Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical alpha- and two identical gamma-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the alpha- and gamma-subunits of HbF were fused in order to stabilize the alpha-subunits and facilitate balanced expression of alpha- and gamma-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes

Towards New Generation of Hemoglobin-Based Blood Substitutes

Blood transfusion is a clinically significant and crucial process, which saves millions of lives every year. However,shortage of donated blood and the risk of virus transmission through transfusible blood seriously affect theavailability of the blood. Hemoglobin (Hb), owing to its oxygen carrying capacity, has been studied as a startingmaterial for the development of artificial blood substitutes/Hb-based oxygen carrier (HBOC). Several kinds ofHBOC products have been developed and tested for their safety at different stages of clinical trials with minimalsuccess. The failure of such products is mainly associated with intrinsic toxicity of cell-free Hb which damageslipids, proteins, DNA and surrounding tissues. This thesis describes two approaches aiming to gain furtherknowledge of potential side effects of Hb molecules on genetic material. Additionally, genetic engineeringapproach was used as an alternative to chemical modification of Hb molecule, which is essential for theperformance of HBOC product in cell-free environment.Using the comet assay, we have evaluated the genotoxic effect of the penultimate tyrosine residues of the alphaand beta chains. Replacement of a tyrosine residue with phenylalanine, in the alpha chain (α-Y140F) has shown40% higher DNA damage compared to wildtype HbA. However, a similar mutant on the beta chain had negligibleeffect on the genotoxicity of Hb molecule.In a plasmid DNA cleavage assay, we have demonstrated that Hb itself can interact with DNA molecules andinitiate their cleavage. Conversion of supercoiled plasmid DNA (sc pDNA) into open circular (ocDNA) or linearDNA (LDNA) was used to determine the DNA cleavage activity of Hb. Our investigation revealed that fetalhemoglobin (HbF) was three-fold less active than adult hemoglobin (HbA). Thus, we have proposed HbF as apotential starting material for creation of safe HBOC product.In a second approach, we have demonstrated beneficial effects of a polypeptide tag (dubbed XTEN) geneticallyattached to fusion fetal hemoglobin (fHbF), forming XTEN-HbF. The main purpose of this XTEN polymer is toavoid the chemical processing such as PEGylation, which often increases the production cost. Additionally,PEGylation also impair the structural and functional properties of Hb molecule. Using XTEN polypeptide, thefunctional properties of a fHbF remains largely unchanged, reflected by identical oxygen affinity and absorptionspectra. XTEN-HbF was produced as a homogenous mixture of product and increased the molecular size offHbF by a factor of 2.2 folds.In addition, we have produced fluorescent Hb, referred as GFP-HbF. It is composed of green fluorescent protein(GFP) linked to fHbF at the DNA level. The primary results suggest that the purified protein is fully functional, asreflected by spectral properties of fHbF and characteristic fluorescence of the GFP molecule. Furthermore, theadsorption properties of the molecularly imprinted polymers (MIP) have been estimated using fHbF, with orwithout GFP. These MIPs have a capacity to facilitate the separation and purification of Hb molecules

Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

Hemoglobin (Hb) is well protected inside the red blood cells (RBCs). Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA). Hb induced cleavage of supercoiled pDNA (sc pDNA) which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2) and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA) could easily induce complete pDNA cleavage while fetal hemoglobin (HbF) was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals

Trapping of human hemoglobin by haptoglobin: Molecular mechanisms and clinical applications.

Significance: Haptoglobin (Hp) is an abundant plasma protein controlling the fate of hemoglobin (Hb) released from red blood cells after intravascular hemolysis. The complex formed between Hp and Hb is extraordinary strong, and once formed, this protein-protein association can be considered irreversible. Recent advances: A model of the Hp-Hb complex has been generated and the first steps towards understanding the mechanism behind the shielding effects of haptoglobin have been taken. The clinical potential of the complex for modulating inflammatory reactions and for functioning as an HBOC (hemoglobin-based oxygen carrier) have been described. Critical Issues: The three-dimensional structure of the Hp-Hb complex is unknown. Moreover, Hp is not a homogeneous protein. There are two common alleles at the Hp genetic locus denoted Hp1 and Hp2, which when analyzed on the protein levels result in differences between their physiological behavior, particularly in their shielding against hemoglobin-driven oxidative stress. Additional cysteine residues on the alpha-subunit allow Hp2 to form a variety of native multimers, which influence the biophysical and biological properties of Hp. The multimeric conformations in turn also modulate the glycosylation patterns of Hp by steric hindrance. Future Directions: A detailed analysis of the influence of Hp glycosylation will be instrumental to generate a deeper understanding of its biological function. Several pathological conditions also modify the glycan compositions allowing Hp to be potentially used as a marker protein for these disorders