Lysosomaalisen kalvoproteiinin kloonaus ja ilmentäminen hiiren sidekudossoluissa

Abstract

Opinnäytetyötä tehtiin Helsingin yliopistossa osana laajempaa projektia, missä tutkittiin lysosomin pintaproteiini (LAMP) vaikutusta solun kolesterolikertymille. Lysosomilla tarkoitetaan hajottavaa soluosastoa, mikä on muun muassa solunpinnalta kuroutuvan endosytoosi-rakkulan sisällön loppusijoitus/hajotuspaikka. Oma osaprojektini keskittyi LTL3-kimeerin kloonaamiseen ja ilmentymiseen hiiren sidekudossoluissa. Kloonatessa geenijuosteita tai osia geeneistä liitetään yhteen biotekniikan menetelmillä aikaan saaden kimeerisiä yhdistelmägeenejä. Yhdistin fluoresoivat merkkiproteiineja koodaavat geenit RFP-eGFP ja kalvon läpäisevää osaa koodaavan palan geenistä TGN38 yhteen LAMP1 proteiinin solulimassa sijaitsevan C-terminaalista häntää koodaavan geenin osan kanssa, muodostaen yhdistelmägeenin LTL3. Lisäksi kimeeriin kuului LAMP-2 geenin signaalisekvenssi, mikä ohjaa yhdistelmägeenin proteiinituotteen solukalvostolle ja sitä kautta eritysreitille. Ilmentymisen paikallistumiseen aitotumallisessa solussa käytin fluoresoivaa proteiinia RFP, mikä emittoi punaista ja eGFP, mikä emittoi vihreää valoa eksitoituessaan fluoresenssimikroskoopissa. Näillä reportteriproteiineilla voidaan seurata kimeeriproteiinin liikkumista soluissa. eGFPn fluoresenssin tiedetään sammuvan lysosomien happamassa pH:ssa, kun RFPn fluoresenssi kestää happamia oloja kauemmin. Kimeeriä ilmennettiin LAMP1- ja LAMP2- poistogeenisissä hiiren alkion sidekudossoluissa ja merkkiproteiinikertymistä seurattiin fluoresenssimikroskoopilla eri ajankohtina ilmentymisen aikana. LAMP-proteiinin paikallistumista solussa myöhäiseen endosomiin ja lysosomiin ohjaavat osat sijaitsevat C-terminaalisessa hännässä. Kimera olikin havaittavissa endosyyttisissä osastoissa perustuen vihreän fluoresenssin himmenemiseen punaisena fluoresoivilla alueilla, mitkä yhteispaikantuvat myöhäisen endosomin lipidin BMP ja LIMP2; endosomiproteiinin kanssa. Yhteispaikantuminen oli runsaampi BMPn kanssa perustuen Image Pro mittauksiin. LTL3n kalvonläpäisevä vesipakoisen osaproteiinin pituus aminohapoissa oli lyhyempi kuin alkuperäisen LAMP-proteiinin ja aminohapposekvenssi oli erilainen. Kimeerin havaittiin kertyvän huomattavissa määrin Golgiin, minne endogeeniset LAMP-proteiinit eivät normaalisti kerry. Johtuiko kertyminen lyhyemmästä kalvonläpäisevästä osasta vai lumenalisen LAMP-proteiinin N-terminaalisen pään puutteesta ei tutkittu. Teorian mukaan proteiinit saattavat muodostaa Golgissa lauttoja tunnistautumalla Golgin sisäisillä proteiini-proteiini interaktioilla, mitkä yhteisvaikutukseltaan nopeuttavat Golgista poistumista tai vahvistavat Golgiin jäämistä. Tämä, kuten myös kimeerin vaikutus kolesterolin kertymiseen ovat avoimia kysymyksiä, joihin vastaus saadaan jatkotutkimuksista.

This Theses was carried out at the University of Helsinki as part of a larger project, which studied how the membrane protein of the lysosome (LAMP) affects the cholesterol that is stored in the cell. The lysosome means degrading cell compartment, which contents of the vesicle endocytosis-pinch off of, inter alia, from the cell surface repository/cleavage.

My sub-project focused on LTL3 chimera cloning and expression of mouse fibroblastic cells. In cloning gene strands or parts of gene are joined by methods of biotechnology providing a combi-nation of chimeric genes. The combiner fluorescent marker proteins encoded by genes such as RFP-eGFP and transmembrane parts of the gene encoding TGN38 one piece with LAMP1 in the cytoplasm of the protein c-terminal tail of the coding portion of the gene, to form a recombinant gene LTL3. In addition, the chimera was the signal sequence LAMP2 gene which controls the re-combinant gene protein product to the cell membrane and through the secretory pathway. Expres-sion localization in eukaryotic cell I used a fluorescent protein RFP, which emits red light and eGFP, which emits green light as it comes out of the fluorescence microscopy. With these reporter proteins chimeric protein can follow the movement of the in cells. eGFP’s fluorescence is known to turn off the lysosomal acidic pH, the fluorescence RFP’s will last longer in acidic conditions. Chi-mera was expressed on LAMP1- and LAMP2- knockout mouse embryonic fibroblastic (MEF) – cells and accumulation of marker protein was monitored by fluorescence microscopy at different time points during expression.

LAMP localization of the protein in the cell and the late endosome to the lysosome guiding ele-ments are located in the C-terminal tail. Chimera was a noticeable at endocytic compartments based on red fluorescent areas of green fluorescent obscuration, which both localized at the late endosomal lipid BMP and LIMP2, with the endosome protein. Co localization was abundant based on the BMP’s Image Pro measurements. Part of the LTL3’s protein’s transmembrane hydrophobic amino acids was shorter than the original LAMP protein and amino acid sequence was different. The chimera was found to accumulate a considerable extent Golgi, where endogenous LAMP pro-teins do not normally accumulate. Was the result due to the N-terminus of the lumenal LAMP pro-tein was not investigated. The theory is that proteins may form ferries in the Golgi by identifying intra-Golgi protein-protein interactions, which takes the effect to accelerate the Golgi exit or confirm the Golgi residues. This, as well as the chimera effect on the accumulation of cholesterol are both open questions to which require further studies.

This Theses was carried out at the University of Helsinki as part of a larger project, which studied how the membrane protein of the lysosome (LAMP) affects the cholesterol that is stored in the cell. The lysosome means degrading cell compartment, which contents of the vesicle endocytosis-pinch off of, inter alia, from the cell surface repository/cleavage.

My sub-project focused on LTL3 chimera cloning and expression of mouse fibroblastic cells. In cloning gene strands or parts of gene are joined by methods of biotechnology providing a combi-nation of chimeric genes. The combiner fluorescent marker proteins encoded by genes such as RFP-eGFP and transmembrane parts of the gene encoding TGN38 one piece with LAMP1 in the cytoplasm of the protein c-terminal tail of the coding portion of the gene, to form a recombinant gene LTL3. In addition, the chimera was the signal sequence LAMP2 gene which controls the re-combinant gene protein product to the cell membrane and through the secretory pathway. Expres-sion localization in eukaryotic cell I used a fluorescent protein RFP, which emits red light and eGFP, which emits green light as it comes out of the fluorescence microscopy. With these reporter proteins chimeric protein can follow the movement of the in cells. eGFP’s fluorescence is known to turn off the lysosomal acidic pH, the fluorescence RFP’s will last longer in acidic conditions. Chi-mera was expressed on LAMP1- and LAMP2- knockout mouse embryonic fibroblastic (MEF) – cells and accumulation of marker protein was monitored by fluorescence microscopy at different time points during expression.

LAMP localization of the protein in the cell and the late endosome to the lysosome guiding ele-ments are located in the C-terminal tail. Chimera was a noticeable at endocytic compartments based on red fluorescent areas of green fluorescent obscuration, which both localized at the late endosomal lipid BMP and LIMP2, with the endosome protein. Co localization was abundant based on the BMP’s Image Pro measurements. Part of the LTL3’s protein’s transmembrane hydrophobic amino acids was shorter than the original LAMP protein and amino acid sequence was different. The chimera was found to accumulate a considerable extent Golgi, where endogenous LAMP pro-teins do not normally accumulate. Was the result due to the N-terminus of the lumenal LAMP pro-tein was not investigated. The theory is that proteins may form ferries in the Golgi by identifying intra-Golgi protein-protein interactions, which takes the effect to accelerate the Golgi exit or confirm the Golgi residues. This, as well as the chimera effect on the accumulation of cholesterol are both open questions to which require further studies.