Ekblom, Jonas; Holmgren, Erik; Doverskog, Magnus; Abrahmsén, Lars; Nilsson, Joakim

doi:10.1016/j.pep.2005.10.027

Cited by 4 publications

(9 citation statements)

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“…The yield of sVAP-1 after purification using size exclusion chromatography ranged from 10 to 59 mg/liter of culture medium (n ϭ 6 purifications). The expression of sVAP-1 was markedly higher than what had been reported by others (27). To calculate the amount of active sVAP-1, the concentration of TPQ cofactor was determined by titrating the enzyme against phenylhydrazine, which forms a stable covalent adduct with the cofactor.…”

Section: Resultsmentioning

confidence: 87%

“…The calculated mass of sVAP-1 is 81,571.9 g/mol, which does not correspond to the apparent mass derived from SDS-PAGE analysis. However, it has been proposed that glycosylation of sVAP is likely responsible for this discrepancy (27). The expressed sVAP-1 was found to be virtually inactive (0.3 unit/ ml) because of the low concentration of copper in the medium.…”

Section: Resultsmentioning

confidence: 99%

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Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

Heuts

Gummadova

Pang

et al. 2011

Journal of Biological Chemistry

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Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pK a values that defined a bell-shaped plot of turnover number k cat,app as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (k cat /K m ) app on pH revealed a single pK a value (ϳ9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (k cat /K m ) app over the pH range of 6 to 10 was observed with d 2 -benzylamine. Over the same pH range, the KIE on k cat was found to be close to unity. The unusual KIE values on (k cat / K m ) app were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on k cat,app (8.01 ؎ 0.28 with phenylethylamine), indicating that C-H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.Copper amine oxidases or semicarbazide-sensitive amine oxidases (CAOs/SSAOs 3 ; EC 1.4.3.21/EC 1.4.3.22) comprise a group of copper-dependent enzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes with concomitant release of hydrogen peroxide and ammonia. (R is a phenyl moiety in case of benzylamine and a benzyl moiety in case of phenylethylamine.)From the viewpoint of structure and mechanism, copper-dependent amine oxidases have been researched extensively for several decades, but relatively little is known about their physiological function.Human vascular adhesion protein-1 (VAP-1) is a dimeric membrane bound and circulating protein that is encoded by the AOC3 gene and is expressed mainly in endothelial cells of blood vessels, adipocytes, and smooth muscle cells (1-3). VAP-1 is a protein comprising two structural domains: an adhesive domain that targets leukocytes for transmigration and an amine oxidase domain (2, 4). At sites of inflammation on the endothelial cell surface, VAP-1 is involved in the recruitment and extravasation of lymphocytes and neutrophils. VAP-1 activity generates important signaling compounds such as hydrogen peroxide (5, 6). SSAO activity is also involved in glucose transport in adipose cells (1...

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Section: Resultsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

Heuts

Gummadova

Pang

et al. 2011

Journal of Biological Chemistry

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“…To confirm the newly identified protein interaction, it was attempted to purify AOC3 from E. coli . Since all expression conditions failed, a modified method using HEK293 cells as expression host was chosen [48]. Using lentivirus, secretable forms of GST-AOC3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Leukocyte transmigration in AOC3 k.o. mice is massively hampered, leading to abnormal leukocyte traffic [47] and strongly reduced leukocyte infiltration into adipose tissue [48]. AOC3 appears to have both catalytic and adhesive functions, although the molecular mechanism mediating leukocyte migration is incompletely understood.…”

Section: Discussionmentioning

confidence: 99%

“…An expression plasmid, pSpexMax, was constructed as shown in Figure 9 for the production of both murine AOC3 and murine ZAG in mammalian cells. The leader sequence of Ig kappa light chain was taken from Ohman et al [48], which directs the protein into the medium. The SP163 translational enhancer sequence was incorporated upstream of the leader sequence to promote recombinant protein translation, while the GST-tag, equipped with a cleavage site (recognised by PreScission Protease; GE Healthcare), was inserted downstream of the leader to facilitate affinity purification.…”

Section: Methodsmentioning

confidence: 99%

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Zinc-α2-Glycoprotein Is An Inhibitor Of Amine Oxidase Copper-Containing 3

Romauch¹

2019

Preprint

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6Zinc-alpha2-glycoprotein (ZAG) is a major plasma protein whose levels increase in 7 chronic energy-demanding diseases and thus serves as an important clinical biomarker 8 in the diagnosis and prognosis of the development of cachexia. Current knowledge 9 suggests that ZAG mediates progressive weight loss through β-adrenergic signaling in 10 adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through 11 crosslinking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a 12 novel ZAG binding partner. AOC3 -also known as vascular adhesion protein 1 (VAP-1) 13 and semicarbazide sensitive amine oxidase (SSAO) -deaminates primary amines, 14 thereby generating the corresponding aldehyde, H2O2 and HN3. It is an ectoenzyme 15 largely expressed by adipocytes and induced in endothelial cells during inflammation. H2O2 has an insulinogenic effect. The observations described here suggest that ZAG acts 18 as an allosteric inhibitor of AOC3 and interferes with the associated pro-inflammatory 19 and anti-lipolytic functions. Thus, inhibition of the deamination of lipolytic hormone 20 octopamine by AOC3 represents a novel mechanism by which ZAG might stimulate 21 lipolysis. Furthermore, experiments involving overexpression of recombinant ZAG 22 reveal that its glycosylation is co-regulated by oxygen availability and that the pattern of 23 glycosylation affects its inhibitory potential. The newly identified protein interaction 24 2 between AOC3 and ZAG highlights a previously unknown functional relationship, which 25 may be relevant to inflammation, energy metabolism and the development of cachexia. 26 27 93 provide much-needed insight into the mechanism of ZAG function and stimulate future work 94 in basic and clinical research on ZAG.95 96 5 2 Results 97 2.1 ZAG binds to ectoenzyme AOC3 98To attempt to identify ZAG interaction partners, purified recombinant ZAG and freshly 99 prepared adipocyte plasma membranes were co-incubated and any physical interactions 100 between them were stabilized by a photoactivatable crosslinker molecule (Fig. 11). Both 101 human and murine ZAG (without leader sequence) were produced in E. coli after cloning in 102 the expression plasmid pGEX-6P-2 and affinity purified by GST (glutathione-S-transferase)-103 tag. Both purified human and mouse proteins (GST-hZAG and GST-mZAG, respectively) and 104 GST-tag aloneserving as a controlwere labeled with the photoactivatable crosslinker 105

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Zinc-α2-glycoprotein as an inhibitor of amine oxidase copper-containing 3

Romauch

2020

Open Biol.

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Zinc-α2-glycoprotein (ZAG) is a major plasma protein whose levels increase in chronic energy-demanding diseases and thus serves as an important clinical biomarker in the diagnosis and prognosis of the development of cachexia. Current knowledge suggests that ZAG mediates progressive weight loss through β-adrenergic signalling in adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through cross-linking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a novel ZAG binding partner. AOC3—also known as vascular adhesion protein 1 (VAP-1) and semicarbazide sensitive amine oxidase (SSAO)—deaminates primary amines, thereby generating the corresponding aldehyde, H 2 O 2 and NH 3 . It is an ectoenzyme largely expressed by adipocytes and induced in endothelial cells during inflammation. Extravasation of immune cells depends on amine oxidase activity and AOC3-derived H 2 O 2 has an insulinogenic effect. The observations described here suggest that ZAG acts as an allosteric inhibitor of AOC3 and interferes with the associated pro-inflammatory and anti-lipolytic functions. Thus, inhibition of the deamination of lipolytic hormone octopamine by AOC3 represents a novel mechanism by which ZAG might stimulate lipolysis. Furthermore, experiments involving overexpression of recombinant ZAG reveal that its glycosylation is co-regulated by oxygen availability and that the pattern of glycosylation affects its inhibitory potential. The newly identified protein interaction between AOC3 and ZAG highlights a previously unknown functional relationship, which may be relevant to inflammation, energy metabolism and the development of cachexia.

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Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells

Cited by 4 publications

References 45 publications

Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

Zinc-α2-Glycoprotein Is An Inhibitor Of Amine Oxidase Copper-Containing 3

Zinc-α2-glycoprotein as an inhibitor of amine oxidase copper-containing 3

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