Biokemijske lastnosti in delovanje ricinu B podobnega lektina iz gobe Macrolepiota procera : doktorska disertacija

Žurga, Simon (author), Kos, Janko (mentor), Sabotič, Jerica (comentor)

Višje prostotrosnice oziroma njihova plodišča so vir mnogih biološko zanimivih učinkovin, med drugim tudi lektinov. Lektini so proteini, ki specifično in reverzibilno brez kovalentne modifikacije vežejo ogljikove hidrate in imajo pomembno vlogo pri celičnih in medceličnih procesih. V okviru doktorske disertacije smo pripravili rekombinantno obliko lektina, imenovanega MpL, ki je bil izoliran iz gobe orjaški dežnik (Macrolepiota procera). Pripravili smo tudi pet mutant lektina s spremenjenimi vezavnimi mesti in dve njegovi fuziji s proteaznima inhibitorjema klitocipinom in cistatinom C. Iz kristalne strukture lektina smo ugotovili, da gre za ricinu B podoben lektin z β-triperesnim vzorcem zvitja, ki je sestavljen iz treh strukturno podobnih ponovitev: α, β in γ. Lektin vsebuje na α- in γ-ponovitvi dve hipotetični mesti za vezavo sladkorjev. Najmočnejše vezavno mesto je na α-podenoti, kjer smo potrdili vezavo treh ligandov, in sicer galaktoze, laktoze in di-N-acetillaktozamina. V vezavnem mestu na γ-ponovitvi je vezan glicerol, ki deluje kot mimetik galaktoze. Lektin tvori strukturno nedefinirane dimere, dokazane z nativno poliakrilamidno gelsko elektroforezo in premreženjem z glutaraldehidom. Testiranje na glikanskih mikromrežah je pokazalo, da je vezavno mesto na α-ponovitvi lektina najbolj specifično za N-acetillaktozamin (Galβ1,4GlcNAc) in njegove ponovitve, mesto na γ-ponovitvi pa za Galβ1,3GlcNAc. Konstanta disociacije za vezavo mono- in disaharidov je v mikromolarnem območju. Nadalje smo ugotovili, da lektini, med njimi tudi MpL, in proteazni inhibitorji z β-triperesnim vzorcem zvitja iz gob medsebojno interagirajo in vplivajo na medsebojne funkcije z morebitno fiziološko vlogo obrambe gobe pred škodljivci. Lektin MpL se različno izraža v posameznih delih gobe in je najbolj izražen v delih, ki so najbolj izpostavljeni morebitnim škodljivcem. Poleg tega je lektin zelo toksičen za ogorčico Caenorhabditis elegans. Lektin rMpL je toksičen tudi za mutante ogorčice C. elegans, ki imajo spremenjeno oziroma okrnjeno sintezo glikanskih struktur na glikoproteinih in glikolipidih. Za lektin rMpL smo dokazali netoksičnost za različne tipe humanih celic. Lektin vpliva na diferencirane celice histiocitnega limfoma U937 z makrofagnimi lastnostmi, in sicer zmanjša njihovo adhezijo na glikoprotein fibronektin in zveča njihovo adhezijo na fibrinogen. Fluorescenčna analiza celic po dodatku lektina je pokazala, da lektin rMpL aktivno vstopa v celice preko endocitoze s klatrinskimi mešički in se nato preko retrogradnega transporta usmeri v trans-Golgijev aparat. Lektin rMpL smo uporabili kot orodje za vnos proteaznih inhibitorjev v notranjost celice. Fuziji lektina rMpL s proteaznima inhibitorjema klitocipinom in cistatinom C aktivno vstopata v celico na enak način kot nemodificiran lektin rMpL. Tekom našega dela smo temeljito proučili biokemijske lastnosti lektina MpL in njegove biološke aktivnosti. S pripravo in uporabo fuzij lektina s proteaznima inhibitorjema klitocipinom in cistatinom C smo dokazali, da je lektin MpL primeren za ciljano dostavo proteinskih učinkovin v celice.

• Language: Slovenian
• Keywords: glive, orjaški dežnik, lektini, rekombinantni proteini, biokemijske lastnosti, biološka aktivnost, disertacije
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [S. Žurga]
• Year: 2015
• Number of pages: XIV, 126 str.
• PID: 20.500.12556/RUL-143734
• UDC: 606:615.322(043.3)
• COBISS.SI-ID: 279208192
• Publication date in RUL: 11.01.2023

Secondary language

• Language: English
• Title: Biochemical properties and function of ricin B like lectin from mushroom Macrolepiota procera

Abstract

Fruiting bodies of higher fungi are a well-known source of biologically active ingredients, including lectins. These are proteins that specifically and in a non-modifying way bind carbohydrates and have major roles in cellular processes as well as in cell-cell interactions. During our research we prepared recombinant lectin named MpL which was found in parasol mushroom (Macrolepiota procera). We also designed and produced five mutants of this lectin with modified carbohydrate-binding sites and two fusion proteins of lectin MpL with protease inhibitors clitocypin and cystatin C. Cristal structure shows that it belongs to the ricin B-like group of lectins and possesses β-trefoil fold composed of three structurally similar repeats α, β and γ. We discovered that lectin has two hypothetical binding sites for glycans. Strong binding site on α-repeat is occupied in crystal structure with galactose, lactose and di-N-acetyllactosamine. The binding site on γ-repeat is occupied only with glycerol which can act as a mimetic of galactose. Native polyacrylamide gel electrophoresis and glutaraldehyde crosslinking show that MpL forms structurally undefined dimers. The binding site on α-repeat of MpL is most specific to glycans terminating with one or more repeats of N-acetyllactosamine (Galβ1,4GlcNAc) and the binding site on γ-repeat for Galβ1,3GlcNAc, as determined by glycan microarray analysis. Constant of dissociation for binding of mono- and disaccharides is in a micromolar range. β-Trefoil fold lectins (including MpL) and protease inhibitors from mushrooms interact and regulate their biological functions which could have a broader physiological effect of this proteins in defense against antagonists. Lectin MpL is differentially expressed throughout the fruiting body and has the highest expression in parts that are most likely to be attacked by antagonists. Lectin MpL is highly toxic to Caenorhabditis elegans. Lectin MpL is also toxic to C. elegans mutatnts with affected glycan structures present on glycoproteins and glycolipids. On the other hand, MpL is not toxic to any human cell line tested. However, MpL affects the adhesion of differentiated histiocytic lymphoma cell line U937, with macrophage-like properties. In the presence of lectins the adhesion on fibronectin is decreased and on fibrinogen it is increased. Upon addition to the cells, MpL lectin undergoes clathrin-mediated endocytosis and is translocated from early endosomes to the trans-Golgi network via retrograde transport mechanism. Lectin MpL was used as a tool for targeted delivery of protease inhibitors into cells. Two fusion proteins of lectin with protease inhibitors clitocypin and cystatin C actively enter human cells in the same way as unmodified rMpL. By studying the biochemical properties and biological activities of lectin MpL and by preparing and using the fusion proteins of lectin with protease inhibitors clitocypin and cystatin C we proved that lectin MpL is suitable for targeted delivery of proteins into cells.