Engineering of Human Fetal Hemoglobin

Forskningsoutput: AvhandlingDoktorsavhandling (sammanläggning)

62 Nedladdningar (Pure)

Sammanfattning

In blood, the oxygen-transporting protein hemoglobin (Hb) governs the oxygenation of cells and tissues. Naturally, this protein has claimed a place in the center of the research field of artificial oxygen therapeutics. Such Hb-based products have used cell-free Hb purified from human, bovine, invertebrate, or recombinant sources to create hemoglobin-based oxygen carriers (HBOCs). However, administration of these cell-free Hb products into the bloodstream initiates several unwanted adverse events. The inherent toxic reactivity of Hb related to heme-mediated oxidation, nitric oxide scavenging, and heme release give rise to serious side effects. These issues have persisted despite attempts at finding a formulation strategy to tame native Hb outside the red blood cell. This dissertation describes work regarding the engineering of human fetal Hb (HbF) for screening of beneficial protein design strategies on the protein itself, both in terms of retaining oxidative stability and from a production perspective.
Oxidative side effects are central to the extracellular toxicity exhibited by Hb. We examined the effect of modifying redox active cysteine residues in HbF by removing and/or adding cysteine at the conserved hotspot γCys93 and a surface-located site on the α-subunit. The conserved cysteine was important for the oxidative stability of the protein and removal produced a more unstable Hb molecule. In contrast, the addition of cysteine on the surface of the α-subunit alleviated damaging reactions during oxidative conditions by providing an alternative oxidation hotspot.
As the surface of the α-subunit appeared to be promising as a target area for mutagenesis, we explored a set of mutants where alanine residues were substituted into negatively charged aspartic acid. This lowered the pI of HbF and reduced the DNA cleavage rate without affecting the overall structural integrity. We also observed an extended half-life in vivo as well as unchanged oxidation and heme loss rates, indicating that improved functions could be attained with modification of the net surface charge without adversely affecting key functions and stability.
We continued to focus on the surface of HbF and created mutants with more dramatically changed net surface charge by replacing positive surface residues with negatively charged residues. We improved Hb yields in the crude extracts during recombinant expression in E. coli with this strategy, but non-target Hb fractions were present in significant quantities in two of the three mutant samples. This indicated an unbalanced assembly of subunits in the HbF variants carrying the γ-subunit mutations, leading to the formation of homotetramers. In addition, the chosen γ-subunit mutations contributed to a more oxidatively unstable Hb molecule, as seen by increased autoxidation rates.
The best-performing negatively charged mutant was subjected to a more in-depth characterization study. The crystal structure of this mutant was solved and thus confirmed the surface-exposed locations of the mutations. The mutant showed no significant differences in oxidation rate reactions but differed in reduction and heme loss rates from wild-type HbF in reactions governed by the α-subunit. In contrast to a previously studied HbF mutant, this mutant did not show any increased effect on retention time in vivo. However, a dramatic decrease in DNA cleavage rate was seen, indicating a much less damaging behavior towards important cellular components.
We conclude that there are strategies for modifying HbF itself for the improvement of protein properties towards a better, and more easily produced Hb for HBOC development. The suggested modifications to the HbF protein presented in this work could be used in combination with other protein engineering strategies for Hb development.
Originalspråkengelska
KvalifikationDoktor
Handledare
  • Bülow, Leif, handledare
Tilldelningsdatum2021 juni 11
Förlag
ISBN (tryckt)978-91-7422-814-4
ISBN (elektroniskt)978-91-7422-815-1
StatusPublished - 2021

Bibliografisk information

Defence details
Date: 2021-06-11
Time: 13:00
Place: Lecture hall Marie Curie, Kemicentrum, Naturvetarvägen 14, Faculty of Engineering LTH, Lund University, Lund. Zoom: https://lu-se.zoom.us/j/64373925491
External reviewer(s)
Name: Fago, Angela
Title: Prof.
Affiliation: Aarhus University, Denmark.
---

Ämnesklassifikation (UKÄ)

  • Biokemi och molekylärbiologi

Fingeravtryck

Utforska forskningsämnen för ”Engineering of Human Fetal Hemoglobin”. Tillsammans bildar de ett unikt fingeravtryck.

Citera det här