Glycosylation of proteins in endoplasmic reticulum during horseradish (Armoracia lapathifolia Gilib.) cellular differentiation in in vitro culture

Abstract

U ovom radu uspoređivane su transformirane linije hrena (tumorska i teratomska) s kontrolnim tkivom lista u elektroforetskoj slici i glikozilaciji ukupnih staničnih proteina. Poseban naglasak stavljen je na usporedbu navedenih tkiva u strukturi i sastavu N-glikana proteina endoplazmatskog retikuluma. Osim središnje uloge u metabolizmu, endoplazmatski retikulum je i početno mjesto glikozilacije proteina u eukariotskoj stanici. N-glikozilacija je glavna modifikacija proteina u biljnim stanicama. Prema supstituentima vezanim na šećernu jezgru, biljne N-glikane možemo podijeliti u četiri osnovne grupe: visoko manozni tip, složeni tip, paucimanozni tip te hibridni tip N-glikana. Dok je Nglikozilacija relativno dobro opisan proces, O-glikozilacija u biljaka je slabo istražena. U ovom radu glikoproteini su odvojeni SDS-PAG elektroforezom te preneseni na nitroceluloznu membranu. N-glikozilirani proteini na membranama otkriveni su lektinom Con A. Glikani su dalje analizirani obradom membrana različitim lektinima (GNA, MAA, SNA, PNA, DSA, RCA I i UEA). Rezultati prikaza glikoziliranih proteina lektinima dokazali su da je glikozilacija ukupnih staničnih proteina i proteina endoplazmatskog retikuluma promijenjena tijekom diferencijacije stanica hrena. Elektronskom mikroskopijom uočene su velike razlike u unutrašnjoj građi stanica tkiva lista, teratoma (organiziranog i neorganiziranog) te tkiva tumora. U svim transformiranim linijama primijećeni su nepravilno građeni plastidi. Velika sličnost u građi stanica uočena je kod tumora i neorganiziranog teratoma. Ova dva tipa tkiva sadrže velike nepravilne jezgre i male plastide. U navedenim tkivima nisu uočene razlike u strukturi endoplazmatskog retikuluma.In this work different horseradish transformed lines (tumour and teratoma) were compared with control samples of leaf tissue in electrophoretic pattern of total cell proteins and their glycosylation. Emphasis was put on the comparison of these tissues in structure and composition of N-glycans of endoplasmic reticulum proteins. Despite its central metabolic role, the endoplasmic reticulum is also a starting point of protein glycosylation in eukaryotic cells. Two forms of protein glycosylation are known, N- and Oglycosylation. N-glycosylation is a major modification of proteins in plant cells. Plant N-glycans can be classified into four basic groups: high-mannose-type, complex-type, paucimannosidic-type and hybrid-type. N-glycosylation is a well described process whereas O-glycosylatin is not well characterized. Glycoproteins were separated on SDS-PAGE and subsequently transferred onto nitrocellulose membrane. Glycosylated proteins were detected with Con A. N-glycans were further characterized by treating the membranes with different lectins (GNA, MAA, SNA, PNA, DSA, RCA I and UEA). The obtained results showed that glycosylation of total cell proteins and proteins of endoplasmic reticulum had been changed during horseradish cellular differentiation. Electron microscopy showed great differences in cell structure between leaf tissue, teratoma tissue (organized and unorganized) and tumour tissue. In all transformed horseradish lines plastids with irregular structure were noticed. The great similarity between tumour tissue and unorganized teratoma tissue was also observed. These two types of tissues had large abnormal nuclei and small plastids. In all types of tissues no differences in structure of endoplasmic reticulum were noticed