猪-人异种移植相关基因CMAH的克隆、表达及功能生物信息学分析
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摘要
【目的】研究版纳微型猪近交系(Banna mini-pig inbred line,BMI)CMAH基因特性,探索该基因在猪-人异种器官移植免疫排斥中的作用。【方法】采用实时荧光定量PCR技术检测BMI 27个重要组织的CMAH m RNA表达水平,采用Western blot检测BMI在9个组织中的CMAH蛋白表达特征,并对CMAH编码的氨基酸进行生物信息学预测,分析CMAH蛋白质的功能。【结果】扩增得到BMI CMAH编码区序列长1 734 bp,编码577个氨基酸,序列已提交Gen Bank,基因登录号KM098147,对应的氨基酸登录号AIS36193。多组织相对荧光定量表达分析表明CMAH基因在颌下腺、脾及舌下腺中呈相对高表达水平,在其他组织中呈相对中、低表达水平。Western Blot分析表明CMAH蛋白在除脊髓和大脑外的其他组织都有表达。功能生物信息学分析表明CMAH蛋白质包含Rieske和Ula G保守结构域,二级结构以α螺旋为主,N末端亲水,C末端疏水,有6类功能活性位点。【结论】明确了CMAH基因的结构以及在多种组织中差异化表达情况,为深入研究其在异种器官移植方面的功能积累了基础资料。
【Objective】The aim of this study was to obtain the properties and function of CMAH in Banna mini-pig inbred line(BMI) based on m RNA expression and protein level,providing theoretical reference for further study of the potential role of CMAH in the immune rejection caused by xeno-transplantation.【Method】Real-time quantitative PCR was applied to analyze the CMAH gene expression profiles of 27 important tissues. The expression of CMAH protein in 9 tissues was detected by Western blot and the amino acids encoded by CMAH were carried out by bioinformatics analysis.【Result】A c DNA clone of 1734 bp(Gen Bank accession number: KM098147) from CMAH was obtained,which encodes a protein of 577 amino acids(Gen Bank accession number AIS36193). Real-time quantitative PCR in multi-tissues indicated that CMAH was expressed exclusively in the submandibular gland tissue,spleen and sublingual gland. The middle and lower expression levels were detected in the other tissues. Western blot analysis showed that CMAH protein was expressed in all tissues except spinal cord and brain. Further functional bioinformatics analysis demonstrated that CMAH protein contained two conserved domains,Rieske and Ula G. Its secondary structure is predominantly α-helices with hydrophilic N-terminus and hydrophobic C-terminus. The CMAH protein has six kinds of functional active sites. 【Conclusion】The structure of CMAH gene and its differentiation in various tissues were clarified,providing basic data for further researches focused on xeno-transplantation.
【Objective】The aim of this study was to obtain the properties and function of CMAH in Banna mini-pig inbred line(BMI) based on m RNA expression and protein level,providing theoretical reference for further study of the potential role of CMAH in the immune rejection caused by xeno-transplantation.【Method】Real-time quantitative PCR was applied to analyze the CMAH gene expression profiles of 27 important tissues. The expression of CMAH protein in 9 tissues was detected by Western blot and the amino acids encoded by CMAH were carried out by bioinformatics analysis.【Result】A c DNA clone of 1734 bp(Gen Bank accession number: KM098147) from CMAH was obtained,which encodes a protein of 577 amino acids(Gen Bank accession number AIS36193). Real-time quantitative PCR in multi-tissues indicated that CMAH was expressed exclusively in the submandibular gland tissue,spleen and sublingual gland. The middle and lower expression levels were detected in the other tissues. Western blot analysis showed that CMAH protein was expressed in all tissues except spinal cord and brain. Further functional bioinformatics analysis demonstrated that CMAH protein contained two conserved domains,Rieske and Ula G. Its secondary structure is predominantly α-helices with hydrophilic N-terminus and hydrophobic C-terminus. The CMAH protein has six kinds of functional active sites. 【Conclusion】The structure of CMAH gene and its differentiation in various tissues were clarified,providing basic data for further researches focused on xeno-transplantation.
引文
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[2]KWON D N,LEE K,KANG M J,et al.Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs[J].Scientific Reports,2013(3):1981.
[3]YANG Y G,SYKES M.Xenotransplantation:current status and a perspective on the future[J].Nature Reviews Immunology,2007,7(7):519-531.
[4]BAUMANN B C,STUSSI G,HUGGEL K,et al.Reactivity of human natural antibodies to endothelial cells from Gal-alpha(1,3)Gal-deficient pigs[J].Transplantation,2007,83(2):193-201.
[5]ZENG R,ZENG Y Z.Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line[J].Animal Genetics,2005,36(3):267-269.
[6]HUO J L,WANG P,ZHAO Y,et al.Molecular cloning,m RNAexpression and characterization of a novel FAIM1 gene from Chinese Banna mini-pig inbred line(BMI)[J].Journal of Animal and Veterinary Advances,2012,11(8):1080-1086.
[7]WANG P,HUO H L,WANG S Y,et al.Cloning,sequence characterization,and expression patterns of members of the porcine TSSK family[J].Genetics and Molecular Research,2015,14(4):14908-14919.
[8]LI S,WAER M,BILLIAU AD.Xenotransplantation:role of natural immunity[J].Transplant Immunology,2009,21(2):70-74.
[9]朱圣明,王艳萍,曾养志,等.中国近交系版纳微型猪1,3-半乳糖基转移酶基因的克隆及其真核表达载体的构建[J].生物医学工程学杂志,2009,26(2):360-365.
[10]LUO V,WEN J,LUO C,et al.Pig xenogeneic antigen modification with green coffee bean alpha-galatosidase[J].Xenotransplamation,1999,6(4):238-248.
[11]LAI L,KOLBER-SIMONDS D,PARK K W,et al.Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning[J].Science,2002,295(5557):1089-1092.
[12]LUTZ A J,LI P,ESTRADA J L,et al.Double knockout pigs deficient in N-glycolylneuraminic acid and galactoseα-1,3-galactose reduce the humoral barrier to xenotransplantation[J].Xenotransplantation,2013,20(1):27-35.
[13]PARK H M,KIM Y W,KIM K J,et al.Comparative N-linked glycan analysis of wild-type andα1,3-galactosyltransferase gene knock-out pig fibroblasts using mass spectrometry approaches[J].Molecules and Cells,2015,38(1):65-74.
[14]PHELPS C J,KOIKE C,VAUGHT T D,et al.Production of alpha 1,3-galactosyltransferase-deficient pigs[J].Science,2003,299(5605):411-414.
[15]KELM S,SCHAUER R.Sialic acids in molecular and cellular interaction[sJ].International Review of Cytology,2007,175:137-240.
[16]TRAVING C,SCHAUER R.Structure,function and metabolism of sialic acids[J].Cellular and Molecular Life Sciences,2008,54(12):1330-1349.
[17]BYRNE G W,STALBOERGER P G,DAVILA E,et al.Proteomic identification of non-Gal antibody targets after pig-to-primate cardiac xenotransplantation[J].Xenotransplantation,2008,15(4):268-276.
[18]NGUYEN D H,TANGVORANUNTAKUL P,VARKI A.Effects of natural human antibodies against a nonhuman sialic acid that metabolically incorporates into activated and malignant immune cell[sJ].Journal of Immunology,2005,175(1):228-236.
[19]YIN J,HASHIMOTO A,IZAWA M,et al.Hypoxic culture induces expression of sialin,a sialic acid transporter,and cancer-associated gangliosides containing non-human sialic acid on human cancer cells[J].Cancer Research,2006,66(6):2937-2945.
[20]SONG K H,KANG Y J,JIN U H,et al.Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation[J].Biochemical Journal,2010,427(1):179-188.
[21]SCHMIDT C L,SHAW L.A comprehensive phylogenetic analysis of Rieske and Rieske-type iron-sulfur proteins[J].Journal of Bioenergetics and Biomembranes,2001,33(1):9-26.
[22]GAO H,ZHAO C,XIANG X,et al.Production ofα1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning[J].Journal of Reproduction and Development,2017,63(1):17-26.
[23]SALAMA A,EVANNO G,HARB J,et al.Potential deleterious role of anti-Neu5Gc antibodies in xenotransplantation[J].Xenotransplantation,2015,22(2):85-94.
[24]HURH S,KANG B,CHOI I,et al.Human antibody reactivity against xenogeneic Nglycolylneuraminic acid and galactose-a-1,3-galactose antigen[J].Xenotransplantation,2016,23(4):279-292.
[25]BOUHOURS D,BOUHOURS J F.Tissue-specific expression of GM3(NeuGc)and GD3(NeuGc)in epithelial cells of the small intestine of strains of inbred rats.Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rat[sJ].Journal of Biological Chemistry,1988,263(30):15540-15545.
[26]DISWALL M,ANGSTR魻M J,KARLSSON H,et al.Structural characterization of alpha1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodie[sJ].Xenotransplantation,2010,17(1):48-60.
[27]BERGFELD A K,PEARCE O M,DIAZ S L,et al.Metabolism of vertebrate amino sugars with N-glycolyl groups:elucidating the intracellular fate of the non-human sialic acid N-glycolylneuraminic acid[J].Journal of Biological Chemistry,2012,287(34):28865-28881.
[28]DUVAL S,SANTINI J M,NITSCHKE W,et al.The small subunit AroB of arsenite oxidase:lessons on the[2Fe-2S]Rieske protein superfamily[J].Journal of Biological Chemistry,2010,285(27):20442-20451.
[29]MARCHLER-BAUER A,BO Y,HAN L,et al.CDD/SPARCLE:functional classification of proteins via subfamily domain architecture[sJ].Nucleic Acids Research,2017,45(D1):200-203.
[30]MARCHLER-BAUER A,DERBYSHIRE M K,GONZALES NR,et al.CDD:NCBI's conserved domain database[J].Nucleic Acids Research,2015,43(D):222-226.
[31]LI L,SHAKHNOVICH E I,MIRNY L A.Amino acids determining enzyme-substrate specificity in prokaryotic and eukaryotic protein kinases[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(8):4463-4468.