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Fetuin O-glikanlarının, Bioaktif N-Glikanların Yeni Endo-B-N-asetilglukozaminidaz Tarafından İzole Edilmesindeki Katkısının Belirlenmesi

Year 2018, Volume: 21 Issue: 3, 286 - 291, 15.06.2018

Sercan Karav

https://doi.org/10.18016/ksudobil.335396
Cited By: 1

Abstract

İnek fetuini N- ve O- glikanları içerdiğinden
dolayı farklı glikosidazların aktivitesini test etmek için kullanılan model bir
proteindir. Yeni bir glikosidaz olan endo-B-N-asetilglukozaminidaz enzimi
(EndoBI-1) bebek bağırsaklarında bulunan Bifidobakteri
infantis’ten tarafımızca daha önceden izole edilmiştir. Bu enzim farklı
protein yapılarında bulunan N-glikan merkezlerini kesebilmektedir. Enzimin
yüksek aktivite ve geniş substrat aralığından dolayı, peynir altı suyu
proteinleri gibi karmaşık yapılarda aktivite gösterebilmektedir. Ayrıca, bu
enzim aktivitesini yüksek sıcaklıklarda koruyabildiği için, pastörizasyon gibi
ısıl işlem gerektiren süreçlerde de kullanılabilmektedir. İnek peynir altı
suyu, yıllık milyonlarca ton üretilen bir glikoprotein kaynağıdır. Fakat,
EndobI-1 enziminin bu substrata uygulanması, peynir altı proteinlerinin
O-glikanları tarafından bloke edilip aktiviteyi düşürdüğü düşünülmemektedir.
O-glikanların, proteinleri bir  korucuyu
görevi ile koruyarak, yeni bir prebiyotik kaynağı olarak kabul edilen
N-glikanların izolasyonunu zorlaştırmakta olduğu sanılmaktadır. Bu çalışmada,
O-glikanlar fetuinden ayrılarak, bunun N-glikanların enzimatik olarak EndoBI-1
tarafından ayrılmasına olan katkısı incelenmiştir. Ayrılan glikanlar kütle
spektrometresi ile analiz edilmiş ve izomerler dahil 22 farklı yapı
gözlemlenmiştir. Sonuçlara göre, O-glikanların fetuinden ayrılması, N-glikan
izolasyon etkinliğini (Kcat/Km) 0.52 ‘den 1.54 ml/mg x min-1’a çıkarırken, Km
değerini 0.32 ‘den 0.22 mg/ml’ye düşürmüştür. Bu sonuçlara göre, EndobI-1 süt
endüstrisinde çok daha etkili bir şekilde kullanabilmesinin yolu açılmıştır.

Keywords

Fetuin Glikanlar Enzim Kinetiği

References

  • Altmann F, Schweiszer S, Weber C 1995. Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity. Glycoconjugate Journal, 12(1):84-93.
  • Barboza M, Pinzon J, Wickramasinghe S, Froehlich JW, Moeller I, Smilowitz JT, Ruhaak LR, Huang J, Lonnerdal B, German JB 2012. Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation enhancing its role in pathogenic bacteria-host interactions. Molecular and Cellular Proteomics, 11(6):M111 015248.
  • Bode L 2006. Recent advances on structure, metabolism, and function of human milk oligosaccharides. The Journal of Nutrition, 136(8):2127-2130.
  • Green, Eric D 1988. The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. Journal of Biological Chemistry, 30(4):18253-18268.
  • Hamosh M 2001. Bioactive factors in human milk. Pediatric Clinics of North America, 48(1):69-86.
  • Hejtmánková A, Pivec V, Trnková E, Dragounová H 2012. Differences in the composition of total and whey proteins in goat and ewe milk and their changes throughout the lactation period. Czech J. Anim. Sci., 57(7):323-31.
  • Holman RC, Stoll BJ, Curns AT, Yorita KL, Steiner CA, Schonberger LB 2006. Necrotising enterocolitis hospitalisations among neonates in the United States. Paediatric and Perinatal Epidemiology, 20(6):498-506.
  • Jenness R 1988. Composition of milk. Fundamentals of dairy chemistry: Springer, 30(4) :1-38.
  • Moore SA, Anderson BF, Groom CR, Haridas M, Baker EN 1997. Three-dimensional structure of diferric bovine lactoferrin at 2.8 Å resolution. Journal of Molecular Biology, 274(2):222-236.
  • Morgan B, Winick M 1980. Effects of administration of N-acetylneuraminic acid (NANA) on brain NANA content and behavior. The Journal of Nutrition, 110(3):416-424.
  • Nuck R, Zimmermann M, Sauvageot D, Josi D, Reutter W 1990. Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase from Flavobacterium meningosepticum. Glycoconjugate Journal, 7(4):279-86.
  • Nwosu CC, Aldredge DL, Lee H, Lerno LA, Zivkovic AM, German JB, Lebrilla CB 2012. Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry. Journal of Proteome Research, 11(5):2912-2924.
  • O'Riordan N, Kane M, Joshi L, Hickey RM 2014. Structural and functional characteristics of bovine milk protein glycosylation. Glycobiology, 24(3):220-236.
  • Parham J, A and Deng SP 2000. Detection, quantification and characterization of β-glucosaminidase activity in soil. Soil Biology and Biochemistry, 32(8), 1183-1190.
  • Sojar HT, Bahl OP 1987. A chemical method for the deglycosylation of proteins. Archives of Biochemistry and Biophysics, 259(1):52-57.
  • Spik G, Coddeville B, Mazurier J, Bourne Y, Cambillaut C, Montreuil J 1994. Primary and three-dimensional structure of lactotransferrin (lactoferrin) glycans. Lactoferrin: Springe, 33(3) p 21-32.
  • Stavenhagen K, Plomp R, Wuhrer M 2015. Site-specific protein N-and O-glycosylation analysis by a C18-porous graphitized carbon–liquid chromatography-electrospray ionization mass spectrometry approach using pronase treated glycopeptides. Analytical Chemistry, 87(23):11691-11699.
  • Sun S, Shah P, Eshghi ST, Yang W, Trikannad N, Yang S, Chen L, Aiyetan P, Höti N, Zhang Z 2016. Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides. Nature Biotechnology, 34(1):84-88.
  • Takahashi N 1977. Demonstration of a new amidase acting on glycopeptides. Biochemical and Biophysical Research Communications, 76(4):1194-201
  • Windwarder M, Altmann F 2014. Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry. Journal of Proteomics, 10(8):258-268.
  • Yolken R, Peterson J, Vonderfecht S, Fouts E, Midthun K, Newburg D 1992. Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. Journal of Clinical Investigation, 90(5):1984.

Understanding of the Contribution of Fetuin O-glycans for the Release of New Bioactive Compounds by a Novel Endo-β-N-acetylglucosaminidase

Year 2018, Volume: 21 Issue: 3, 286 - 291, 15.06.2018

Sercan Karav

https://doi.org/10.18016/ksudobil.335396
Cited By: 1

Abstract

Bovine fetuin is a model
protein to study the activity of various glycosidases since it contains both N-
and O- glycans attached to the polypeptide chain. We recently showed a novel
glycosidase, endo-β-N-acetylglucosaminidase isolated from an
infant gut microbe, Bifidobacterium
infantis. This enzyme is capable of cleaving the N-N’-diacetyl chitobiose moiety found in the N-glycan core of a wide variety of proteins. It is considered a
promising approach to release N-glycans from complex substrates such as whey
proteins due to its high activity and wide substrate specificity. Moreover, it
also maintains its activity at high temperatures enabling the use of this
enzyme in thermal dairy processes such as during the pasteurization. Bovine
whey is a potential source of glycans providing million tons of glycoproteins
annually. Application of EndoBI-1 on bovine whey is challenging due to the
complexity of the whey proteins and their O-glycosylation pattern. O-glycans
are considered to be a protective agent for N-deglycosylation that hinders the
isolation of these recently found novel compounds. In this study, O-glycans
were removed from fetuin (both O- and N- glycosylated model glycoprotein) and
the contribution of O-glycans to the accessibility of EndoBI-1 to bovine fetuin
N-glycans were tested. Released glycans were characterized by advanced mass
spectrometry and 22 different N-glycans (including isomers) were monitored.
According to the results, it was shown that removing O-glycans from Fetuin
increases the Kcat/Km value 0.52 to 1.54 ml/mg x min-1and the affinity of EndoBI-1 (Km value from 0.32
to 0.22 mg/ml) to target N-glycans enabling more feasible application of this
enzyme in dairy streams.

Keywords

Fetuin glycans enzyme kinetics

References

  • Altmann F, Schweiszer S, Weber C 1995. Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity. Glycoconjugate Journal, 12(1):84-93.
  • Barboza M, Pinzon J, Wickramasinghe S, Froehlich JW, Moeller I, Smilowitz JT, Ruhaak LR, Huang J, Lonnerdal B, German JB 2012. Glycosylation of human milk lactoferrin exhibits dynamic changes during early lactation enhancing its role in pathogenic bacteria-host interactions. Molecular and Cellular Proteomics, 11(6):M111 015248.
  • Bode L 2006. Recent advances on structure, metabolism, and function of human milk oligosaccharides. The Journal of Nutrition, 136(8):2127-2130.
  • Green, Eric D 1988. The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. Journal of Biological Chemistry, 30(4):18253-18268.
  • Hamosh M 2001. Bioactive factors in human milk. Pediatric Clinics of North America, 48(1):69-86.
  • Hejtmánková A, Pivec V, Trnková E, Dragounová H 2012. Differences in the composition of total and whey proteins in goat and ewe milk and their changes throughout the lactation period. Czech J. Anim. Sci., 57(7):323-31.
  • Holman RC, Stoll BJ, Curns AT, Yorita KL, Steiner CA, Schonberger LB 2006. Necrotising enterocolitis hospitalisations among neonates in the United States. Paediatric and Perinatal Epidemiology, 20(6):498-506.
  • Jenness R 1988. Composition of milk. Fundamentals of dairy chemistry: Springer, 30(4) :1-38.
  • Moore SA, Anderson BF, Groom CR, Haridas M, Baker EN 1997. Three-dimensional structure of diferric bovine lactoferrin at 2.8 Å resolution. Journal of Molecular Biology, 274(2):222-236.
  • Morgan B, Winick M 1980. Effects of administration of N-acetylneuraminic acid (NANA) on brain NANA content and behavior. The Journal of Nutrition, 110(3):416-424.
  • Nuck R, Zimmermann M, Sauvageot D, Josi D, Reutter W 1990. Optimized deglycosylation of glycoproteins by peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase from Flavobacterium meningosepticum. Glycoconjugate Journal, 7(4):279-86.
  • Nwosu CC, Aldredge DL, Lee H, Lerno LA, Zivkovic AM, German JB, Lebrilla CB 2012. Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry. Journal of Proteome Research, 11(5):2912-2924.
  • O'Riordan N, Kane M, Joshi L, Hickey RM 2014. Structural and functional characteristics of bovine milk protein glycosylation. Glycobiology, 24(3):220-236.
  • Parham J, A and Deng SP 2000. Detection, quantification and characterization of β-glucosaminidase activity in soil. Soil Biology and Biochemistry, 32(8), 1183-1190.
  • Sojar HT, Bahl OP 1987. A chemical method for the deglycosylation of proteins. Archives of Biochemistry and Biophysics, 259(1):52-57.
  • Spik G, Coddeville B, Mazurier J, Bourne Y, Cambillaut C, Montreuil J 1994. Primary and three-dimensional structure of lactotransferrin (lactoferrin) glycans. Lactoferrin: Springe, 33(3) p 21-32.
  • Stavenhagen K, Plomp R, Wuhrer M 2015. Site-specific protein N-and O-glycosylation analysis by a C18-porous graphitized carbon–liquid chromatography-electrospray ionization mass spectrometry approach using pronase treated glycopeptides. Analytical Chemistry, 87(23):11691-11699.
  • Sun S, Shah P, Eshghi ST, Yang W, Trikannad N, Yang S, Chen L, Aiyetan P, Höti N, Zhang Z 2016. Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides. Nature Biotechnology, 34(1):84-88.
  • Takahashi N 1977. Demonstration of a new amidase acting on glycopeptides. Biochemical and Biophysical Research Communications, 76(4):1194-201
  • Windwarder M, Altmann F 2014. Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry. Journal of Proteomics, 10(8):258-268.
  • Yolken R, Peterson J, Vonderfecht S, Fouts E, Midthun K, Newburg D 1992. Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis. Journal of Clinical Investigation, 90(5):1984.
There are 21 citations in total.

Details

Primary Language English
Journal Section RESEARCH ARTICLE
Authors

Sercan Karav 0000-0003-4056-1673

Publication Date June 15, 2018
Submission Date August 20, 2017
Acceptance Date December 15, 2017
Published in Issue Year 2018 Volume: 21 Issue: 3

Cite

APA Karav, S. (2018). Understanding of the Contribution of Fetuin O-glycans for the Release of New Bioactive Compounds by a Novel Endo-β-N-acetylglucosaminidase. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Ve Doğa Dergisi, 21(3), 286-291. https://doi.org/10.18016/ksudobil.335396

Cited By

Potential applications of recombinant bifidobacterial proteins in the food industry, biomedicine, process innovation and glycobiology
Food Science and Biotechnology
José A. Morales-Contreras
https://doi.org/10.1007/s10068-021-00957-1
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