Kalorimetrijsko određivanje utjecaja sinergijskog aniona i sijalinizacije na termodinamiku vezanja željeza na ljudski serumski transferin

doctoral thesis

• Title (english): Calorimetric study of the effect of synergistic anion and sialylation on the thermodynamics of the iron binding to human serum transferrin
• Author: Valentina Borko
• Mentor: Tin Weitner (mentor)
• Committee member: Toma Keser (predsjednik povjerenstva)
• Committee member: Mario Gabričević (član povjerenstva)
• Committee member: Branimir Bertoša (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of general and inorganic chemistry); Zagreb
• Defense date and country: 2024-02-16, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Chemistry; Inorganic Chemistry
• Universal decimal classification (UDC): 54 - Chemistry. Crystallography. Mineralogy

Abstract

Ljudski serumski transferin glavni je prenosioc željeza u organizmu. Željezo vezano na transferin doprema se iz probavnog sustava do stanica kojima je ono potrebno i otpušta s transferina promjenom pH. Transferin ima dva mjesta za vezanje željeza u kojem sudjeluje i sinergijski anion. Kod zdravih pojedinaca najčešća glikoforma transferina je glikoforma s dva biantenarna oligosaharidna lanca s ukupno četiri sijalinske kiseline, a sinergijski anion je karbonat. U in vitro uvjetima pokazano je kako drugi strukturno slični karboksilni ioni poput oksalata mogu biti sinergijski anioni. Desijalinizacija transferina primijećena je kod pacijenata s dijagnozom kroničnog alkoholizma, ali i raznih upalnih bolesti te sepse, dok su povećane serumske koncentracije oksalata često rezultat hiperoksalurije ili gastrointestinalnih bolesti. Cilj ovog doktorskog rada bio je odrediti termodinamičke parametre vezanja željeza u formi FeNTA na transferin s različitim indeksom sijalinizacije, u prisutnosti karbonata i oksalata kao sinergijskih aniona. U mjerenjima sa sijaliniziranim transferinom korišten je komercijalno dostupan nativni serumski transferin, dok je desijalinizirani transferin pripremljen djelovanjem enzima neuraminidaze u in vitro uvjetima. Nakon što je desijalinizacija proteina potvrđena i udio sijalinskih kiselina je smanjen za oko 99%, termodinamički parametri određeni su korištenjem izotermne titracijske kalorimetrije. Potvrđeno je kako se dva vezna mjesta razlikuju kinetički i termodinamički te je otkriveno kako desijalinizacija utječe na termodinamičke parametre vezanja željeza u formi FeNTA na transferin tako da efektivne entalpije vezanja postaju egzotermije, a u prisutnosti karbonata pri koncentracijama koje odgovaraj onima u serumu, konstanta vezanja se povećava. Navedeno ide u prilog mogućoj povezanosti desijalinizacije i sekvestracijskog odgovora organizma na infekcije patogenom. Osim desijalinizacije, i oksalat kao sinergijski anion u in vitro uvjetima povećava konstantu vezanja željeza na transferin, a vezanje je egzergonije. Uz jače vezanje oksalata na transferin, navedeno daje osnovu za mogući nastanak ternarnog kompleksa Fe-oksalat-Tf u in vivo uvjetima, pogotovo u stanjima s povećanim koncentracijama oksalata.

Abstract (english)

Human serum transferrin is the major iron transport protein in human organisms. Iron bound to transferrin is transported from the digestive system to cells that require iron. A change in the pH causes the release of iron from transferrin. Transferrin has two iron binding sites that require the presence of the synergistic anion. The most common glycoform of transferrin in healthy humans consists of two biantennary oligosaccharide chains that together contain four sialic acids with carbonate as synergistic anion. Other structurally similar carboxylic ions such as oxalate have shown to act as synergistic anions in vitro. Chronic alcoholic disease, inflammatory diseases and sepsis can cause desialylation of transferrin, and hyperoxaluria or gastrointestinal diseases can lead to higher serum oxalate levels. The aim of this thesis was to determine thermodynamic parameters for the binding of Fe in the form of FeNTA to transferrin with different sialylation index, and in the presence of carbonate and oxalate as synergistic anions. Commercially available native human serum transferrin was used as sialylated transferrin, while the desialylated transferrin was prepared in in vitro using neuraminidase enzyme. After 99% reduction in sialic acid content was confirmed, thermodynamic parameters were determined using isothermal titration calorimetry. Our data have shown that in binding sites differ in both kinetics and thermodynamics of the binding. Moreover, it was discovered that desialylation affects thermodynamic parameters of the iron binding in the form of FeNTA to transferrin, leading to a shift towards exothermic binding enthalpies. Furthermore, in the presence of carbonate at concentrations corresponding to those in human serum, the binding constant increases. These findings are in support of the possible connection between the desialylation and sequestration as an organism’s response to pathogen infections. In addition to desialylation, oxalate as a synergistic anion in in vitro conditions also increases the binding constant of iron to transferrin, and the binding becomes more exergonic. Together with the stronger prebinding constants of oxalate to human serum transferrin, the above provides the basis for the possible formation of the Fe-oxalate-Tf ternary complex in in vivo conditions, especially in conditions with increased concentration of oxalate.

• Keywords: ljudski serumski transferin; sijalinska kiselina; sinergijski anion; glikoproteomika; izotermna titracijska kalorimetrija; termodinamika
• Keywords (english): human serum transferrin; sialic acid; synergistic anion; glycoproteomics; isothermal titration calorimetry; thermodynamics
• Language: croatian
• URN:NBN: urn:nbn:hr:163:153534
• Promotion: 2024
• Study programme: Title: Pharmceutical-biochemical sciences - doctoral study - university study; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti u području biomedicine i zdravstva (doktor/doktorica znanosti u području biomedicine i zdravstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-06-12 11:59:14