Preparation and structure characterization of tumour marker Trop2 using partially glycosylated mutants

Trop2 je transmembranski glikoprotein, ki se pogosto povečano izraža na površini rakastih celic epitelijskih tkiv. Vključen je v celično signaliziranje, katerega začetna stopnja je njegova proteolitska cepitev, sprožitev in pa natančen potek pa še ni popolnoma razjasnjen. Končni učinek prenosa signala vodi k povečani proliferaciji. V okviru magistrskega dela smo želeli podrobneje strukturno okarakterizirati Trop2 in sicer preko določitve kristalne strukture njegovega zunajceličnega dela (Trop2EX). Pripravili smo delno glikozilirane mutirane oblike Trop2EX, da bi z zmanjšanim obsegom heterogene glikozilacije dobili kemijsko bolj homogen vzorec. V luči tega smo z ugasnitvijo izbranih glikozilacijskih mest pripravili štiri delno glikozilirane oblike Trop2EX in se po meritvah DLS odločili za eno izmed njih (Trop2EX(1X3X)). Gre za obliko Trop2EX, ki ima mutirani drugo in četrto glikozilacijsko mesto (mutaciji N120Q in N208Q). Protein smo očistili, končen vzorec pa je vseboval dve različno glikozilirani obliki Trop2EX. Z analizo proteolitske stabilnosti smo pokazali, da naš vzorec ne vsebuje zaznavnih količin kontaminirajočih proteaz, ki bi lahko cepile izpostavljene regije polipeptidne verige in negativno vplivale na integriteto proteina. Izvedli smo presejalne kristalizacijske teste ter identificirali osnovne kristalizacijske pogoje, ki smo jih optimirali z namenom doseganja boljše difrakcijske kvalitete. Ločljivost, do katere sipajo kristali, smo želeli izboljšati tudi s post-kristalizacijsko obdelavo in sicer preko kontrolirane dehidracije kristalov. Najboljše kristale, ki so sipali do ločljivosti 2,8 Å, smo dobili z uporabo kristalizacijskega aditiva EDTA. Prostorska skupina je P 43 2 2. Fazni problem smo rešili z molekulsko zamenjavo z uporabo znane strukture EpCAM (zunajcelični del – EpEX). Ugotovili smo, da so v asimetrični enoti 4 molekule oz. podenote, ki tvorijo dva dimera. Strukturo Trop2EX smo delno rešili in izpililinekatere regije je potrebno ponovno zgraditi, za nekatere pa je elektronska gostota trenutno tako slaba, da njihove strukture ne moremo zgraditi. S primerjavo te strukture s strukturo EpEX smo ugotovili, da sta si strukturi relativno podobni (RMSD poravnanih atomov Cα = 1,94 Å), a v nekaterih regijah so prisotne določene razlike, še posebej v orientaciji N-končne domene glede na preostali del molekule. Ob tem je vzorec disulfidnih vezi v N-končni domeni identičen pri obeh proteinih, isto velja tudi za tiroglobulinsko domeno. Za dimerizacijo so ključne interakcije med aminokislinskimi ostanki β-ploskve in največje zanke tiroglobulinske domene, pri tem pa gre v največji meri za solne mostičkepodobno velja tudi za dimer EpEX. Delno rešena struktura je dobra osnova za končno celotno strukturo zunajceličnega dela Trop2.Trop2 is a transmembrane glycoprotein often overexpressed in cancer cells of epithelial origin. It is involved in cell signaling which starts with it’s proteolytic cleavage, however trigger and detailed mechanism remains poorly understood. The signaling results in enhanced cell proliferation. In order to structurally characterize Trop2 we set on to determine the crystal structure of Trop2 extracellular region (Trop2EX). We prepared four partially glycosylated mutants, aiming to reduce glycosylation-associated heterogeneity, and managed to obtain a protein sample of only two species. The selection of the mutant form Trop2EX(1X3X), which has mutated second and fourth glycosylation sites (mutations N120Q and N208Q), was additionally justified using DLS. Purified protein sample consists of two differently glycosylated forms of Trop2EX(1X3X). Analysis of protein stability shows that protein isn’t a subject of proteolysis by any of the possibly contaminating proteases that could cleave protein and negatively influence protein integrity. By crystallization screening we identified crystallization conditions and optimized them aiming to reach good diffraction quality. In an attempt to improve resolution we also performed controlled post-crystallization dehydration of protein crystals. Best resolution was achieved by adding crystallization additive EDTA. Space group of crystallized protein is P 43 2 2. Phase problem was solved with molecular replacement by using known structure of EpCAM (extracellular region – EpEX). Each asymmetric unit contains four molecules of Trop2EX that form two dimers. We partially solved and refined structure of Trop2EX. Some regions of protein need to be re-built, electron density of other regions is weakly defined, thus the structure of such regions cannot be built. Structures of Trop2EX and EpEX are relatively similar (RMSD of aligned Cα atoms equals 1,94 Å). Biggest difference between two proteins is in orientation of N-terminal domain in comparison to the rest of the molecule. Disulphide bond pattern in N-terminal domain and thyroglobulin domain is identical in both proteins. Important aminoacid residues for formation of Trop2EX dimer are located on β-sheet and thyroglobulin loop. Most significant interactions between those residues are salt bridges, same applies to EpEX. Partially solved structure of Trop2 is good foundation to determine whole structure of protein