HRZZ UIP-2017-05-9537

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Equipment:


a)Funded by the project


1.)  Tecan Spark 10M microplate reader for
       absorbance, fluorescence and fluorescence polarization

2.)  Opentrons OT-2 pipetting robot

3.)  BCN3D SIGMA R19 FFM 3D printer

4.)  Benchmark Scientific PlateFuge microplate microcentrifuge

5.)  SCS 963-NO Benchtop air ioniser

6.)  CAPP CAPPWash plate washer kit


b)Spectrophotometers

1.)  OLIS RSM 1000 UV-Vis Spectrophotometer and
       Spectrofluorimeter with stopped-flow module

2.)  Cary 50 UV-Vis Spectrophotometer with multicell holder and optic-fiber dip probe

3.)  OLIS 8452A UV-Vis Diode Array Spectrophotometer

4.)  Ocean Optics S-2000 UV-Vis Diode Array Spectrophotometer


c) Electrochemistry


1.)  CH Instruments CHI660D Electrochemical Analyser

2.)  ALS CS-3A Cell Stand Faraday cage

3.)  ALS SEC-3F Spectroelectrochemical flow cell


d)Other


1.)  GE Lifesceiences ÄKTA start protein purification system
       with additional de-bubbling vacuum accessory

2.)  VWR Incu-Line incubator with tube rotator

3.)  Mettler Toledo T50 titrator with dual burette drive and conductometric sensor board

4.)  Mettler Toledo MP 220 pH meter with glass electrode

5.)  Sartorius LA 310 S Balance

6.)  VWR CompactStar CS 4 Centrifuge

7.)  Eppendorf 5424 Microcentrifuge

8.)  Ultimaker 2+ FFM 3D printer

9.)  Prusa i3 MK3S FFM 3D printer

10.)  Wanhao Duplicator D7 DLP 3D printer


Objective 1: Determination of the iron-binding equilibrium constants for transferrin glycoforms


The glycosylation pattern of Transferrin (Tf) is altered in numerous physiological and pathological states, mainly by removal of the terminal sialic acids. The influence of the altered glycosylation pattern on iron binding could provide a deeper understanding of the connection between the physiological or pathophysiological changes in the organism and the corresponding post-translational Tf modification. The results will enable us to correlate the changes of Tf glycan structure associated with different conditions to the possible changes in iron binding equilibrium.

The changes in Tf glycosylation during infection as a response to pathogenic bacteria is of particular interest because bacteria have their own iron-scavenging mechanisms:

(i)synthesis of low molecular weight ligands (siderophores) that strongly bind iron and compete with host Tf and

(ii)synthesis of their own Tf receptor and direct use of iron-loaded host Tf. Knowledge of a detailed mechanism of the variable host glycosylation pattern influence on the bacterial supply of iron can provide new ways of influencing their virulence during infection or sepsis.


Objective 2: Determination of the reduction potential of iron bound to transferrin glycoforms


The mechanism of iron binding and release by Tf is redox-mediated. Briefly, upon binding of the Tf molecule to its specific receptor on cell membranes, the receptor-Tf assembly is internalized and iron is liberated from Tf within the endosome and the resulting apotransferrin is released to the circulation for another cycle of iron transport. Therefore, it is of considerable importance in gaining an understanding of the physiological and pathophysiological implications to determine the effect of Tf glycosylation on its’ reduction potential, particularly due to the requirement for reduction of ferric iron, as ferrous iron is transported out of the endosome by a divalent metal transporter, DMT1.

The results will enable us to correlate the changes of Tf glycan structure associated with different conditions to the possible changes in reduction potential of the bound iron, and in turn provide new rationales for the corresponding post-translational Tf modification. Additionally, the results on the reduction potentials of individual Fe(III) ions bound to either N-lobe or C-lobe of Tf should provide insight into the effect of glycosylation on the electronic interaction between the two binding sites.


Objective 3: Determination of the kinetics and mechanism of iron binding and release from transferrin glycoforms


Considering that Tf undergoes significant conformational changes in the processes of iron uptake and release, the effect of relatively large Tf glycan structures may have a significant effect on these pathways. The modifications in the conformation of the protein occurring during iron binding or release may be dependent on the glycosylation pattern and the post-translational Tf modifications might affect the efficiency of cellular iron uptake. The heterogeneous kinetic properties of the iron binding sites are particularly intriguing due to the known 2-minute timeframe of cellular iron uptake cycle.

The possible impact of the Tf glycosylation pattern might therefore be in producing conformational states from which iron can be released either more homogeneously or more heterogeneously, i.e. from which iron release is facilitated or hindered, respectively. However, such an effect could be much more pronounced for the Tf-receptor complex, which has shown a profound effect on the efficiency of endosomal iron release.