Denizkestanesi Paracentrotus lividus'un yumurta jel kılıfı glikoproteinlerinin izolasyonu ve glikan profilinin MALDI-TOF-MS ve LC-MS/MS sisteminde glikomik yaklaşımla belirlenmesi
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Dosyalar
Tarih
2015
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Sperm ve yumurta interaksiyonunda yüzey karbonhidrat içerikleri büyük önem taşımaktadır. Denizkestanesi yumurtasını saran jel tabakada karbohidratça zengin glikoproteinler bulunmaktadır. Henüz çalışmamış olmakla birlikte bunların, omurgalı tanıma molekülleri ile homolog olduğu düşünülmektedir. Bu tabakada yer alan bazı glikoproteinler karakterize edilmiş olmasına karşın, glikan profilleri belirlenmemiştir. Bu nedenle bu tez çalışmasında; memeliler gibi alesital tip yumurtaya sahip olması nedeniyle model organizma olarak seçilen denizkestanesi Paracentrotus lividus'un yumurta jel kılıfında, fertilizasyonda önemi bilinen çeşitli glikoproteinlerin belirlenerek, glikan profillerinin glikomik yaklaşımla aydınlatılması amaçlanmıştır. Bunun için, asidik yöntemle izole edilen yumurta jel kılıflarına diyaliz ve liyofilizasyon yapılmış ve protein miktarı Bradford yöntemiyle ölçülmüştür. Jel kılıfın proteinleri SDS-PAGE ile, bunların hangilerinin glikozilli oldukları Periyodik asit-Schiff (PAS) boyama ile, glikozilli bu proteinlerden glikoprotein olanlarını belirlemek için ise, glikoproteinlerde uç şeker olan sialik aside spesifik LFA ile lektin blotlama yapılmıştır. Glikoproteinlerin glikan zincirlerini oluşturan monosakkaritlerin analizi için yumurta jel kılıfına asit hidrolizi yapılmıştır. Türevlendirme için sialik asit dışındaki monosakkaritlerde PMP reaktifi, sialik asitte DMB flouresan reaktifi kullanılmıştır. Örnekler CapLC-ESI-MS/MS sisteminde analiz edilmiştir. Glikanların glikomik yaklaşımla belirlenmesi için; N-glikanlar protein zincirinden PNGase F ile koparılmış, permetilasyon ile türevlendirilmiş, MALDI-TOF-MS ile N2 lazer eşliğinde analizleri yapılmıştır. Aynı örnekler, CapLC-ESI-MS/MS sisteminde parçalanma iyonları ile karakterize edilmiştir. O-glikan analizi için yumurta jel kılıfına amonyak çözeltisi doğrudan ilave edilmiştir. Ardından PMP ile türevlendirilmiş ve LC-MS/MS sisteminde analiz edilmiştir. Jel kılıf protein/glikoprotein profilinin karakterizasyonu sonucunda; SDS-PAGE ile ~250, 55, 50 ve 15 kDa civarında dört protein bandı ayırt edilmiştir. PAS boyama ile 250, 35 ve 10 kDa civarındaki proteinlerin glikozilli oldukları, bunlardan da sadece 250 kDa civarındaki büyük proteinin glikoprotein olduğu anlaşılmıştır. 35 ve 10 kDa civarındakiler olasılıkla proteoglikandır. Jel kılıf glikoproteinlerinde monosakkarit analizi sonucunda; sırasıyla en fazla fukoz olmak üzere mannoz, arabinoz+ksiloz, galaktoz, glukoz, N-asetilgalaktozamin ve N-asetilglukozamin bulunmuştur. Riboz, genetik materyalden kaynaklanmaktadır. Sialik asit tiplerinden en fazla Neu5Gc bulunmuştur. Ardından sırasıyla Neu5Ac, Neu9Ac5Gc, Neu5,9Ac2, Neu8Ac5Gc, Neu7Ac5Gc, Neu5,7Ac2, Neu5,8Ac2 gelmektedir. Eser düzeyde de sülfat grubu taşıyan Neu5GcS ve Neu5AcS belirlenmiştir. Jel kılıf glikoproteinlerinde glikomik yaklaşımla yüksek mannoz N-glikan ve O-heksoz tipinde O-glikanlar bulunmuştur. Ayrıca çekirdek 1-8 yapıya benzer O-glikanlar da LC-MSn sisteminde belirlenmiştir. MALDI-TOF sisteminde de çekirdek 1-8 yapısı bulunabilmiştir. Sonuç olarak; bu çalışmada, yeni ve modern glikomik tekniklerden olan MALDI-TOF/MS, LC-ESI-MS/MS, lektin blotting sistemleri yanında bilgisayar programları da kullanılarak P.lividus yumurta jel kılıfı glikan profili ilk defa ortaya çıkarılmıştır. Çalışmada model organizma olarak kullanılan P.lividus'un evrim sürecinde diğer türlere göre değişmeksizin günümüze kadar yaşamını sürdürmesi, bu türün özel yapısını ortaya koymaktadır. Bu sayede sadece fertilizasyon değil, onun da ötesinde türleşmenin de moleküler açıklamalarına özellikle glikobiyolojik açıdan cevapların bulunabilmesi ilgi çekicidir. Model organizmada belirlenen detaylar anlaşıldıktan sonra diğer canlılarda da homolog moleküllere bakış açısı etkilenebilir. Bu bağlantıların iyi bilinmesi insanlarda infertilitenin araştırılmasına ve çözülmesine de katkı sağlayabilir
The surface carbohydrate content is crucial in the sperm and egg interactions. There are carbohydrate-rich glycoproteins in the jelly coat which surrounding sea urchin eggs. Although there are no studies yet they are considered to be homologous to the vertebrate recognition molecules. Although some glycoproteins involved in this layer have been characterized, glycan profiles are not determined. Therefore, in this thesis;, it is aimed to identificate various glycoproteins known importance in fertilization and to clarify the glycan profile by glycomics approach in the egg jelly coat of the sea urchin Paracentrotus lividus which is selected as a model organisms due to having alecithal type egg like mammals. Hence, isolated egg jelly coat by acidic method was lyophilized and dialysed and protein content was measured by the Bradford method. Jelly coat proteins determined by SDS-PAGE, glycosylation of proteins identified by periodic acid-Schiff (PAS) staining, glycoproteins were defined by lectin blotting using LFA which is specific for the terminal sugar in the glycoprotein called sialic acid. Analysis of monosaccharides that is forming the glycan chains of glycoproteins is performed using acid hydrolysis to the egg jelly coat. PMP was used for the derivatization of the monosaccharides except sialic acids, DMB flourescent reagent used for the sialic acids. Samples were analysed using CapLC-ESI-MS/MS system. To determine glycans with glycomics approach; N-glycans were released by PNGase F from protein chain, derivatized with permethylation and analysis were performed by MALDI-TOF-MS in the presence of N2 with laser. The same samples were characterized using CapLC-ESI-MS/MS system with fragment ions. Ammonia solution was directly added on to the egg jelly coat for O-glycan analysis. Then derivatized with PMP and analysed by LC-MS/MS system. After the characterisation of protein/glycoprotein of egg jelly; by SDS-PAGE four protein band were observed near 250, 55, 50 ve 15 kDa. It is understood using PAS staining that near 250, 35 ve 10 kDa proteins are glycosylated, those of only near 250 kDa protein is a large glycoprotein. Probably near 35 and 10 kDa bands are proteoglycans. As a result of the monosaccharide analysis of jelly coat glycoproteins; fucose was found at high level and the mannose, arabinose+xylose, galactose, glucose, N-acetylgalactosamine ve N-acetylglucosamine found respectively. Ribose is derived from genetic material. Neu5Gc was found in highest level among sialic acid types. Then Neu5Ac, Neu9Ac5Gc, Neu5,9Ac2, Neu8Ac5Gc, Neu7Ac5Gc, Neu5,7Ac2, Neu5,8Ac2 was found respectively. Neu5GcS and Neu5AcS which substituted with sulphate were determined in trace level. High mannose N-glycan and O-hexose type O-Glycans were determined in the egg jelly coat glycoproteins using glycomics approach. Furthermore, core 1-8 like O-glycans were identified using LC-MSn system. Core 1-8 O-glycans determined in MALDI-TOF-MS, too. In conclusion; in this thesis, glycan profile of the egg jelly of P. lividus revealed for the first time using MALDI-TOF/MS, LC-ESI-MS/MS, lectin blotting and also software which are new and modern glycomics techniques. P.lividus is used as a model organism in this study which survives until today and has been unchanged during the evolution process compared to the other species, reveals the special nature of this species. Therefore, not only fertilization, but also it is interesting to get answers of the molecular explanation of speciation especially from glycobiological view. After the details identified in the model organism is understood, the perspective to the homologous molecules could be affected by other species. It may also contribute to the investigation and deciphering of infertility in humans by knowing these relations well.
The surface carbohydrate content is crucial in the sperm and egg interactions. There are carbohydrate-rich glycoproteins in the jelly coat which surrounding sea urchin eggs. Although there are no studies yet they are considered to be homologous to the vertebrate recognition molecules. Although some glycoproteins involved in this layer have been characterized, glycan profiles are not determined. Therefore, in this thesis;, it is aimed to identificate various glycoproteins known importance in fertilization and to clarify the glycan profile by glycomics approach in the egg jelly coat of the sea urchin Paracentrotus lividus which is selected as a model organisms due to having alecithal type egg like mammals. Hence, isolated egg jelly coat by acidic method was lyophilized and dialysed and protein content was measured by the Bradford method. Jelly coat proteins determined by SDS-PAGE, glycosylation of proteins identified by periodic acid-Schiff (PAS) staining, glycoproteins were defined by lectin blotting using LFA which is specific for the terminal sugar in the glycoprotein called sialic acid. Analysis of monosaccharides that is forming the glycan chains of glycoproteins is performed using acid hydrolysis to the egg jelly coat. PMP was used for the derivatization of the monosaccharides except sialic acids, DMB flourescent reagent used for the sialic acids. Samples were analysed using CapLC-ESI-MS/MS system. To determine glycans with glycomics approach; N-glycans were released by PNGase F from protein chain, derivatized with permethylation and analysis were performed by MALDI-TOF-MS in the presence of N2 with laser. The same samples were characterized using CapLC-ESI-MS/MS system with fragment ions. Ammonia solution was directly added on to the egg jelly coat for O-glycan analysis. Then derivatized with PMP and analysed by LC-MS/MS system. After the characterisation of protein/glycoprotein of egg jelly; by SDS-PAGE four protein band were observed near 250, 55, 50 ve 15 kDa. It is understood using PAS staining that near 250, 35 ve 10 kDa proteins are glycosylated, those of only near 250 kDa protein is a large glycoprotein. Probably near 35 and 10 kDa bands are proteoglycans. As a result of the monosaccharide analysis of jelly coat glycoproteins; fucose was found at high level and the mannose, arabinose+xylose, galactose, glucose, N-acetylgalactosamine ve N-acetylglucosamine found respectively. Ribose is derived from genetic material. Neu5Gc was found in highest level among sialic acid types. Then Neu5Ac, Neu9Ac5Gc, Neu5,9Ac2, Neu8Ac5Gc, Neu7Ac5Gc, Neu5,7Ac2, Neu5,8Ac2 was found respectively. Neu5GcS and Neu5AcS which substituted with sulphate were determined in trace level. High mannose N-glycan and O-hexose type O-Glycans were determined in the egg jelly coat glycoproteins using glycomics approach. Furthermore, core 1-8 like O-glycans were identified using LC-MSn system. Core 1-8 O-glycans determined in MALDI-TOF-MS, too. In conclusion; in this thesis, glycan profile of the egg jelly of P. lividus revealed for the first time using MALDI-TOF/MS, LC-ESI-MS/MS, lectin blotting and also software which are new and modern glycomics techniques. P.lividus is used as a model organism in this study which survives until today and has been unchanged during the evolution process compared to the other species, reveals the special nature of this species. Therefore, not only fertilization, but also it is interesting to get answers of the molecular explanation of speciation especially from glycobiological view. After the details identified in the model organism is understood, the perspective to the homologous molecules could be affected by other species. It may also contribute to the investigation and deciphering of infertility in humans by knowing these relations well.
Açıklama
Anahtar Kelimeler
Monosakkaritler, Glikomiks, MALDI-TOF-MS, LC-MS/MS, Lektin Blotlama, N-glikan, O-glikan, Denizkestanesi Paracentrotus lividus, Monosaccharides, Glycomics, MALDI-TOF-MS, LC-MS/MS, Lectin Blotting, N-glycan, O-glycan, Sea urchin Paracentrotus lividus
