Production and Transduction of a Human Recombinant ß-Globin Chain into Proerythroid K-562 Cells To Replace Missing Endogenous ß-Globin.

Protein replacement therapy (PRT) has been applied to treat severe monogenetic/metabolic disorders characterized by a protein deficiency. In disorders where an intracellular protein is missing, PRT is not easily feasible due to the inability of proteins to cross the cell membrane. Instead, gene therapy has been applied, although still with limited success. ß-Thalassemias are severe congenital hemoglobinopathies, characterized by deficiency or reduced production of the adult ß-globin chain. The resulting imbalance of α-/ß-globin chains of adult hemoglobin (α2ß2) leads to precipitation of unpaired α-globin chains and, eventually, to defective erythropoiesis. Since protein transduction domain (PTD) technology has emerged as a promising therapeutic approach, we produced a human recombinant ß-globin chain in fusion with the TAT peptide and successfully transduced it into human proerythroid K-562 cells, deficient in mature ß-globin chain. Notably, the produced human recombinant ß-globin chain without the TAT peptide, used as internal negative control, failed to be transduced into K-562 cells under similar conditions. In silico studies complemented by SDS-PAGE, Western blotting, co-immunoprecipitation and LC-MS/MS analysis indicated that the transduced recombinant fusion TAT-ß-globin protein interacts with the endogenous native α-like globins to form hemoglobin α2ß2-like tetramers to a limited extent. Our findings provide evidence that recombinant TAT-ß-globin is transmissible into proerythroid K-562 cells and can be potentially considered as an alternative protein therapeutic approach for ß-thalassemias.

Purification, preliminary X-ray crystallography and biophysical studies of triose phosphate isomerase-ß-globin subunit complex.

Triose phosphate isomerase (TIM) is a cytoplasmic enzyme of prime importance in the mammalian glycolytic pathway. It has a major role in the conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We have successfully purified a stable complex of TIM with ß-globin subunit from the sheep kidney using a simple two-step chromatography procedure. It is seen for the first time that TIM is forming a stable complex with ß-globin. The purified protein-protein complex was crystallized and preliminary diffraction data were collected at 2.1Å resolution. We further studied guanidinium chloride (GdmCl)-induced denaturation of TIM-ß-globin complex by monitoring changes in the mean residue ellipticity at 222nm ([θ]222) and difference absorption coefficient at 406nm (Δε406) at pH 7.5 and 25°C. We have observed that GdmCl-induced denaturation is reversible. Coincidence of normalized transition curves of both physical properties ([θ]222 and Δε406) suggests that folding/unfolding of TIM and ß-subunit proteins is a two-state process. Denaturation curves of [θ]222 and Δε406 were used to estimate the stability parameters of the protein-protein complex. This is the first report on the isolation, purification, crystallization and biophysical characterization of the naturally occurring complex of TIM with the ß-globin subunit.

The Primary Structure of ß(I)-Chain of Hemoglobin from Snake Sindhi Krait (Bungarus sindanus sindanus).

The amino acid sequence of ß(I)-globin chain from Sindhi Krait (Bungarus sindanus sindanus) was determined to study the molecular evolution among snakes. The hemoglobin was isolated from the red blood cells and was analyzed by ion-exchange chromatography (IEX). The crude globin was subjected to reversed phased-high performance liquid chromatography (RP-HPLC) using C4 column. The N-terminal sequences of intact globin chains and tryptic peptides were determined by Edman degradation in a pulsed liquid gas phase sequencer using an online Phenylthiohydantoin analyzer. Sindhi Krait is expected to express three hemoglobin components that are composed of ß(II), ß(I), α(D) and α(A)-globin chains, as apparent by IEX, RP-HPLC and N-terminal sequence analyses. Sequence alignment and phylogenetic analyses of ß(I) globin chain from Sindhi Krait showed closest relationship with ß(I) globin chain from Rattlesnake, Water snake and Indigo snake. Interestingly, comparison of primary sequence of ß(I) globin chain of Sindhi Krait with human ß chain revealed 63 % similarity along with the retention of all heme contact points. Variations among the two sequences were prominent at αß contact points and in regions directly not important for function.

High Rate of ß-Globin DNA Detection Validates Self-Sampling in Herpes Simplex Virus Shedding Studies.

BACKGROUND: Self-sampling is a convenient, feasible, and acceptable way of collecting genital specimens, but the veracity of reported self-collection is difficult to verify. We investigated whether a host gene, ß-globin, can be used to confirm adequacy of self-collected mucosal and skin genital specimens in studies of genital herpes simplex virus type 2 (HSV-2) infection. METHODS: Herpes simplex virus type 2-seropositive adults self-collected daily anogenital and oral swabs. Mucosal samples were tested for HSV DNA using a real-time quantitative polymerase chain reaction assay. A real-time Taqman polymerase chain reaction detecting the ß-globin gene was used to quantify host cells. RESULTS: One hundred twelve participants collected 5559 genital and 2002 oral swabs. Sixty (54%) were women, 65% were HSV-2 seropositive, and 35% were HSV-1 and HSV-2 seropositive by Western blot. ß-globin DNA was detected in 99% and 93% of swabs obtained from women and men, respectively. The quantity of ß-globin DNA detected was significantly higher when HSV was present in genital swabs in women (0.1 log10 copies/mL; P = 0.001) and in men (0.6 log10 copies/mL; P < 0.001), but not in oral swabs in women (0.2 log10 copies/mL; P = 0.08) or men (0.0 log10 copies/mL; P = 0.70). CONCLUSIONS: Human ß-globin DNA detection rate was high, and the quantity obtained significantly increased with genital, but not oral HSV shedding. The high rate of ß-globin DNA detection is consistent with high adherence to study procedures in longitudinal studies of genital herpes shedding.

Development of a capillary zone electrophoresis method for rapid determination of human globin chains in α and ß-thalassemia subjects.

Thalassemia is an inherited autosomal recessive blood disorder characterized by the underproduction of globin chains as a consequence of globin gene defects, resulting in malfunctioning red blood cells and oxygen transport. Analysis of globin chains is an important aspect of thalassemia research. In this study we developed a capillary zone electrophoresis (CZE) method for human globin determination in the diagnosis of thalassemia and hemoglobin variants. To demonstrate the utility of this approach, α/ß area ratios were determined for samples from 310 thalassemia patients and healthy controls. The separation was performed on uncoated capillary with simple preparation. Distinct globin peaks were resolved in 17 min, and coefficients of variation (CV) for migration time and areas ranged from 0.37%-1.69% and 0.46%-6.71%, respectively. Receiver operating characteristic (ROC) curve analysis of the α/ß area ratios gave 100% sensitivity and specificity for indicating ß-TI/TM, and 100% sensitivity and 97.4% specificity for Hb H disease. Hemoglobin G-Honolulu (Hb G-Honolulu) and Hb Westmead (Hb WS) were successfully detected using this CZE method. This automated methodology is simple, rapid and cost-effective for the fast determination of human globin chains, which could be an important diagnostic tool in the field of hemoglobinopathies.

In-depth comparative characterization of hemoglobin glycation in normal and diabetic bloods by LC-MSMS.

The glycation level at ß-Val-1 of the hemoglobin ß chain in human blood (HbA1c%) is used to diagnose diabetes and other diseases. However, hemoglobin glycation occurs on multiple sites on different isoforms with different kinetics, but its differential profile has not been clearly demonstrated. In this study, hemoglobin was extracted from the blood of normal and diabetic individuals by protein precipitation. Triplicate solutions prepared from each sample were directly analyzed or digested with multiple enzymes and then analyzed by nano-LC/MS via bottom-up approach for side-by-side characterization. Intact hemoglobin analysis indicated a single glucose-dominant glycation, which showed good correlation with the HbA1c% values. Moreover, full sequence (100%) of α/ß globin was mapped and seven glycation sites were unambiguously assigned. In addition to ß-Val-1, two other major sites at α-Lys-61 and ß-Lys-66, which contain the common sequence HGKK, and four minor sites (<1%) on α-Val-1, ß-Lys-132, α-Lys-127, and α-Lys-40 were identified. All sites were shown to exhibit similar patterns of site distribution despite different glucose levels. Both the intact mass measurement and bottom-up data consistently indicated that the total glycation percentage of the ß-globin was twice higher than the α-globin. Using molecular modeling, the 3D structure of the consensus sequence (HGKK) was shown to contain a phosphate triangle cavity, which helps to catalyze the glycation reaction. For the first time, hemoglobin glycation in normal and diabetic bloods was comparatively characterized in-depth with 100% sequence coverage. The results provide insight about the HbA1c parameter and help define the new and old markers.

SMG6 cleavage generates metastable decay intermediates from nonsense-containing ß-globin mRNA.

mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. The exception to this is nonsense-containing human ß-globin mRNA, where the destabilization of full-length mRNA is accompanied by the cytoplasmic accumulation of 5'-truncated transcripts in erythroid cells of transgenic mice and in transfected erythroid cell lines. The relationship of the shortened RNAs to the decay process was characterized using an inducible erythroid cell system and an assay for quantifying full-length mRNA and a truncated RNA missing 169 nucleotides from the 5' end. In cells knocked down for Upf1 a reciprocal increase in full-length and decrease in shortened RNA confirmed the role of NMD in this process. Kinetic analysis demonstrated that the 5'-truncated RNAs are metastable intermediates generated during the decay process. SMG6 previously was identified as an endonuclease involved in NMD. Consistent with involvement of SMG6 in the decay process full-length nonsense-containing ß-globin mRNA was increased and the Δ169 decay intermediate was decreased in cells knocked down for SMG6. This was reversed by complementation with siRNA-resistant SMG6, but not by SMG6 with inactivating PIN domain mutations. Importantly, none of these altered the phosphorylation state of Upf1. These data provide the first proof for accumulation of stable NMD products by SMG6 endonuclease cleavage.

Locus-specific proteomics by TChP: targeted chromatin purification.

Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive "hook" bound to a specific promoter, we identify transcription factors bound to the repressed γ-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces γ-globin gene expression. Reactivation of γ-globin gene expression ameliorates the symptoms of ß-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.

Prenatal and post-natal screening of ß-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography.

We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common ß-thalassemia mutations found in Thailand. The ß-globin gene was separately amplified by PCR on four different fragments covering eight most common ß-thalassemia mutations including nucleotide -28 A-G, codon 17 (A-T), IVSI-1 (G-T), IVSI-5 (G-C), codon 26 (G-A or Hb E), codons 41/42 (-TTCT), codons 71/72 (+A) and IVSII-654 (C-T). After PCR amplification, heteroduplex was generated by denaturation at 95 °C for 5 min followed by a slow reduction in temperature to 25 °C at 0.03 °C/s. Analysis of heteroduplex was done on an automated WAVE Nucleic Acid Fragment Analysis System. Specific DHPLC profile for each mutation was demonstrated which could be used in screening for all eight ß-thalassemia mutations. Further validation was done on 42 pre- and post-natal DNA samples which demonstrated 100 % accuracy as compared to the result obtained with conventional PCR assays. In a remaining case with an unknown mutation, a different DHPLC profile was noted on one of the amplified fragment. Further DNA sequencing of this fragment revealed a T-G transversion at the IVSI-116, a previously un-described mutation in Thai population. The DHPLC assay developed should prove useful for rapid screening of known and unknown ß-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.

Role of the tertiary and quaternary structure in the formation of bis-histidyl adducts in cold-adapted hemoglobins.

All tetrameric hemoglobins from Antarctic fish, including that from Trematomus bernacchii, HbTb form in the ferric state, promptly and distinctively from all the other tetrameric hemoglobins, a mixture of aquo-met at the α subunits and bis-histidyl adduct (hemichrome) at the ß subunits. The role of the tertiary and quaternary structure in the hemichrome formation is unknown. Here we report the cloning, expression, purification, spectroscopic and computational characterization of the ß-chain of HbTb (ß-HbTb). Similarly to the human ß-chains, ß-HbTb self-assembles to form the homotetramer ß(4)-HbTb; however, the latter quantitatively forms reversible ferric and ferrous bis-histidyl adducts, which are only partially present in the human tetramer (ß(4)-HbA). A molecular dynamics study of the isolated ß subunit of the two Hbs indicates that the ability to form hemichrome is an intrinsic feature of the chain; moreover, the greater propensity of ß-HbTb to form the bis-histidyl adduct is probably linked to the higher flexibility of the CD loop region. On the bases of these experimental and computational results on the isolated chain, the influence of the quaternary structure on the stability of the endogenous ferrous and ferric hexa-coordination is also discussed.