Differential expression of semicrabazide-sensitive amine oxidase (SSAO) in human umbilical arterial tissue, E.coli BL21, HEK and HUAEC

Proceedings of 2013 ICME International Conference on Complex Medical Engineering May 25 - 28, Beijing, China

Differential expression of Semicrabazide-sensitive amine oxidase (SSAO) in human umbilical arterial tissue, E.coli BL21, HEK and HUAEC Kaleem Ullah1,2, Amir Rasool1, Xie Bingjie1, Yulin Deng1* 1

School of Life Science and Technology, Beijing Institute of Technology, 100081 Beijing China Department of Microbiology, University of Balochistan, Quetta 87300 Balochistan, Pakistan *Correspondence Author (E-mail: [email protected])

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cellular differentiation [4, 5] for example deposition of extracellular matrix in smooth muscle cells [6], lipid trafficking in adipocytes [7] and control of muscular tone [8]. The mechanism of participation of SSAO in cellular differentiation is not yet completely clear. SSAO activity level altered in different pathological situations, such as diabetes [9], congestive heart failure [10], atherosclerosis [11], inflammatory condition [12, 13], AD (Alzheimer’s disease) [14] and others [15, 16]. So, keeping in view the higher activity of SSAO in different diseases, it is assumed that excessive SSAO contributes and play important role in above pathologies. Evidences have already been reported such as; an increased SSAO activity was involved in vascular tissue damage and others [17, 18]. It is therefore, research on modifying SSAO activity for therapeutic purpose is focused and progressively increasing in number [19]. While on other hand, mechanisms regarding excessive SSAO expression and connection to pathological consequences are uncertain. One reason of this uncertainty could be due to the difficulty of studying SSAO in cell or tissue systems [20]. Therefore, we present a comparative study of SSAO expression and enzymatic activity in natural source like human umbilical tissue, universal recombinant expression system like in E.coli, wildly used cell line such as HEK and naturally SSAO expressive cell like HUAEC. This comparison has allowed us for the first time to describe SSAO expression in different models and will be helpful for the evaluation of pharmaceutical compounds that could alter its activity for therapeutic concerns.

Abstract—Semicarbazide-sensitive amine oxidase (SSAO) is multifunctional enzyme which is highly express in vasculature, smooth muscle and adipose tissues. Clinical and experimental studies suggest that increased activity and the catalytic product of deamination may be toxic and cause the tissue damage. Correlation between the SSAO activity and severity of different serious pathological condition such as Diabetes, Alzheimer’s disease and atherosclerosis have been widely reported but pathophysiological meaning of this enzyme is not yet discovered. Therefore, research related to SSAO as therapeutic objective are becoming more important. We present liquid chromatography electrospray ionization mass spectrometry (LC-MS/MS) based comparative enzymatic activity and expression study in human umbilical tissue, universal recombinant expression system E.coli, commonly used cell line i.e., human embryonic kidney cell (HEK) and naturally SSAO expressive primary vascular cell line i.e., human umbilical arterial endothelial cell (HUAEC). Our results indicate that HEK:SSAO is best model among these while the second most abundant activity was observed in E.coli BL21:SSAO, with a rough estimation HEK:SSAO shows two to three fold higher activity than HUAEC. The concept of comparison provides new insight on this enzyme expression. Our models also open new possibilities for the evaluation of potential compounds that could modify SSAO activity for therapeutic determinations. Keywords- Semicarbazide-sensitive Amine Oxidase (SSAO); human umbilical artery; human embryonic kidney cell (HEK); E.coli BL21; human umbilical arterial endothelial cells (HUAEC)

I.

INTRODUCTION

SSAOs widely exist in plants, microorganisms and mammals. It converts primary amines to aldehydes (except histamine). During this biochemical process hydrogen peroxide and ammonium are also formed. There are two isoforms of SSAOs are identified in mammals: the membrane bound and soluble (plasma) isoforms [1]. The majority of the SSAOs can be found in the vascular walls of brain and other tissue including smooth muscle, adipocytes, retina, placenta and bone marrow etc. Surprisingly, after cloning the primary structure of SSAO, it was found identical to another protein, which was mapped before, the vascular adhesion protein (VAP-1) [2]. To the best of our knowledge, up till now full length cDNA sequences are available from seven mammalian SSAOs. Regarding human, SSAO enzyme has been cloned [2, 3] and found that it contains 762 amino acids. In addition, SSAO also take part in 978-1-4673-2971-2/13/$31.00 ©2013 IEEE

II. A.

MATERIALS AND METHODS

Chemicals and Reagents Sodium dodecyl sulfate (SDS) was purchased from SigmaAldrich (Steinheim, Germany). The protease inhibitor was supplied from Roche (Mannheim, Germany). E.coli BL21 (DE3) was purchased from Novagen (Beijing, China). HUAEC cells purchase from ScienCell (San Diego, CA). HEK and SH-SY5Y cells were stored in our Lab. Monoclonal human SSAO antibodies were bought from R&D Systems (Minneapolis, MN).

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H. Measurement of SSAO activity in HEK and HUAEC SSAO activity was checked by LC-MS/MS analysis developed by our Lab group [24]. SSAO activity was defined by the production of formaldehyde (pmol) per minute/mg protein intervened by SSAO from MS detector response. Briefly, an amount of 300 ȝg of Cell lysates or sample protein were used in each reaction and triplicates were made for each sample. Proteins with respective controls were pre-incubated with 50 uL clorgyline (10 mM) and 200 ml PBS (50 mM, pH 6.8) for 20 min at room temperature to block the MAO activity completely. Followed by incubation with or without (50 ȝL) Semicarbazide (10 mM) at room temperature for 20 min before the addition of substrate. The enzymatic reaction was begun in the presence of 20 ml methylamine (25 mM) and 20 ȝl dopamine (10 mM) at 37°C for 30 min. The reactions were then terminated by addition of perchloric acid (PCA, 1 M); here we used isoproterenol (10 mM) as internal standard the reaction mixture were then injected into the HPLC-ESIMS directly.

B. Tissue Sample preparation The human umbilical artery was provided by the XuanWu Hospital, Beijing, China and prepared as described by [21]. Briefly the tissue homogenate was centrifuged at 1,000×g for 10 min. The supernatant was collected and centrifuged at 17,000×g for 30 min. The resulting supernatant was and quantified by Bradford assay. C. Construction of expression vector for E.coli BL21 Full-length human SSAO was amplified from plasmid pCMV SPORT6- ssao (Invitrogen, CA) and cloned into the corresponding site of pGEX-6p-2 (Invitrogen) or as described by [22]. Briefly, the resulting PCR product was cloned into pGEX-6p-2 vector and transformed to E.coli BL21 (TransGen, Beijing-China) in the presence of 50 mg mLí1 ampicillin. Positive transformants were identified by restriction digestion and sequenced. D. SSAO Induction and expression in E.coli BL21 Positive E.coli BL21 was grown in shaking incubator for 16 h at 37°C and further E.coli cells were induced to express SSAO as described by [22] briefly total cell protein were used and protein concentration was determined by Bradford assay.

I.

Liquid chromatography and mass spectrometry LC-MS/MS analysis was done by the method used by [24] briefly mobile phase was consisted of methanol–water (25/75 v/v) with 10 mM ammonium formate (pH 3.5) and delivered at flow rate of 0.15 ml/min. The injection volume was 20 ȝl. The [M+H] + precursor ions/ product ions for the MRM scans were used for N-methyl-salsolinol (m/z166/117.1) and isoproterenol (m/z 212.1/107.1)

E. Construction of expression vector for HEK and HUAEC Amplified Full-length human SSAO was also cloned into the corresponding site of pcDNA3.1-myc-His (-) (Invitrogen), according to manufacturer instruction. Plasmid DNA was confirmed by restriction enzyme digestion and sequencing.

J.

F. Cell culture and transfection The HUAEC and HEK were cultured according to the standard protocol, pcDNA3.1(-) vector containing human SSAO was transfected in HUAEC using jetPEI (Polyscience, Europe) and lipofectamine2000 (Invitrogen, China) was used in HEK cell. After 24 hours cell were harvested. Total cell lysates were obtained by homogenizing cells in 50 mmol/L Tris/HCl, pH 7.5, 1% Triton X-100, 10 mmol/L EDTA and protease inhibitors. The protein concentration of the total cell lysates was determined by the Bradford method.

Statistics

Results were expressed as the mean standard deviation (±SD). Statistical evaluations were performed using Student’s t-test. P-Values of less than 0.05 were considered statistically significant and for multiple comparisons the results were assessed using analytical software of SPSS 11.0 followed by multiple comparisons (Newman-Keuls). III.

RESULTS

A. SSAO expression in Human Umbilical Artery, E.coli BL21, HUAEC and HEK

G. Immunoblot analyses Equal amounts of protein (20ȝg/lane) of tissue sample, E.coli BL21, HUAEC and HEK were separated by SDSPAGE 7.5% gel (Bio-Rad mini-protein III apparatus) in triplicate. The gels were electroblotted onto a 0.45-ȝm PVDF membrane. Membranes were blocked for 1 h with TBS/0.1% Tween buffer plus 5% (w/v) non-fat dried skimmed milk powder and incubated overnight at 4°C with antibodies raised against human SSAO and mouse anti-ȕ-actin (1:2000) (Sigma–Aldrich). After incubation with the corresponding secondary antibodies, blots were developed using ECL® Chemoluminiscent detection reagents and high performance Chemiluminiscence Films (GE Healthcare). For the negative control we used SH-SY5Ycells. The results were quantitated by densitometric analysis.

In order to evaluate tissue model for SSAO expression and compare with other models, tissue protein was prepared and quantified. The SSAO protein was found to be present in tissue lysates of umbilical artery which were analyzed in triplicate by protein immunoblot (Fig. 1A). Banding pattern of SSAO activity in human umbilical artery was more than HUAEC:SSAO, but this expression was less than HEK:SSAO, while lack of protein expression and activity in negative control (SH-SY5Y) was also clear. Moreover, the stability and reliability of SSAO quantification was assessed by repeating experiment as triplet. No significance variations were observed in case of SSAO expression although a very low band pattern variation was noticed while beta action used for the loading control. Universal expression model, the E.coli was used as a model to study SSAO activity and compare with other models.

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SSAO quantification was assessed in the same way used for other models.

(A)

B. SSAO Enzymatic activity differences in Human Umbilical Artery, E.coli BL21 HUAEC and HEK LC-MS/MS method [24] was successfully applied to determine SSAO activity in human umbilical tissue, E.coli BL21:SSAO, HUAEC:SSAO and HEK:SSAO cells. In our experiment the linearity was obtained from the regression of the peak area ratio against the concentration of formaldehyde from 0.5 to 5.00 μM. Regression analysis resulted in the equation y=0.38x+0.01 and the r value was 0.989, SSAO activity was defined by the production of formaldehyde (pmol) per minute/mg protein mediated by SSAO as obtained from a series of detector response analyses. As shown in the (Fig. 2A) SSAO activity of HEK was found increased comparatively to human umbilical tissue, E.coli BL2:SSAO, HUAEC:SSAO. The SSAO activity was 570.53±18.04, 223.99±20.8, 471±10.4 and 516.66±11.5 in HUVEC:SSAO, HEK:SSAO, umbilical artery tissue and E.coli BL21:SSAO respectively.

(B)

(A) Fig. 1 Differential SSAO expression in HUAEC, HEK, umbilical tissue and E.coli BL21 (A) Determination of SSAO analysis by immunoblotting, Lane 1& 3 indicates Negative Control (SH-SY5Y cell), Lane 2 shows protein Molecular Weight Marker and Lane 5, 7 & 10 represent Control cell (HUAEC, HEK and E.coli BL21 respectively without ssao transfection). Lane 4 & 6 represent HUAEC & HEK transfected with pcDNA.3.1/myc-His(-):SSAO, Lane 9 indicates E.coli BL21 transfected with pGEX-6p-2:SSAO. down from Lane 1,3-8 represent the beta-actin. (B) Densitometric analysis of SSAO expression by Western Blotting, Numerical ratios between samples are plot. Results were expressed as the mean (±SD) of three independent experiments

After induction of SSAO by IPTG, E.coli BL21 total proteins lysate were centrifuged and proteins were quantified. The SSAO protein was found in lysates of E.coli BL21:SSAO which were analyzed in triplicate by protein immunoblot (Fig. 1A). SSAO activity of recombinant expression in E.coli BL21:SSAO was quite prominent than all other model except HEK:SSAO, which showed more SSAO activity in banding pattern. The lack of protein expression and activity in negative control of E.coli BL21 without transfection was also clear. Moreover, the stability of SSAO quantification was assessed by repeating experiment in triplet. Very low band variations were seen and no significance variations were observed. To compare SSAO expression and activity with other models we developed two cell line model i.e., HEK and HUAEC, after transfection with pcDNA3.1(-):SSAO vector in HUAEC and HEK, cell were harvested and homogenized in lysis buffer (mention earlier) followed by protein quantification. The SSAO protein was found in total cell lysates of HUAEC:SSAO and HEK:SSAO which were also in triplicate by protein immunoblot (Fig. 1A). According to banding pattern of SSAO activity in HUAEC:SSAO cell were quite less than HEK:SSAO and all other models, the lack of protein expression and activity in negative controls like HEK, HUAEC and SH-SY5Y was also clear. The reliability of

(B)

Fig. 2 Comparisons of SSAO activity in HUAEC, HEK, umbilical tissue and E.coli BL21 (A) Determination of SSAO activity in by LC MS/MS method. (B) The transfected SSAO in HEK:SSAO is sensitive to inhibition by Sc (10mM). The data are presented as the mean (±SD) of three independent experiments

Moreover, the successful inhibition of positive HEK cell lysate containing SSAO was achieved by Semicarbazide. As can be seen in (Fig. 2B), the levels of SSAO activity in HEK control or cell containing SSAO with or without

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Semicarbazide were 4±3.5, 535±13.2 respectively.

28.33±17.3

and,

expression is different. Higher SSAO expression in our models could be of much important in a pathophysiological condition where increase SSAO activity have been seen. Taken together, result indicates that our models could be used for the in vitro study of this enzyme. HUAEC naturally express SSAO in vivo, So, that is considered to provide the proper micro environment for SSAO expression, although we found less activity in these cell when compared with HEK and E.coli BL21, a possible reason could be that these cell are hard to transfect and can’t achieved a good percentage of transfection efficiency with conventional method of transfection. HEK and E.coli BL21 models which are missing naturally SSAO expression are still widely using for basic research on diseases. Moreover, SSAO is a multifunctional protein contributes in large number of processes and could not be entirely understood by only cell models. In conclusion, all the four models compare here could provide suitable experimental tools to determine the SSAO functions clearly. Our models will also be helpful for elucidating the role of SSAO in related pathologies and for the assessment of possible pharmacological compounds that could modify its pathophysiological activity.

14±5.29,

C. Comparative SSAO activity values and Densitometric Unit of Immunoblot Quantification After comparing all models together by western blotting and enzymatic activity, results showed that HEK:SSAO is best model among human umbilical arterial tissue, E.coli BL21 and HUAEC. The Second most abundant expression and activity were observed in E.coli BL21:SSAO, E.coli was also found appropriate in expressing human SSAO. With a rough estimation HEK:SSAO shows 2-3 fold higher activity then HUAEC. Moreover, HEK cell were found easy to grow and handle then HUAEC. ß-actin expression was very useful as indicator, reflecting the protein concentration among cell lines but in case of human umbilical tissue ß-actin banding pattern was quite prominent then cellular models, it was may be due to difference in the gene expression in tissue and laboratory cell line. IV.

DISCUSSION

In order to explore the role of SSAO in cerebrovascular and other tissues related to serious pathologies such as Alzheimer’s disease and also its contribution to vascular damage, it was important to work with SSAO. Although SSAO have been widely cloned using different expression systems but the expression is not achieved consistent, could be due to the effect of its enzymatic product or respond to gene expression in vitro culturing where SSAO expression lost progressively. The main reason for comparison study was due to involvement and contribution of SSAO in various severe pathological situations. Moreover, reported data indicated that SSAO plays a very important role such as leucocyte trafficking during inflammation condition, tissue maturation, and endothelial cell damage via aliphatic amine metabolism in different pathologies but the exact mechanism involved is yet not explored. This could be due to the lack of suitable expression models. Here, we have reported SSAO from different sources in a comparison way to elucidate its expression and enzymatic activity in human umbilical tissue, E.coli BL21, HEK and HUAEC models. In order to assess SSAO activity from direct human tissue, prokaryotic expression system and two cell lines with immortal and mortal property were focused and demonstrated here. In this respect different level of SSAO expression were found from very efficient in HEK:SSAO cell model to comparatively less efficient expression in HUAEC:SSAO model and in agreement with the hypothesis that SSAO expression and activity controlling mechanism responds differentially in different expression system [20]. Protein expression of SSAO varies in our models. These findings are not in full agreement with the activities 91.9±11.5 pmol/min per mg of protein in HUVEC hSSAO/VAP-1 compared with 133±43 in lung [23]. The substantial argue for this reason is uncertain, but it could be either due to the use of different method to determine the SSAO activity or explained in the way that the susceptibility of these models to SSAO gene

V.

ACKNOWLEDGMENT

The authors are thankful for the support from Ministry of Science and Technology of China (2009BAK59B02) and the Higher Education Commission of Pakistan (HEC).

VI. [1]

[2]

[3]

[4]

[5]

[6]

[7]

[8]

511

REFERENCES

Abella A, Garcia-Vicente S, Viguerie N, Ros-Baro A, Camps M, Palacin M, et al. “Adipocytes release a soluble form of VAP-1/SSAO by a metalloprotease-dependent process and in a regulated manner,” Diabetologia, 2004,47(3), pp. 429-438. Smith DJ, Salmi M, Bono P, Hellman J, Leu T, Jalkanen S. “Cloning of vascular adhesion protein 1 reveals a novel multifunctional adhesion molecule,” J Exp Med, 1998, 188(1), pp.17-27. Zhang X, McIntire WS. “Cloning and sequencing of a coppercontaining, topa quinone-containing monoamine oxidase from human placenta,” Gene, 1996, 179(2), pp. 279-286. Mercier N, Moldes M, El Hadri K, Feve B. “Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells,” Biochem J, 2001, 358(Pt 2), pp. 335-342. El Hadri K, Moldes M, Mercier N, Andreani M, Pairault J, Feve B. “Semicarbazide-sensitive amine oxidase in vascular smooth muscle cells: differentiation-dependent expression and role in glucose uptake,” Arterioscler Thromb Vasc Biol, 2002, 22(1), pp.89-94. Langford SD, Trent MB, Boor PJ. “Semicarbazide-sensitive amine oxidase and extracellular matrix deposition by smooth-muscle cells,” Cardiovasc Toxicol, 2002, 2(2), pp.141-150. Souto RP, Vallega G, Wharton J, Vinten J, Tranum-Jensen J, Pilch PF. “Immunopurification and characterization of rat adipocyte caveolae suggest their dissociation from insulin signaling,” J Biol Chem, 2003, 278(20), pp.18321-18329. Conklin DJ, Cowley HR, Wiechmann RJ, Johnson GH, Trent MB, Boor PJ. “Vasoactive effects of methylamine in isolated human blood vessels: role of semicarbazide-sensitive amine oxidase, formaldehyde, and hydrogen peroxide,” Am J Physiol Heart Circ Physiol, 2004, 286(2), pp. H667-676.

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

Boomsma F, Derkx FH, van den Meiracker AH, Man in 't Veld AJ, Schalekamp MA. “Plasma semicarbazide-sensitive amine oxidase activity is elevated in diabetes mellitus and correlates with glycosylated haemoglobin,” Clin Sci (Lond), 1995, 88(6), pp.675-679. Boomsma F, van Veldhuisen DJ, de Kam PJ, Man in't Veld AJ, Mosterd A, Lie KI, et al. “Plasma semicarbazide-sensitive amine oxidase is elevated in patients with congestive heart failure,” Cardiovasc Res., 1997,33(2), pp.387-391. Meszaros Z, Szombathy T, Raimondi L, Karadi I, Romics L, Magyar K. “Elevated serum semicarbazide-sensitive amine oxidase activity in noninsulin-dependent diabetes mellitus: correlation with body mass index and serum triglyceride,” Metabolism, 1999, 48(1), pp.113-117. Kurkijarvi R, Adams DH, Leino R, Mottonen T, Jalkanen S, Salmi M. “Circulating form of human vascular adhesion protein-1 (VAP-1): increased serum levels in inflammatory liver diseases,” J Immunol, 1998, 161(3), pp. 1549-1557. Madej A, Reich A, Orda A, Szepietowski JC. “Vascular adhesion protein-1 (VAP-1) is overexpressed in psoriatic patients,” J Eur Acad Dermatol Venereol, 2007, 21(1), pp.72-78. del Mar Hernandez M, Esteban M, Szabo P, Boada M, Unzeta M. “Human plasma semicarbazide sensitive amine oxidase (SSAO), betaamyloid protein and aging,” Neurosci Lett, 2005, 384(1-2), pp.183-187. Nemcsik J, Szoko E, Soltesz Z, Fodor E, Toth L, Egresits J, et al. “Alteration of serum semicarbazide-sensitive amine oxidase activity in chronic renal failure,” J Neural Transm, 2007, 114(6), pp.841-843. Hernandez-Guillamon M, Garcia-Bonilla L, Sole M, Sosti V, Pares M, Campos M, et al. “Plasma VAP-1/SSAO activity predicts intracranial hemorrhages and adverse neurological outcome after tissue plasminogen activator treatment in stroke. Stroke” 2010, 41(7), pp.1528-1535. Hernandez M, Sole M, Boada M, Unzeta M. “Soluble semicarbazide sensitive amine oxidase (SSAO) catalysis induces apoptosis in vascular smooth muscle cells,” Biochim Biophys Acta, 2006, 1763(2), pp.164173. Sole M, Hernandez-Guillamon M, Boada M, Unzeta M. “p53 phosphorylation is involved in vascular cell death induced by the catalytic activity of membrane-bound SSAO/VAP-1,” Biochim Biophys Acta, 2008, 1783(6), pp.1085-1094. Iffiu-Soltesz Z, Wanecq E, Lomba A, Portillo MP, Pellati F, Szoko E, et al. “Chronic benzylamine administration in the drinking water improves glucose tolerance, reduces body weight gain and circulating cholesterol in high-fat diet-fed mice,” Pharmacol Res, 2010, 61(4), pp.355-363. Jaakkola K, Kaunismaki K, Tohka S, Yegutkin G, Vanttinen E, Havia T, et al. “Human vascular adhesion protein-1 in smooth muscle cells,” Am J Pathol, 1999,155(6),pp.1953-1965. Zhang Y, Xiao S, Wang L, Wang H, Zhu Y, Li Y, et al. “Absolute quantification of semicarbazide-sensitive amine oxidase in human umbilical artery by single-reaction monitoring with electrospray tandem mass spectrometry,” Anal Bioanal Chem, 2010, 397(2), pp.709-715. Zhang Y, Lai C, Duan J, Guan N, Ullah K, Deng Y. “Simulated microgravity affects semicarbazide-sensitive amine oxidase expression in recombinant Escherichia coli by HPLC-ESI-QQQ analysis,” Appl Microbiol Biotechnol, 2011. Andres N, Lizcano JM, Rodriguez MJ, Romera M, Unzeta M, Mahy N. “Tissue activity and cellular localization of human semicarbazidesensitive amine oxidase,” J Histochem Cytochem ,2001, 49(2). pp. 20917. Wang L, Zhang Y, Xiao S, Hu G, Che B, Qing H, Li Y, Zhuang L, Deng Y (2012) LC-MS method for determining the activity of semicarbazidesensitive amine oxidase in rodents. Analytical Methods, 2012, 4 (5):1383-1388

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