Glycosylated proteins of the gastric pathogen Helicobacter pylori

Bu çalışmada, sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) ve iki boyutlu jel elektroforez (2-DE) ile ayrıştırılan Helicobacter pylori (H. pylori) glikozile proteinlerinin tayin edilmesi amaçlandı. H. pylori’nin protein analizi denatüre %8 poliakrilamid jel elektroforezi (SDS-PAGE) ile gerçekleştirildi. H. pylori 26695 hücre proteinleri ayrıca 2-DE tekniği ile jel üzerinde yüzlerce spot şeklinde ayrıştırıldı. SDS-PAGE ve 2-DE ile ayrıştırılan H. pylori hücre proteinleri poliakrilamid jel’den ‘‘yarı-kuru blotting’’ tekniği ile polivinilidin florid (PVDF) membranı üzerine transfer edildi. Blotting ile membran üzerine aktarılan protein bantları ya da spotlarında glikozile proteinler DIG-Glikan tayin kiti (glikozile proteinlerin genel tayini için) ve DIG-Glikan Differensiasyon (lektin) kitleri (Roche Diagnostics, Germany) ile belirlendi. Peanut agglutinin (PNA) lectinini içeren DIG-Glikan Differensiasyon (lektin) kiti ile glikozile proteinlerin karakterizasyonu gerçekleştirildi. SDS-PAGE’den sonra jel’den membrana ‘‘yarı-kuru blotting’’ ile aktarılan proteinler üzerinde DIG-Glikan tayin kiti kullanılarak, H. pylori glikozile protein bantları belirlendi. İlginç bir şekilde, H. pylori’nin 26695 (PA4), PR20 ve P12 suşlarına ait proteinler SDS-PAGE ile %8’lik jel üzerinde ayrıştırılmasından sonra ‘‘yarı-kuru blotting’’ ile PVDF membrana aktarıldı ve suşlar arasında farklı sayıda glikozile protein bantlarının bulunduğu belirlendi. Ayrıca, H. pylori’nin dış membran proteinleri içerisinde yaklaşık 54 kDa molekül ağırlığında çok belirgin bir glikozile protein bantının varlığı gözlendi. H. pylori’nin 26695 suşuna ait proteinlerin 2-DE ile ayrıştırılmasından sonra blotting ile PVDF membrana aktarılan proteinler üzerinde yapılan PNA lektin analizi sonuçları bu bakterinin yaklaşık oniki kadar glikozile proteinin O-glikozidik bağları içeren karbonhidrat zincirlerine (galaktoz–β(1-3)-N-asetilgalaktozamin) sahip olduğunu gösterdi. Elde edilen bu sonuçlara göre potansiyel olarak bazı glikozile proteinlerin H. pylori’de mevcut olduğu kanısına varıldı.

Anahtar Kelimeler:

elektroforez, patojenler, mide hastalıkları, Helicobacter pylori, SDS-PAGE

Gastrik patojen Helicobacter pylori nin glikozile proteinleri

The aim of this study was to detect the glycosylated proteins of Helicobacter pylori (H. pylori) separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two dimensional gel electrophoresis (2-DE). Protein analysis of the H. pylori strains was performed using denaturing 8% SDS-PAGE. H. pylori 26695 cell proteins were also separated by 2-DE into hundreds of spots. Separated proteins of H. pylori strains by SDSPAGE and 2-DE were transferred onto the Polyvinylidene Fluoride (PVDF) membrane by semi-dry blotting. Detection of glycosylated proteins of the protein bands or spots on blotted membranes were determined by overlay reactions with Digoxigenin (DIG)-Glycan Detection and Differentation kits (Roche Diagnostics, Germany). The Roche DIG-Glycan Differentiation Kit including peanut agglutinin (PNA) lectin was utilized to further characterize the glycosidic modifications. Analysis of protein bands on a blotted membrane with DIG Glycan Detection kit after SDS-PAGE analysis gave the general pattern of glycosylated proteins of H. pylori; interestingly PA4, PR20 and P12 strains of H. pylori gave the different patterns of protein glycosylation on 8% polyacrilamide gels. Moreover, a glycosylated protein band (~54 kDa) was also detected dominantly on the outer membrane part of H. pylori. 2-DE analysis of H. pylori proteins showed about twelve clear spots indicating the O-glycosidically linked carbohydrate chains (galactose–β(1-3)-N-acetylgalactosamine) determined by PNA lectin staining of the blotted membrane. Obtained results suggest the presence of some potentially glycosylated proteins in H. pylori.

Keywords:

electrophoresis, pathogens, stomach diseases, Helicobacter pylori, SDS-PAGE,

PDF

___

Lis H, Sharon N: Protein glycosylation: structural and functional aspects. Eur J Biochem, 218, 1-27, 1993.
Kornfeld R, Kornsfeld S: Assembly of Asparagine-Linked Oligosaccharides. Annu Rev Biochem, 54, 631-64, 1985.
Sleytr UB, Messner P, Pum D, Sara M: Crystalline Bacterial Cell Surface Proteins. Austin: R.G. Landes/Academic Pres, 1996.
Kandler O: Archaea (Archaebacteria). Progr Botany, 54: 1-24, 1993.
Messner P: Bacterial glycoproteins. Glycoconjugate Journal, 14 (1): 3-11, 1997.
Upreti RK, Kumar M, Shankar V: Bacterial glycoproteins: Functions, biosynthesis and applications. Proteomics, 3 (4): 363-379, 2003.
Montreuil J: Spatial conformation of glycans and glycoproteins. Biol Cell, 51,115-31, 1984.
Mescher MF, Strominger JL: Structural (shape-maintaining) role of the cell surface glycoprotein of Halobacterium salinarium. Proc Natl Acad Sci, USA 73 (8): 2687-2691,1976.
Wieland F, Lechner J, Sumper M: The cell wall glycoprotein of halobacteria: Structural, functional and biosynthetic aspects. Zbl Bakt Hyg I Abt Orig, C3, 161-170, 1982.
Benz I, Schmidt MA: Never say never again: Protein glycosylation in pathogenic bacteria. Molecular Microbiology, 45 (2): 267-276, 2002.
Odenbreit S, Wieland B, Haas R: Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain. J Bacteriol, 178, 6960–6967, 1996.
Jungblut PR, Seifert R: Analysis by high-resolution twodimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells. J Biochem Biophys Meth, 21 (1): 47-58, 1990.
Laemmli UK: Claveage of the structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685, 1970.
Klose J, Kobalz U: Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis, 16, 1034-1059, 1995.
Jungblut PR, Bumann, D, Haas G., Zimny-Arndt U, Holland P, Lamer S, Siejak F, Aebischer A, Meyer TF: Comparative proteome analysis of Helicobacter pylori. Mol Microbiol, 36, 710-725, 2000.
Doherty NS, Littman BH, Reilly K, Swindell AC, Buss JM, Anderson NL: Analysis of changes in acute-phase plasma proteins in an acute inflammatory response and in rheumatoid arthritis using two-dimensional gel electrophoresis, Electrophoresis, 19, 355–363, 1998.
Jungblut P, Eckerskorn C, Lottspeich F, Klose J: Blotting efficiency investigated by using two-dimensional electrophoresis, hydrophobic membranes and proteins from different sources. Electrophoresis, 11 (7): 581-588,1990.
Abu-Qarn M, Eichler J, Sharon N: Not just for Eukarya anymore: Protein glycosylation in Bacteria and Archaea. Curr Opin Struct Biol, 18 (5): 544-550, 2008.
Patterson SD, Aebersold RH: Proteomics: In the first decade and beyond. Nature Genet, 33, 311-323, 2003.
Jungblut PR, Holzhütter HG, Apweiler R, Schlüter H: The speciation of the proteome. Chem Cent J, 2,16, 2008.