N-Glikom Membranskih Proteina i Receptora za Insulin i Faktore Rasta Slične Insulinu, Izolovanih iz Humane Placente u Različitim (Pato)Fiziološkim Stanjima

Abstract

Funkcije membranskih proteina su brojne: međućelijska komunikacija, adhezija, signalna transdukcija. Većina membranskih proteina je glikozilovana i N-glikani imaju važnu ulogu u formiranju trodimenzionalne strukture membranskih proteina, kao i u ispoljavanju njihove funkcije. Receptori za insulin (IR) i faktor rasta sličan insulinu tip 1 (IGF1R) su transmembranske tirozin-kinaze sa visokim stepenom homologije (u nekim domenima čak i 80%). Familija IGF receptora, pored IR i IGF1R, uključuje i receptor za faktore rasta slične insulinu tip 2 (IGF2R). Sva tri receptora (IR, IGF1R i IGF2R) su glikozilovani i obilno prisutni u placenti, gde imaju važne uloge u njenom razvoju i funkcionisanju.

Placenta raste i razvija se da bi ispunila različite potrebe fetusa, pa se struktura i funkcija placente menjaju tokom gestacije. Znajući da su proteini odgovorni za biološke funkcije placente pretpostavljeno je da tokom gestacije može doći i do promene u sadržaju različitih tipova N-glikana prisutnih na membranskim proteinima. U ovoj tezi analizirani su tipovi N-glikana koji se mogu naći u sastavu membranskih glikoproteina, odnosno membranski N-glikom proteina humane placente. Ispitana je podložnost membranskog N-glikoma individualnim varijacijama i uticaju starosti majke/trudnice, kao i uticaj gestacije na membranski N-glikom. Znajući da je izmenjena glikozilacija često povezana sa izmenjenom funkcijom, kao i da izmenjena funkcija jednog ili više proteina može biti uzrok bolesti, ispitane su potencijalne promene membranskog N-glikoma u patološkim trudnoćama (kod preeklampsije) i trudnoćama komplikovanim patologijom majki (dijabetes). Uporedo je ispitan i uticaj starosti, gestacije i patologije na tip i zastupljenost različitih N-glikana koji ulaze u sastav receptora IGF sistema. Cilj je bio da se ispita da li promene na ukupnim membranskim proteinima (na N-glikomu) prate promene N-glikana prisutnih na pojedinačnim membranskim glikoproteinima važnim za rast i funkcionisanje placente.

Pokazano je da je membranski N-glikom bogat različitim N-glikanskimstrukturama, od kojih je jedanaest struktura najzastupljenije, i to su: biantenarni N-glikani: bigalaktozilovani glikan bez fukoze (Fuc) u jezgru - NA2, bigalaktozilovaniglikan sa Fuc u jezgru NA2F, bigalaktozilovani glikan sa Fuc u jezgru i sa umetnutimostatkom N-acetilglukozamina (GlcNAc) - NA2FB, agalakto glikan sa Fuc u jezgru -NGA2, triantenarni N-glikani sa antenarnom Fuc - Na3Fb, triantenarni N-glikani sa Fucu jezgru - NA3F i tetraantenarni N-glikani sa antenarnom Fuc - Na4Fb. Strukturepreostalih N-glikana koji se eluiraju na pozicijama max1, max2 i max6 pretpostavljenesu na osnovu retencionih vremena RNaze B, dok struktura max4 nije mogla bitiidentifikovana. Pokazano je da starost žena nema uticaja na membranski N-glikomhumane placente. U uzorcima placenti različite gestacijske starosti (placenta prvog iplacenta trećeg trimestra) prisutno je svih jedanaest N-glikanskih struktura, ali jenjihova zastupljenost različita, i razlika se javlja kod devet od jedanaest N-glikanskihfrakcija. Tokom gestacije sadržaj paucimanoznih, NA2F i NA3F N-glikana sepovećava, dok se sadržaj NGA2, max4, NA2, max6 i NA2FB smanjuje. Nađeno je dapreeklampsija dovodi do značajnog povećanja zastupljenosti manjih N-glikana(paucimanozne strukture, NGA2 i max4) i smanjenja zastupljenosti većih N-glikana(NA2, max6 i NA3F). Sa druge strane, u slučaju dijabetesa nađeno je da postoje razlikeu odnosu na kontrolnu grupu, mada nije ustanovljena statistička značajnost.

Ispitivanjem uticaja starosti žena, gestacijske starosti placente i patoloških stanjana tip i zastupljenost N-glikana prisutnih na receptorima IGF sistema nađeno je: i) dastarost žena ne utiče na tip i zastupljenost N-glikana prisutnih na IR i IGF2R, dok kodIGF1R sa starošću žena, u prvom trimestru trudnoće, dolazi do smanjenja sadržajabiantenarnih N-glikana, i povećanja sadržaja tri- i tetraantenarnih N-glikana; ii) da setokom gestacije kod IR smanjuje sadržaj N-glikana koji nose ostatke L-Fuc, zatimbiantenarnih N-glikana, i N-glikana koji se završavaju ostacima α2,6-Sia, da se kodIGF1R tokom gestacije smanjuje sadržaj fukozilovanih i N-glikana koji se završavajuostacima α2,6-Sia, i povećava zastupljenost N-glikana sa jezgarnom Fuc, dok se kodIGF2R tokom gestacije povećava zastupljenost fukozilovanih N-glikana; iii) dadijabetes i preeklampsija ne dovode do promena tipa i zastupljenosti N-glikana prisutnihna IR, dok dijabetes ne dovodi do promena tipa i zastupljenosti N-glikana prisutnih naIGF1R i IGF2R.

Rezultati ove teze ukazuju da je N-glikozilacija proteina regulisana tokomrazvoja, kao i da trend promena membranskog N-glikoma nije praćen istim trendompromena na nivou N-glikana membranskih receptora odgovornih za rast, proliferaciju idiferencijaciju (IR, IGF1R i IGF2R).

Alternate abstract:

Membrane proteins have numerous functions such as cell-cell communication, adhesion and signal transduction. Most of them are glycosylated and N-glycans play important role in the formation of three-dimensional structure and function of membrane proteins. Receptors for insulin (IR) and the type 1 insulin-like growth factors receptor (IGFIR) are transmembrane tyrosine kinases with high degree of homology (in some domains even 80%). Together with IR and IGFIR, the family of IGF receptors includes the type 2 insulin-like growth factor receptor (IGF2R). All three receptors (IR, IGFIR and IGF2R) are glycosylated and abundantly present in placenta, where they play important roles in placental development and functions.

Placenta grows and differentiates to fulfil different needs of a foetus, so its structure and function change during gestation. Knowing that proteins are responsible for placental biological functions, it was assumed that during gestation, changes could occur in the content of different types of N-glycans present on membrane proteins. The types of N-glycans commonly found attached to membrane glycoproteins were examined, which resulted in an overview of the membrane N-glycome of human placental proteins. The susceptibility of membrane N-glycome to individual variations and to the effect of maternal/women age was investigated, as well as the influence of the gestational stage on membrane N-glycome. Knowing that altered glycosylation is often related to altered function, and that altered function of one or more proteins can cause a disease, potential changes in membrane N-glycome were analysed in pathological pregnancies (preeclampsia) and in pregnancies complicated by pathology of a mother (diabetes). The impact of aging, gestation and pathology on the type and abundance of different N-glycans on receptors belonging to the IGF system was investigated in parallel. The aim was to resolve whether changes on the entire membrane N-glycome coincide with changes on specific membrane glycoproteins important for placental growth and functions.

It was found that membrane N-glycome is rich in different N-glycan structures, among which eleven are the most abundant: biantennary N-glycans: bigalactosylated glycans without core fucosylation - NA2, bigalactosylated glycan with core fucosylation - NA2F, bigalactosylated glycan with core fucosyation and bisected N- acetylglucosamine (GlcNAc) - NA2FB, agalacto glycan with core fucosylation - NGA2, triantennary N-glycan with antennary fucose (Fuc) - Na3Fb, triantennary N-glycan with core fucosylationNA3F, and tetraantennary N-glycan with antennary Fuc Na4Fh. Structures of glycans eluting at position of maxl, max2 i max6 are suggested according to the retention times of RNase B glycans, while N-glycan eluting at position of max4 was not identified. It was found that maternal age does not affect N-glycome of human placental membrane. Investigating gestational influence on placental membrane N- glycome it was found that same eleven structures appear in all analyzed samples, except that their abundance differes in nine out of eleven glycan structures. While some glycans are more abundant at term (paucimannosidic, NA2F and NA3F), abundance of other glycans appears to be higher in the first trimester of gestation (NGA2, max4, NA2, max6 and NA2FB). It was found that preeclampsia significantly increases the abundance of smaller N-glycan structures (paucimannosidic, NGA2 and max4) and decreases the abundance of larger N-glycans (NA2, max6 and NA3F). In the case of diabetes, it was found that disease affects placental membrane N-glycome but obtained data were not subjected to statistical analysis.

Influence of maternal age, gestation and pathology on the type and abundance of N-glycans found on the receptors of IGF system showed that: i) maternal age does not affect the type and abundance of N-glycans present on IR and IGF2R, while in the case of IGFIR it was found that with maternal age, in first trimester of gestation content of biantennary N-glycans decreases and the content of tri- and tetraantennary N-glycans increases; ii) during gestation in the case of IR content of N-glycans with L-Fuc residues decreases as well as the content of biantennary N-glycans and N-glycans terminating with a2,6-Sia, in the case of IGFIR during gestation content of fucosylated and N-glycans terminating with 02,6-Sia decreases, while the content of N-glycans with core fucosylation increases, and in the case of IGF2R content of fucosylated N-glycans increases during gestation: iii) diabetes and preeclampsia do not affect type and abundance of N-glycans present on placental IR, and diabetes does not affect type and abundance of N-glycans present on placental IGFIR and IGF2R.

Data obtained in this PhD thesis revealed strong gestational impact on placental N-glycome, showing that membrane protein N-glycosylation is developmentaly regulated. It was also found that changes on the membrane N-glycome are not in accordance with the pattern observed for N-glycans present on membrane receptors responsible for growth, proliferation and differentiation (IR, IGFIR and IGF2R).