The impact of glycosylation and phosphorylation on cellular localization of cystatin F active form

Cistatin F je inhibitor cisteinskih peptidaz in spada v družino cistatinov tipa II. Od drugih cistatinov tega tipa se razlikuje po tem, da ima tri glikozilacijska mesta (na asparaginih na mestih 61, 62 in 115) in da se precejšen delež cistatina F nahaja v endosomih/lizosomih celic. Sintetizira se kot neaktiven dimer, povezan z dvema disulfidnima vezema. Cepitev 15 aminokislin na N-koncu olajša njegovo monomerizacijo in pretvorbo v aktivno obliko, ki inhibira cisteinske katepsine C, H in L, ki se nahajajo v endosomih/lizosomih. Pokazano je bilo, da stopnja glikozilacije vpliva na izločanje in privzem neaktivne oblike cistatina F in da je prehod cistatina F v celico in v endosome/lizosome omogočen z vezavo fosforiliranih glikanov na M6P-receptor. Namen magistrske naloge je bil ugotoviti ali stopnja glikozilacije vpliva na privzem, izločanje iz celice ter lokalizacijo tudi aktivne oblike cistatina F. Za ta namen smo naredili enojno, dvojno in trojno neglikozilirane mutante aktivne oblike cistatina F in preverili njihovo izražanje, izločanje in prevzem v celice HeLa, ki normalno ne izražajo cistatina F. S prenosom western smo določali ali se neglikozilirane aktivne oblike proteina izločajo iz transfeciranih celic, in obratno, ali se iz gojišča lahko privzemajo v celice. Lokalizacijo v celičnih organelih smo ugotavljali s pomočjo fluorescenčne konfokalne mikroskopije. Kot kontrolo smo uporabili polno glikoziliran aktivni cistatin F, ki po izražanju prehaja v endosome/lizosome ter se po transfekciji izloča v gojišče in tudi privzema nazaj v celico. Z uporabo neglikoziliranih mutant smo ugotovili, da se v celice privzema le skrajšan, na mestu 115 neglikoziliran cistatin F. To potrjujejo rezultati konfokalne mikroskopije, ki kažejo, da ta mutanta enako kot polno glikozilirana oblika, po transfekciji prehaja v endosome/lizosome. Druge neglikozilirane mutante, ki smo jih uporabili, se ne privzemajo v celice in se v celici po transfekciji ne nahajajo v endosomih/lizosomih. Se pa v gojišče poleg skrajšanega, na mestu 115 neglikoziliranega cistatina F, izločata tudi skrajšani in na mestih 61 in 62 neglikozilirani mutanti, kar kaže na to, da lahko glikozilacija na asparaginu 115 pripomore k prehodu proteina iz celice. Ugotovili smo, da ima glikozilacija tudi na asparaginih na mestih 61 in 62 velik vpliv na izločanje in privzem ter tudi na lokalizacijo aktivne oblike cistatina F v celicah HeLa, saj neglikozilirane mutante v veliko manjši meri ali pa sploh ne prehajajo membrane. Rezultati kažejo tudi na to, da morebitna O-glikozilacija ali fosforilacija nimata vpliva na prehod aktivne oblike cistatina F preko membran.Cystatin F is a cysteine peptidase inhibitor that belongs to the type II cysteine family. Among other differences that distinguish it from other members of type II family, cystatin F possesses three glycosylation sites (on asparagines 61, 62 and 115) and is localized in endosomal/lyososomal compartments of cells. Cleavage of 15 N-terminal amino acids enhances monomerization and turns cystatin F into an active form in which it becomes a potent inhibitor of cysteine cathepsines C, H and L, localized in the endo/lysosomal compartments. It has been shown that degree of glycosylation influences secretion and uptake of cystatin F inactive form and that transition is facilitated by binding of phosphorylated glycans to M6P-receptor. The aim of this thesis was to determine whether the degree of glycosylation has an effect on secretion, re-uptake and cellular localization of active form of cystatin F. For this purpose, we prepared single, double and triple non-glycosylated mutants of N-terminally truncated cystatine F (active cystatin F) and examined their expression, secretion and reuptake on HeLa cells, that normaly do not express cystatin F. We determined whether mutants of active form are secreted and taken up by the cells by western blot analysis. Using fluorescent confocal microscopy, we analysed the intracellular localization of non-glycosylated mutants. As a control, glycosylated N-terminally truncated form of cystatin F was used, which is secreted from cells as well as localized in endosomal/lysosomal compartments. Our results show that only active cystatin F, non-glycosylated on asparagine 115 can be taken up to the cell. This result was confirmed by confocal microscopy, showing the localization of this mutant after transfection in endo/lysosomal compartments. Other non-glycosylated mutants are not localized in the endosomal/lysosomal compartments and can not be taken up by the cells. Secretion of mutants that are not glycosylated on asparagines 61 and 62 shows us that glycosylation on asparagine 115 affects secretion of the protein from the cell. Glycosylation, especially on asparagines 61 and 62 has a big impact on secretion, protein uptake and localization of active form of cystatin F in HeLa cells. Probably because non-glycosylated mutants are crossing the membrane to a much lesser extent or not at all. Results also indicate that the transfer of the active form of cystatin F across membranes is not affected by possible O-glycosylation or phosphorylation on glycosylation sites