粪肠球菌源EndoE的重组表达及酶学性质研究
|
摘要
内切-β-N-乙酰氨基葡萄糖苷酶(Endo-β-N-acetylglucosaminidase, ENGase, EC3.2.1.96)是糖蛋白去糖基化重要的工具酶,其可以切割糖蛋白上N-连接的聚糖。本研究基于粪肠球菌(Enterococcus faecalis)基因组数据,通过PCR扩增ENGase基因endoE (endoglycosidase E),并以同源重组方式构建表达载体pET-28a-endoE,实现了EndoE在大肠杆菌(Escherichia coli) BL21 Star (DE3)中的高效表达,并对EndoE去糖化的催化特性等进行了较为系统的分析。研究结果显示,重组EndoE在大肠杆菌中以可溶形式表达,经钴离子螯合层析与阴离子交换层析可获得高纯度的目标蛋白,产量为45.6 mg/L。去糖基化活性分析表明,重组EndoE能去除天然及变性核糖核苷酸酶B (ribonuclease B, RNase B)、鸡卵白蛋白(ovalbumin, Ova)和免疫球蛋白G (immune globulin G, IgG) N-连接的糖基侧链。基质辅助激光解吸/电离串联飞行时间质谱证实了EndoE对RNase B N-连接糖链的水解活性。Endo E在20～50℃和pH 4.0～6.0的范围内均具有良好的水解活性,并对100 mmol/L二硫苏糖醇(DL-dithiothreitol, DTT)、1 mol/L NaCl和2%Triton X-100具有耐受性。EndoE较已报道的粪肠球菌来源的ENGase具有更为广泛的底物作用范围,是蛋白质糖基化分析中有潜在应用价值的工具酶。
Endo-β-N-acetylglucosaminidase(ENGase) can cleave the N-linked oligosaccharides of glycoprotein and is an essential tool enzyme for protein deglycosylation. This study obtained the genome data of Enterococcus faecalis from the NCBI database and amplified the ENGase gene endoE(endoglycosidase E) by PCR. Then the expression vector pET-28 a-endoE was constructed by homologous recombination and transformed into Escherichia coli BL21 star(DE3) competent cells. The recombinant protein was efficiently expressed in E. coli, and the deglycosylation properties of EndoE was systematically analyzed. The results showed that recombinant protein was expressed in soluble form in E. coli, and the yield was 45.6 mg/L after purified by Co2+affinity chromatography and anion-exchange chromatography. The deglycosylation activity analysis indicated that the recombinant EndoE could hydrolyze the N-linked glycan of both natural and denatured ribonuclease(RNase B), ovalbumin(Ova) and immune globulin G(IgG). Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) proved that recombinant EndoE could remove the N-linked oligosaccharides in RNase B. In addition, in the temperature range of 20～50 ℃ or pH range of 4.0～6.0, EndoE had ideal hydrolytic activity. It also had tolerance up to 100 mmol/L DLdithiothreitol(DTT), 1 mol/L NaCl, and 2% Triton X-100. Compared to the other reported ENGase from Enterococcus faecalis, EndoE has a broader range of substrates and is a prospective tool enzyme in protein deglycosylation study.
Endo-β-N-acetylglucosaminidase(ENGase) can cleave the N-linked oligosaccharides of glycoprotein and is an essential tool enzyme for protein deglycosylation. This study obtained the genome data of Enterococcus faecalis from the NCBI database and amplified the ENGase gene endoE(endoglycosidase E) by PCR. Then the expression vector pET-28 a-endoE was constructed by homologous recombination and transformed into Escherichia coli BL21 star(DE3) competent cells. The recombinant protein was efficiently expressed in E. coli, and the deglycosylation properties of EndoE was systematically analyzed. The results showed that recombinant protein was expressed in soluble form in E. coli, and the yield was 45.6 mg/L after purified by Co2+affinity chromatography and anion-exchange chromatography. The deglycosylation activity analysis indicated that the recombinant EndoE could hydrolyze the N-linked glycan of both natural and denatured ribonuclease(RNase B), ovalbumin(Ova) and immune globulin G(IgG). Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) proved that recombinant EndoE could remove the N-linked oligosaccharides in RNase B. In addition, in the temperature range of 20～50 ℃ or pH range of 4.0～6.0, EndoE had ideal hydrolytic activity. It also had tolerance up to 100 mmol/L DLdithiothreitol(DTT), 1 mol/L NaCl, and 2% Triton X-100. Compared to the other reported ENGase from Enterococcus faecalis, EndoE has a broader range of substrates and is a prospective tool enzyme in protein deglycosylation study.
引文
黄云娜,吴今人,郑蓓,等.2018.粪肠球菌来源内切-β-N-乙酰氨基葡萄糖苷酶的重组表达及催化特性[J].农业生物技术学报,26(4):698-710.(Huang Y N,Wu J R,Zheng B,et al.2018.Recombinant expression and characterization of Endo-β-N-acetyglucosaminidase from Enterococcus faecalis[J].Journal of Agricultural Biotechnology,26(4):698-710.)
李军,杜鑫,Hosseini Moghaddam,等.2009.蛋白质糖基化修饰研究进展[J].科技通报,25(6):773-778.(Li J,Du X,Hosseini Moghaddam,et al.2009.The research progress in protein glycosylation modification[J].Bulletin of Science and Technology,25(6):773-778.)
刘国琴,吴玮,陈鹏.2011.现代蛋白质实验技术[M].北京:中国农业大学出版社,pp.138-142.(Liu G Q,Wu W,Chen P.2011.Modern protein experiment technology[M].China Agricultural University Press,Beijing,pp.138-142.)
王晓龙,王秀然,卢天成.2017.蛋白质糖基化修饰的研究进展[J].基因组学与应用生物学,36(10):468-472.(Wang X L,Wang X R,Lu T C.2017.The research progress of protein glycosylation modification[J].Genomics and Applied Biology,36(10):468-472.)
曾嵘,邵晓霞,王克夷,等.1999.液相色谱-电喷雾质谱法分析牛胰核糖核酸酶B酶解肽谱,糖基化位点及糖型[J].生物化学与生物物理学报,31(6):82-87.(Zeng R,Shao X X,Wang K Y,et al.1999.Analysis of peptide mapping,glycosylated site and glycan structure in ribonuclease B by liquid chromatography-electrospray mass spectrometry[J].Acta Biochimica et Biophysica Sinica,31(6):82-87.)
支艳艳,唱韶红,巩新,等.2014.Endo-H在毕赤酵母中的表达、纯化及其在N-糖基化分析中的应用[J].军事医学,38(3):193-197.(Zhi Y Y,Chang S H,Gong X,et al.2014.Expression of endo-beta-N-acetylglucosaminidase H in Pichia pastoris and its application to N-glycosylation analysis[J].Military Medical Sciences,38(3):193-197.)
An H J,Peavy T R,Hedrick J L,et al.2003.Determination of N-glycosylation sites and site heterogeneity in glycoproteins[J].Analytical Chemistry,75(20):5628-5637.
Apweiler R,Hermjakob H,Sharon N.2000.On the Frequency of protein glycosylation,as deduced from analysis of the SWISS-PROT database[J].Biochimica et Biophysica Acta,1473(1):4-8.
Clerc F,Reiding K R,Jansen B C,et al.2016.Human plasma protein N-glycosylation[J].Glycoconjugate Journal,33(3):309-343.
Collin M,Olsén A.2001.Endos,a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human igg[J].The Embo Journal,20(12):3046-3055.
Collin M,Fischetti V A.2004.A novel secreted endoglycosidase from Enterococcus faecalis with activity on human immunoglobulin G and ribonuclease B[J].Journal of Biological Chemistry,279(21):22558-22570.
Garbe J,Collin M.2012.Bacterial hydrolysis of host glycoproteins-powerful protein modification and efficient nutrient acquisition[J].Journal of Innate Immunity,4(2):121-131.
Garbe J,Sj?gren J,Cosgrave E F,et al.2014.EndoE from En?terococcus faecalis hydrolyzes the glycans of the biofilm inhibiting protein lactoferrin and mediates growth[J].Plos One,9(3):e91035.
Garrido D,Nwosu C,Ruizmoyano S,et al.2012.Endo-β-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins[J].Molecular&Cellular Proteomics,11(9):775-785.
Huang Y,Higuchi Y,Kinoshita T,et al.2018.Characterization of novel endo-β-N-acetylglucosaminidases from Sphin?gobacterium species,Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG[J].Scientific Reports,8(1):246.
H?gglund P,Matthiesen R,Elortza F P,et al.2007.An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins[J].Journal of Proteome Research,6(8):3021-3031.
Leisner J J,Larsen M H,Ingmer H,et al.2009.Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583[J].Journal of Applied Microbiology,107(6):2080-2087.
Masuda A,Xu J,Mitsudome T,et al.2015.Improvement of endo-β-N-acetylgluco aminidase H production using silkworm-baculovirus protein expression system[J].Journal of Asia-Pacific Entomology,18(2):175-180.
Mitsudome T,Xu J,Nagata Y,et al.2014.Expression,purification,and characterization of endo-β-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system[J].Applied Biochemistry and Biotechnology,172(8):3978-3988.
Nuck R,Zimmermann M,Sauvageot D,et al.1990.Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum[J].Glycoconjugate Journal,7(4):279-286.
Rao V,Guan C,Van Roey P.1995.Crystal structure of endobeta-N-acetylglucosaminidase h at 1.9?resolution:Active-site geometry and substrate recognition[J].Structure,3(5):449-457.
Roberts G,Homer K A,Tarelli E,et al.2001.Distribution of endo-β-N-acetylglucosaminidase amongstenterococci[J].Journal of Medical Microbiology,50(7):620-626.
Spiro R G.2002.Protein glycosylation:Nature,distribution,enzymatic formation,and disease implications of glycopeptide bonds[J].Glycobiology,12(4):43R-56R.
Strasser R.2016.Plant protein glycosylation[J].Glycobiology,26(9):926-939.
Tarentino A L,Maley F.1974.Purification and properties of an endo-β-N-acetylglucosaminidase from Streptomyces griseus[J].Journal of Biological Chemistry,249(3):811-817.
Thobhani S,Yuen C T,Bailey M J A,et al.2009.Identification and quantification of N-linked oligosaccharides released from glycoproteins:An inter-laboratory study[J].Glycobiology,19(3):201-211.
Trimble R B,Tarentino A L.1991.Identification of distinct endoglycosidase(endo)activities in Flavobacterium me?ningosepticum:Endo F1,endo F2,and endo F3[J].Journal of Biological Chemistry,266(3):1646-1651.
Varki A,Cummings R D,Esko J D,et al.,2009.Essentials of glycobiology:Glycan-binding proteins[M].Cold Spring Harbor(NY):Cold Spring Harbor Laboratory Press,New York,United States,pp.143-161.
Wang F,Wang X,Yu X,et al.2015.High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins[J].Plos One,10(3):e0120458.
Wlliams R L,Greene S M,Mcpherson A.1987.The crystal structure of ribonuclease B at 2.5-?resolution[J].Journal of Biological Chemistry,262(33):16020-16031.
李军,杜鑫,Hosseini Moghaddam,等.2009.蛋白质糖基化修饰研究进展[J].科技通报,25(6):773-778.(Li J,Du X,Hosseini Moghaddam,et al.2009.The research progress in protein glycosylation modification[J].Bulletin of Science and Technology,25(6):773-778.)
刘国琴,吴玮,陈鹏.2011.现代蛋白质实验技术[M].北京:中国农业大学出版社,pp.138-142.(Liu G Q,Wu W,Chen P.2011.Modern protein experiment technology[M].China Agricultural University Press,Beijing,pp.138-142.)
王晓龙,王秀然,卢天成.2017.蛋白质糖基化修饰的研究进展[J].基因组学与应用生物学,36(10):468-472.(Wang X L,Wang X R,Lu T C.2017.The research progress of protein glycosylation modification[J].Genomics and Applied Biology,36(10):468-472.)
曾嵘,邵晓霞,王克夷,等.1999.液相色谱-电喷雾质谱法分析牛胰核糖核酸酶B酶解肽谱,糖基化位点及糖型[J].生物化学与生物物理学报,31(6):82-87.(Zeng R,Shao X X,Wang K Y,et al.1999.Analysis of peptide mapping,glycosylated site and glycan structure in ribonuclease B by liquid chromatography-electrospray mass spectrometry[J].Acta Biochimica et Biophysica Sinica,31(6):82-87.)
支艳艳,唱韶红,巩新,等.2014.Endo-H在毕赤酵母中的表达、纯化及其在N-糖基化分析中的应用[J].军事医学,38(3):193-197.(Zhi Y Y,Chang S H,Gong X,et al.2014.Expression of endo-beta-N-acetylglucosaminidase H in Pichia pastoris and its application to N-glycosylation analysis[J].Military Medical Sciences,38(3):193-197.)
An H J,Peavy T R,Hedrick J L,et al.2003.Determination of N-glycosylation sites and site heterogeneity in glycoproteins[J].Analytical Chemistry,75(20):5628-5637.
Apweiler R,Hermjakob H,Sharon N.2000.On the Frequency of protein glycosylation,as deduced from analysis of the SWISS-PROT database[J].Biochimica et Biophysica Acta,1473(1):4-8.
Clerc F,Reiding K R,Jansen B C,et al.2016.Human plasma protein N-glycosylation[J].Glycoconjugate Journal,33(3):309-343.
Collin M,Olsén A.2001.Endos,a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human igg[J].The Embo Journal,20(12):3046-3055.
Collin M,Fischetti V A.2004.A novel secreted endoglycosidase from Enterococcus faecalis with activity on human immunoglobulin G and ribonuclease B[J].Journal of Biological Chemistry,279(21):22558-22570.
Garbe J,Collin M.2012.Bacterial hydrolysis of host glycoproteins-powerful protein modification and efficient nutrient acquisition[J].Journal of Innate Immunity,4(2):121-131.
Garbe J,Sj?gren J,Cosgrave E F,et al.2014.EndoE from En?terococcus faecalis hydrolyzes the glycans of the biofilm inhibiting protein lactoferrin and mediates growth[J].Plos One,9(3):e91035.
Garrido D,Nwosu C,Ruizmoyano S,et al.2012.Endo-β-N-acetylglucosaminidases from infant gut-associated bifidobacteria release complex N-glycans from human milk glycoproteins[J].Molecular&Cellular Proteomics,11(9):775-785.
Huang Y,Higuchi Y,Kinoshita T,et al.2018.Characterization of novel endo-β-N-acetylglucosaminidases from Sphin?gobacterium species,Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG[J].Scientific Reports,8(1):246.
H?gglund P,Matthiesen R,Elortza F P,et al.2007.An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins[J].Journal of Proteome Research,6(8):3021-3031.
Leisner J J,Larsen M H,Ingmer H,et al.2009.Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583[J].Journal of Applied Microbiology,107(6):2080-2087.
Masuda A,Xu J,Mitsudome T,et al.2015.Improvement of endo-β-N-acetylgluco aminidase H production using silkworm-baculovirus protein expression system[J].Journal of Asia-Pacific Entomology,18(2):175-180.
Mitsudome T,Xu J,Nagata Y,et al.2014.Expression,purification,and characterization of endo-β-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system[J].Applied Biochemistry and Biotechnology,172(8):3978-3988.
Nuck R,Zimmermann M,Sauvageot D,et al.1990.Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase from Flavobacterium meningosepticum[J].Glycoconjugate Journal,7(4):279-286.
Rao V,Guan C,Van Roey P.1995.Crystal structure of endobeta-N-acetylglucosaminidase h at 1.9?resolution:Active-site geometry and substrate recognition[J].Structure,3(5):449-457.
Roberts G,Homer K A,Tarelli E,et al.2001.Distribution of endo-β-N-acetylglucosaminidase amongstenterococci[J].Journal of Medical Microbiology,50(7):620-626.
Spiro R G.2002.Protein glycosylation:Nature,distribution,enzymatic formation,and disease implications of glycopeptide bonds[J].Glycobiology,12(4):43R-56R.
Strasser R.2016.Plant protein glycosylation[J].Glycobiology,26(9):926-939.
Tarentino A L,Maley F.1974.Purification and properties of an endo-β-N-acetylglucosaminidase from Streptomyces griseus[J].Journal of Biological Chemistry,249(3):811-817.
Thobhani S,Yuen C T,Bailey M J A,et al.2009.Identification and quantification of N-linked oligosaccharides released from glycoproteins:An inter-laboratory study[J].Glycobiology,19(3):201-211.
Trimble R B,Tarentino A L.1991.Identification of distinct endoglycosidase(endo)activities in Flavobacterium me?ningosepticum:Endo F1,endo F2,and endo F3[J].Journal of Biological Chemistry,266(3):1646-1651.
Varki A,Cummings R D,Esko J D,et al.,2009.Essentials of glycobiology:Glycan-binding proteins[M].Cold Spring Harbor(NY):Cold Spring Harbor Laboratory Press,New York,United States,pp.143-161.
Wang F,Wang X,Yu X,et al.2015.High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins[J].Plos One,10(3):e0120458.
Wlliams R L,Greene S M,Mcpherson A.1987.The crystal structure of ribonuclease B at 2.5-?resolution[J].Journal of Biological Chemistry,262(33):16020-16031.