GALNT14与MT2A相互作用验证

doi:10.3969/j.issn.1000-1565.2016.01.011

摘要/Abstract

摘要： 利用免疫共沉淀(Co-IP)和蛋白沉降实验(GST pull-down),分别从体内外对N-乙酰氨基半乳糖转移酶(GALNT14)与金属硫蛋白2A(MT2A)之间的相互作用进行了验证.将MT2A全长cDNA片段克隆到pET-DsbA和pCMV-Myc载体上.表达并纯化了His-Dsba-MT2A和GST-GALNT14蛋白,在体外通过GST pull-down技术分析,显示MT2A能与GALNT14结合,证实了二者在体外的直接相互作用.继而,共转染pCMV-Myc-MT2A和pCDNA3.1-Flag-GALNT14到人乳腺癌细胞株MCF-7中,通过免疫共沉淀实验发现抗Myc抗体可以将GALNT14从细胞裂解液中沉淀下来,证实了它们在胞内的相互作用.结果显示了GALNT14和MT2A在体内外条件下能够发生直接相互作用,这为进一步明确GALNT14的功能及作用机制奠定了基础.

关键词: N-乙酰氨基半乳糖转移酶14(GALNT14), 金属硫蛋白2A(MT2A), 蛋白沉降实验(GST pull-down), 免疫共沉淀(Co-IP)

Abstract: Co-immunoprecitipation assay and GST pull-down assay were applied to validate the interaction of GALNT14 and MT2A in vivo and in vitro.The cDNA fragment of MT2A was cloned into pET-DsbA and pCMV-Myc.His-Dsba-MT2A and GST-GALNT14 were expressed and purified,and the results of GST pull-down assay showed that MT2A could bound with GALNT14,which confirmed the directly interaction between them in vitro.Then pCMV-Myc-MT2A and pCDNA3.1-Flag-GALNT14 were co-transfected into human breast cancer cell MCF-7.The results of Co-IP illustrated that anti-Myc antibody can precipitated GALNT14 from cell lysates,which revealed MT2A interacted with GALNT14 in vivo.The results showed that GALNT14 could bind MT2A and interact with each other directly in vivo and in vitro,which provided some promising clue for the further exploration into the function of GALNT14.

Key words: N-acetylgalactosaminyltransferase 14(GALNT14), metallothionein 2A(MT2A), GST pull-down assay(GST pull-down), co-immunoprecitipation analysis(Co-IP)

中图分类号:

左涛,刘学敏,单金帅,汤静珍,吴琛. GALNT14与MT2A相互作用验证[J]. 河北大学学报(自然科学版), 2016, 36(1): 65-71.

ZUO Tao,LIU Xuemin,SHAN Jinshuai,TANG Jingzhen,WU Chen. Validation of the interaction of GALNT14 and MT2A[J]. Journal of Hebei University (Natural Science Edition), 2016, 36(1): 65-71.

参考文献

[1] PEGRAM M D,BORGES V F,IBRAHIM N,et al.Phase I dose escalation pharmacokinetic assessment of intravenous humanized anti-MUC1 antibody AS1402 in patients with advanced breast cancer[J].Breast Cancer Res,2009,11(5):R73.DOI:10.1186/bcr2409.
[2] BURCHELL J M,MUNGUL A,TAYLOR-PAPADIMITRIOU J.O-Linked glycosylation in the mammary gland:changes that occur during malignancy[J].J Mammary Gland Biol Neoplasia,2001,6(3):355-364.DOI:10.1023/A:1011331809881.
[3] RöTTGER S,WHITE J,WANDALL H H,et al.Localization of three humanpolypeptide GalNAc-transferases in HeLa cells suggests initiation of O-linked glycosylation throughout the Golgi apparatus[J].J Cell Sci,1998,111(Pt1):45-60.DOI:10.1242/jeb.01346.
[4] PERRINE C L,GANGULI A,WU P,et al.Glycopeptide-preferring polypeptide GalNAc transferase 10(ppGalNAc T10),involved in mucin-type O-glycosylation,has a unique GalNAc-O-Ser/Thr-binding site in its catalytic domain not found in ppGalNAc T1 or T2[J].J Biol Chem,2009,284(30):20387-20397.DOI:10.1074/jbc.M109.017236.
[5] TEN HAGEN K G,FRITZ T A,TABAK L A.All in the family:the UDP-GalNac:polypeptide N-acetylgalactosaminyltransferases[J].Glycobiology,2003,13(1):1R-16R.DOI:10.1093/glycob/cwg007.
[6] 沈宏杰,吴士良.多肽:N-乙酰氨基半乳糖转移酶的结构和特性[J].生命的化学,2004,24(6):472-474.DOI:10.3969/j.issn.1000-1336.2004.06.009. SHEN Hongjie,WU Shiliang.Structure and characteristic of polypeptide:N-acetylgalactosaminyltransferases[J].Chemistry of Life,2004,24(6):472-474.DOI:10.3969/j.issn.1000-1336.2004.06.009.
[7] 郭晓丹,吴琛,康现江.UDP-GalNAc:多肽N-乙酰氨基半乳糖转移酶-14[J].生命的化学,2010,30(1):27-32. GUO Xiaodan,WU Chen,KANG Xianjiang. UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 14[J].Chemistry of Life,2010,30(1):27-32.
[8] SHIBAO K,IZUMI H,NAKAYAMA Y,et al.Expression of UDP-N-acetyl-alpha-D-galactosamine-polypeptide galNAc N-acetylgalactosaminyl transferase-3 in relation to differentiation and prognosis in patients with colorectal carcinoma[J].Cancer,2002,94(7):1939-1946.DOI:10.1002/cncr.10423.
[9] OLAUSON H,KRAJISNIK T,LARSSON C,et al.A novel missense mutation in GALNT3 causing hyperostosis-hyperphosphataemia syndrome[J].Eur J Endocrinol,2008,158(6):929-934.DOI:10.1530/EJE-08-0011.
[10] GOK F,CHEFETZ I,INDELMAN M,et al.Newly discovered mutations in the GALNT3 gene causing autosomal recessive hyperostosis-hyperphosphatemiasyndrome[J].Acta Orthop,2009,80(1):131-134.DOI:10.1080/17453670902807482.
[11] BEROIS N,MAZAL D,UBILLOS L,et al.UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker[J].J Histochem Cytochem,2006,54(3):317-328.DOI:10.1369/jhc.5A6783.2005.
[12] FREIRE T,BEROIS N,SOÑORA C,et al.UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 6(ppGalNAc-T6)mRNA as a potential new marker for detection of bone marrow-disseminated breast cancer cells[J].Int J Cancer,2006,119(6):1383-1388.DOI:10.1002/ijc.21959.
[13] PARK J H,NISHIDATE T,KIJIMA K,et al.Critical roles of mucin 1 glycosylation by transactivated polypeptide N-acetylgalactosaminyltransferase 6 in mammary carcinogenesis[J].Cancer Res,2010,70(7):2759-2769.DOI:10.1158/0008-5472.CAN-09-3911.
[14] BEROIS N,BLANC E,RIPOCHE H,et al.ppGalNAc-T13:a new molecular marker of bone marrow involvement in neuroblastoma[J].Clin Chem,2006,52(9):1701-1712.DOI:10.1373/clinchem.2006.067975.
[15] 岳爱环,行书丽,唐春花,等.N-乙酰氨基半乳糖转移酶2对胃癌细胞SGC7901黏附迁移能力的影响[J].江苏大学学报(医学版),2008,18(3):223-226.DOI:10.3969/j.issn.1671-7783.2008.03.010. YUE Aihuan,XING Shuli,TANG Chunhua,et al.Polypeptide:Nacetylgalactosaminyltransferase2’s role during adhesion and migration of gastric cancer cell SGC7901 at cell level[J].Journal of Jiangsu University(Medicine Edition),2008,18(3):223-226.DOI:10.3969/j.issn.1671-7783.2008.03.010.
[16] WU Y M,LIU C H,HU R H,et al.Mucin glycosylating enzyme GALNT2 regulates the malignant character of hepatocellular carcinoma by modifying the EGF receptor[J].Cancer Res,2011,71(23):7270-7279.DOI:10.1158/0008-5472.CAN-11-1161.
[17] WANG H,TACHIBANA K,ZHANG Y,et al.Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase,pp-GalNAc-T14[J].Biochem Biophys Res Commun,2003,300(3):738-744.DOI:0.1016/S0006-291X(02)02908-X.
[18] WU C,YAO G,ZOU M J,et al.N-Acetylgalactosaminyltransferase 14,a novel insulin-like growth factor binding protein-3 binding partner[J].Biochem Biophys Res Commun,2007,357(2):360-365.DOI:10.1016/j.bbrc.2007.03.153.
[19] WU C,SHAN Y J,LIU X X,et al.GalNAc-T14 may be involved in the regulation of apoptotic action of IGFBP-3[J].J Biosci,2009,34(3):389-395.DOI:10.1007/s12038-009-0045-z.
[20] WU C,GUO X D,WANG W N,et al.UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-14 as a potential new immunohistochemical marker for breast cancer[J].BMC Cancer.2010,10:123.DOI:10.1186/1471-2407-10-123.
[21] 吴琛,田焕娜,王媛媛,等.GALNT14对人乳腺癌MCF-7细胞迁徙的影响[J].中国生物工程杂志,2012,32(7):8-15. WU Chen,TIAN Huanna,WANG Yuanyuan,et al.Effect of GALNT14 on the migration of Human breast cancer cells MCF-7[J].China Biotechnology,2012,32(7):8-15.
[22] KARIN M,RJCHARDS R I.Human metallothionein genes-primary structure of the metallothionein-lI gene and a related processed gene[J].Nature,1982,299(5886):797-802.DOI:10.1038/299797a0.
[23] KARIN M.Metallothionelns:protons in search of function[J].Cell,1985,41(1):9-10.DOI:10.1016/0092-8674(85)90051-0.
[24] BREMNER I.Nutritional and physiological significance of metallothionein[J].Experientia Suppl,1987,52:81-107.DOI:10.1007/978-3-0348-6784-9_5.
[25] NATH R,KAMBADUR R,GULATI S,et al.Molecular aspects,physiologieal function,and clinical significance ofmetallothioneins[J].Crit Rev Food Sci Nutr,1988,27(1):41-85.DOI:10.1080/10408398809527477.
[26] NORDBERG G F.Modulation of metal toxieity by metallothionein[J].Biol Trace Elem Res,1989,21:131-135.DOI:10.1007/BF02917245.
[27] SATO M,BREMNER I.Oxygen free radicals and metallothionein[J].Free Radic Biol Med,1993,14(3):325-333.DOI:10.1016/0891-5849(93)90029-T.
[28] HUSSAIN S,SLIKKER W J,ALI S F.Role of metallothionein and other antioxidants in scavenging superoxide radicals and their possible role in neuroprotection[J].Neurochem Int,1996,29(2):145-152.DOI:10.1016/0197-0186(95)00114-X.
[29] SHIMODA R,ACHANZAR W E,QU W,et a1.Metallothionein is a potential negative regulator of apoptosis[J].Toxieol Sei,2003,73(2):294-300.DOI:10.1093/toxsci/kfg095.
[30] CHERIAN M G,APOSTOLOVA M D.Nuclear localization of metallothionein during cell proliferation and differentiation[J].Cell Mol Biol(Noisy-le-grand),2000,46(2):347-356.
[31] SATOH M,CHERIAN M G,LMURA N,et al.Modulation of resistance to anticancer drugs by inhibition of metallothionein synthesis[J].Cancer Res,1994,54(20):5255-5257.
[32] JIN R,CHOW V T,TAN P H,et al.Metallothionein 2A expression is associated with cell proliferation in breast caner[J].Carcinogenesis,2002,23(1):81-86.DOI:10.1093/carcin/23.1.81.
[33] JIN R,BAY B H,CHOW V T,et a1.Significance of metallothionein expression in breast myoepithelial cells[J].Cell Tissue Res,2001,303(2):221-226.DOI:10.1007/s004410000310.
[34] BOON-HUAT B,RONGXIAN J,ANITA J.Analysis of metallothionein expression in human cancers[J].Acta Histochem Cytoc,2001,34(3):171-176.DOI:10.1267/ahc.34.171.