Effect of point mutation on structure-function correlation of hemoglobin variants, HbE and HbD Punjab.

Hemoglobinopathies are examples of autosomal recessive disorders of human hemoglobin. Hemoglobin E (HbE) and Hemoglobin D Punjab (HbD Punjab) are two of the most common hemoglobin variants geographically spread across Asian continent. These two variants differ from normal human hemoglobin (HbA) at a single amino acid residue caused by the point mutation of ß globin gene. The presence of the mutated amino acid residue causes perturbation in the function of both variants. However, the structure-function correlation of these variants has not been established yet. In the present study, we analyzed the conformational changes associated with oxygenation of hemoglobin variants using hydrogen/deuterium exchange-based mass spectrometry of backbone amide hydrogens of α and ß globin chains in the tetrameric hemoglobin molecule. We also performed the functional assay of these variants using oxygen dissociation equilibrium curve. Compared to HbA, both variants showed reduced oxygen affinity, as reported earlier. The functional perturbations exhibited by these variants were correlated well with their structural alterations with respect to the reported changes in the residue level interactions upon oxygenation of normal hemoglobin, monitored through the hydrogen/deuterium exchange kinetics of several peptic peptides originated from the isotopically exchanged oxy and deoxy forms of HbE and HbD Punjab.

Glycation profile of minor abundant erythrocyte proteome across varying glycemic index in diabetes mellitus.

PURPOSE: Long-term glycemic index in patients with diabetes mellitus (DM) is measured by glycated hemoglobin (HbA1c) besides blood glucose. In DM, the primary amino groups of proteins get glycated via non-enzymatic post-translational modification. This study aims at identifying and characterizing site-specific glycation of erythrocyte proteome across varying glycemic index in patients with DM. EXPERIMENTS: We isolated the glycated erythrocyte proteome devoid of hemoglobin from control and diabetic samples using boronate affinity chromatography. Proteomic analysis was performed using nanoLC/ESI-MS proteomics platform. The site-specific modification on different proteins was deciphered using a customized database. RESULTS: We report 37 glycated proteins identified and characterized from samples with HbA1c of 6%, 8%, 12%, and 16%. Our results show that both extent and site-specific modification of proteins increased with increasing HbA1c. The observed residue-specific modifications of catalase, peroxiredoxin, carbonic anhydrase, lactate dehydrogenase B and delta-aminolevulinic acid dehydratase were correlated with the literature report on their functional disorder in DM. CONCLUSIONS: and clinical relevance: 37 glycated erythrocyte proteins apart from hemoglobin were characterized from DM patient samples with varying HbA1c values. We correlated the site-specific glycation and associated functional disorder of five representative proteins. However, the clinical correlation with the observed modifications needs further investigation.

Molecular insights of inhibition in sickle hemoglobin polymerization upon glutathionylation: hydrogen/deuterium exchange mass spectrometry and molecular dynamics simulation-based approach.

In sickle cell anemia, polymerization of hemoglobin in its deoxy state leads to the formation of insoluble fibers that result in sickling of red blood cells. Stereo-specific binding of isopropyl group of ßVal6, the mutated amino-acid residue of a tetrameric sickle hemoglobin molecule (HbS), with hydrophobic groove of another HbS tetramer initiates the polymerization. Glutathionylation of ßCys93 in HbS was reported to inhibit the polymerization. However, the mechanism of inhibition in polymerization is unknown to date. In our study, the molecular insights of inhibition in polymerization were investigated by monitoring the conformational dynamics in solution phase using hydrogen/deuterium exchange-based mass spectrometry. The conformational rigidity imparted due to glutathionylation of HbS results in solvent shielding of ßVal6 and perturbation in the conformation of hydrophobic groove of HbS. Additionally, molecular dynamics simulation trajectory showed that the stereo-specific localization of glutathione moiety in the hydrophobic groove across the globin subunit interface of tetrameric HbS might contribute to inhibition in polymerization. These conformational insights in the inhibition of HbS polymerization upon glutathionylation might be translated in the molecularly targeted therapeutic approaches for sickle cell anemia.

Analysis of the Quaternary Structure of Hemoglobin Beckman Variant and Molecular Interpretation of Its Functional Abnormality: A Mass-Spectrometry-Based Approach.

Electrostatic attraction between α and ß globin chains holds the subunits together in a tetrameric human hemoglobin molecule (α2 ß2 ). Compared to normal globin chains, the affinity of a mutant chain to its partner globin might be different in genetic variants of hemoglobin. This leads to an unequal abundance of normal and variant hemoglobin in heterozygous samples, even though the rates of synthesis of both the normal and variant chains are the same. The aforementioned affinities across various globin chains might be assessed by quantification of the different forms of the tetramers present in a variant hemoglobin sample. In the present study, by exploiting mass differences between globin chains, differently populated hemoglobin tetramers present in hemoglobin (Hb) Beckman, a ß variant (ßA135D), were structurally characterized. The relative populations of dissymmetric tetramers (α2 ß2 , α2 ßßV , and α2 ßV2 ) indicated that both ß and ßV have different affinities towards the α globin chain. Conformational dynamics analyzed from hydrogen/deuterium exchange kinetics of the three peptide fragments of Hb Beckman in its oxy state displayed molecular insight into its functional abnormality. However, in comparison to normal hemoglobin (α2 ß2 ), the point mutation did not show any change in the collision cross-sections of the functionally active conformers of the variant hemoglobin molecules (α2 ßßV and α2 ßV2 ).

Application of isotope exchange based mass spectrometry to understand the mechanism of inhibition of sickle hemoglobin polymerization upon oxygenation.

Sickle hemoglobin (HbS) polymerization initiates in the deoxy state with the binding of hydrophobic patch formed by the isopropyl group of ßVal6 residue of a hemoglobin tetramer with the hydrophobic pocket of another tetramer, whose hydrophobic patch binds to the hydrophobic groove of a third molecule. Subsequent elongation of a single stranded polymer followed by the formation of a double strand and finally combination of seven such pairs of double strands results in a fourteen stranded fibrous polymer. Precipitation of this fiber inside the erythrocytes results in sickling of red blood cells. Surprisingly, the polymerization does not occur in the oxy state of HbS. Due to the unavailability of crystal structure of oxy form of HbS, the molecular basis of inhibition of polymerization in the oxy state is unknown to date. In the present study, we have attempted to understand the molecular mechanism of inhibition of polymerization by exploiting the exchange of backbone amide hydrogens of HbS with deuterated solvent. Hydrogen/deuterium exchange kinetics of peptide amide hydrogens of both oxy and deoxy form of HbS were monitored through ESI mass spectrometry. Upon oxygenation changes in the conformational flexibility across different regions of α and ß globin chains in the tetrameric HbS molecule were investigated. It was observed that oxygenation led to perturbation in the conformation of several residues around the hydrophobic patch, groove of a tetramer and axial, lateral contacts across the double strands that are involved in HbS polymerization.

Assessment of Cysteine Reactivity of Human Hemoglobin at Its Residue Level: A Mass Spectrometry-Based Approach.

In general, the reactivity of cysteine residues of proteins is measured by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) kinetics using spectrophotometry. Proteins with several cysteine residues may exhibit varying DTNB kinetics but residue level information can only be obtained with the prior knowledge of their three-dimensional structure. However, this method is limited in its application to the proteins containing chromophores having overlapping absorption profile with 2-nitro-5-thiobenzoic acid, such as hemoglobin (Hb). Additionally, this method is incapable of assigning cysteine reactivity at the residue levels of proteins with unknown crystal structures. However, a mass spectrometry (MS)-based platform might provide a solution to these problems. In the present study, alkylation kinetics of cysteine residues of adult human Hb (Hb A; α2ß2) and sickle cell Hb (Hb S; HBB: c.20A>T) were investigated using matrix-assisted laser desorption/ionization (MALDI) MS. Differential site-specific reactivities of cysteine residues of Hb were investigated using alkylation kinetics with iodoacetamide (IAM). The observed reactivities corroborated well with the differential surface accessibilities of cysteine residues in the crystal structures of human Hb. The proposed method might be used to investigate cysteine reactivities of all the genetic and post-translational variants of Hb discovered to date. In addition, this method can be extended to explore cysteine reactivities of proteins, irrespective of the presence of chromophores and availability of crystal structures.

Mass Spectrometry-Based Diagnosis of Hemoglobinopathies: A Potential Tool for the Screening of Genetic Disorder.

Hemoglobinopathies are caused by point mutation in globin gene that results in structural variant of hemoglobin. While 7 % of world populations are carrier of hemoglobinopathies, the prevalence of the disease varies between 3 to 17 % across different population groups in India. In a diagnostic laboratory, alkaline gel electrophoresis and cation exchange-based HPLC (CE-HPLC) are most widely used techniques for characterization of hemoglobin variants. In the above methods, the differential surface charge of hemoglobin molecule in variants is exploited for their characterization. Sometime, co-migration of variants in gel electrophoresis and co-elution or elution with unknown retention time in automated CE-HPLC might lead to ambiguity in the analysis of hemoglobinopathies. Under such circumstances, it is necessary to use other analytical methods that provide unambiguous results. Mass spectrometry-based proteomics approach and DNA sequence analysis are examples of such alternative methods. In the present study, liquid chromatography coupled to mass spectrometry has been used for three commonly observed variants in India, e.g., HbE, HbQ India and HbD Punjab that appeared with inappropriate results in the conventional analysis. A customized hemoglobin variant database has been used in the mass spectrometry-based analysis of those three variants. Mass spectrometry-based proteomics approach was used to analyze above variant sample accurately.

Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA(1c) sample.

Glycated hemoglobin (HbA1c) is a 'gold standard' biomarker for assessing the glycemic index of an individual. HbA1c is formed due to nonenzymatic glycosylation at N-terminal valine residue of the ß-globin chain. Cation exchange based high performance liquid chromatography (CE-HPLC) is mostly used to quantify HbA1c in blood sample. A few genetic variants of hemoglobin and post-translationally modified variants of hemoglobin interfere with CE-HPLC-based quantification, resulting in its false positive estimation. Using mass spectrometry, we analyzed a blood sample with abnormally high HbA1c (52.1%) in the CE-HPLC method. The observed HbA1c did not corroborate the blood glucose level of the patient. A mass spectrometry based bottom up proteomics approach, intact globin chain mass analysis, and chemical modification of the proteolytic peptides identified the presence of Hb Beckman, a genetic variant of hemoglobin, in the experimental sample. A similar surface area to charge ratio between HbA1c and Hb Beckman might have resulted in the coelution of the variant with HbA1c in CE-HPLC. Therefore, in the screening of diabetes mellitus through the estimation of HbA1c, it is important to look for genetic variants of hemoglobin in samples that show abnormally high glycemic index, and HbA1c must be estimated using an alternative method.

Characterization of a Double Heterozygote HbE/ß(+) Thalassemia IVS 1-1 [G>T] in a Juvenile Diabetic.

The present case report describes the molecular and proteomic based study of Hb variant HbE associated with ß(+) thalassemia IVS 1-1 G>T, in a juvenile diabetic patient. Given the ethnic origin and mobility of the variant hemoglobin at alkaline pH, HbE would be suspected. But hematologically and clinically abnormality being detected, HPLC and Electrophoresis not being able to characterize due to retention time and band being in region of HbA2, respectively, further characterization of hemoglobinopathy was made using MALDI and IVS 1-1 G>T being validated by reverse dot blot hybridization. Capillary electrophoresis was also employed in order to separate HbE and HbA2 bands. This case report being first of its kind, wherein a HbE/ß(+) thalassemia has been characterized using multiple techniques.

Automated analysis of hemoglobin variants using nanoLC-MS and customized databases.

Unambiguous analysis of hemoglobin variants is critical in the diagnosis of hemoglobinopathies. In diagnostic laboratories, alkaline gel electrophoresis and automated HPLC are used in identifying variants. In specific instances, comigration of hemoglobin variant bands in gel and coelution of different variants or elution of variants with unmatched library information in HPLC can result in ambiguities in interpretation. Hemoglobin variants mostly arise from point mutations leading to very high sequence homology between normal and variant hemoglobin. In addition, unavailability of a variant database compatible with proteomics data analysis software makes mass spectrometry based variant analysis very challenging. In the present study, we standardized a nanoLC-MS based method for variant analysis to achieve substantially high sequence coverage. We developed three hemoglobin variant databases, specific to three different proteolytic enzymes, compatible with proteomics search engine software. The above nanoLC-MS method and the compatibility of the customized databases were validated by analysis of a sickle hemoglobin variant. Six other hemoglobin variants were characterized wherein diagnosis reports based on conventional tools were ambiguous. The novelty of our method lies in its simplicity and accuracy of the analysis with minimal manual intervention. The presently described method may be used in the future for the routine hemoglobin variant diagnosis.

Pancreatic tissue resident mesenchymal stromal cell (MSC)-like cells as a source of in vitro islet neogenesis.

Insufficient ß-cell mass is a common denominator for both type 1 and type 2 diabetes. In vitro generation of ß-cells from islet precursor cells, exocrine cells or ductal epithelia provide an alternative source of insulin-producing cells. However the presence of multipotent precursor cells within the pancreas is also deliberated. In this study we isolated mesenchymal stromal cell (MSC)-like cells from adult mouse pancreas by collagenase digestion. We used Knockout DMEM for our isolation procedure and the floating islets and acini were removed after 48 h. This strategy permitted the adhesion of stromal cells with typical mesenchymal morphology. These cells not only expressed MSC-specific markers like Sca-1, CD90.2, CD73, and CD44 but also generated osteocytes, adipocytes, and neurons when induced with specific growth media. Upon exposure to islet differentiation serum-free cocktail a significant upregulation of pancreatic markers like Nkx2.2, Nkx6.1, Pdx1, insulin, and somatostatin was seen. The differentiated islet-like cell aggregates (ICAs) secreted insulin which increased over the days in culture in presence of basal glucose levels. Taken together, our data strongly indicate that there is a tissue-resident precursor population within the pancreas that can be exploited for islet neogenesis in vitro.

Vertex-Based Resolvability Parameters for Identification of Certain Chemical Structures.

Chemical graph theory explores chemical phenomena and entities through the conceptual framework of graph theory. In chemical graph theory, molecular structures are represented by chemical graphs, where edges and vertices correspond to bonds and atoms, respectively. Chemical graphs serve as fundamental data types in cheminformatics for illustrating chemical structures. The computable properties of graphs form the basis for quantitative structure-property and structure-activity predictions, which are central to cheminformatics. These graphs capture the physical characteristics of molecules and can be further reduced to graph-theoretical indices or descriptors. One extensively studied distance-based graph descriptor is the resolving set Z, which enables the distinction of every pair of distinct vertices in a connected simple graph. Resolving sets were specifically employed in pharmaceutical research to find patterns shared by several different drugs. Since very early times, medicinal drugs have played a significant part in human civilization. In this article, we investigate minimum resolving sets for certain significant drug molecular structures, namely, suramin (S86) and acemannan (A116).

The IVS-II-837 (T>G) appears to be a relatively common 'rare' ß-globin gene mutation in ß-thalassemia patients in Karnataka State, South India.

ß-Thalassemia (ß-thal) is a common single gene autosomal recessive disorder resulting in severe anemia due to reduced or absent ß-globin polypeptide synthesis. The disease is caused by mutations in the ß-globin gene; eight common mutations are proposed to cause the majority of ß-thal in India. However, the occurrence of a region-specific mutation spectrum in India has also been suggested. We had earlier carried out analyses of the ß-globin gene mutation spectrum from southern Indian states of Andhra Pradesh and Karnataka. In the current study, we have analyzed three of 73 transfusion-dependent patients visiting a referral hospital in Karnataka State, South India, who did not carry any of the 22 common ß-globin gene mutations as determined by reverse dot-blot analysis. The IVS-II-837 (T>G) (ß(+)) (HBB:c.316-14TG) mutation was detected in two of the three patients analyzed suggesting a higher occurrence of the mutation in ß-thal patients in Karnataka when compared to other regions of India. The rare polyadenylation (poly A) site (T>C) (AATAAA>AACAAA; ß(+)) mutation was detected in the third patient. The IVS-II-837 mutation was also identified in asymptomatic carrier parents during routine high performance liquid chromatography (HPLC)-based Hb A(1c) screening in suspected diabetes patients. This is the first report of the identification of ß-thal trait through HPLC-based diabetes screening in India, revealing the importance of linking diabetes screening with screening for thalassemia.

Identification of a Rare Hemoglobin Variant HbJ-Rajappen [alpha90 (FG2) Lys → Thr] Using Mass Spectrometry.

Hemoglobin J-Rajappen (alpha)90 Lys â†’ Thr is an alpha chain variant found in heterozygous state and presents normal hematological blood picture. Due to the ambiguity in results obtained while analyzing by HPLC and alkaline gel electrophoresis, we report this rare case of HbJ-Rajappen using non denaturing gel electrophoresis and matrix assisted laser desorption ionization mass spectrometry. Though HbJ-Rajappen has earlier been reported using different techniques, this is the first report being validated using mass spectrometry technique.

Co-inheritance of HbD (Iran)/Beta Thalassemia IVS1-5 (G > C) Trait in a Punjabi Lady with Diabetes.

The present report describes the molecular study of HbD (Iran) (beta) 22 Glu â†’ Gln associated with ß-Thalassemia IVS1-5 (G > C) found in India, and the first case in which mutation has been identified using mass spectrometry. Given the apparent ethnic origin and the mobility of the variant hemoglobin at alkaline pH, hemoglobin D-Punjab would be suspected, but HPLC excluded this possibility. Further characterization of hemoglobinopathy was made by using nondenaturing gel electrophoresis and matrix assisted laser desorption ionization mass spectrometry and IVS1-5 being validated by reverse dot blot hybridization followed by sequencing of the ß-globin gene.

On Degree-Based Topological Indices of Carbon Nanocones.

Topological descriptors are numerical numbers that are assigned to molecular structures and can predict certain physicochemical properties. Because of their importance in nanotechnology and as developing materials with practical uses, the topological properties of nanocones have received a lot of attention. In this paper, we discuss the ev-degree- and ve-degree-based topological indices for the generalized carbon nanocones, CNC r [s]. Furthermore, we find numerical computations for certain types of nanocones and plot these numerical results using Matlab programming.

Analysis of hemoglobin variants using nondenaturing gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry.

Molecular analysis of hemoglobin variants is crucial in the diagnosis of hemoglobinopathies. Routinely used techniques for identifying variants include alkaline gel electrophoresis and automated HPLC. Sometimes comigration of variants in electrophoresis or coelution in HPLC provides ambiguous results. Due to high sequence homology between normal and variant hemoglobin, proteomic analysis using LC/ESI-MS data is also challenging. Here we describe a novel method wherein alkaline gel electrophoresis and MALDI-MS were used in combination to characterize variant samples such as Hb FSD and Hb D-Iran unambiguously. The method is rapid, efficient, and cost effective. In the future, it can be applied as a diagnostic tool.

Characterization of a hemoglobin variant: HbQ-India / IVS 1-1 [G>T]-ß-thalassemia.

Hemoglobin Q- India (alpha) 64 Asp → His is an alpha chain variant which is generally found in heterozygous state and presents normal hematological blood picture. Here we report a rare case of HbQ-India with a thalassemic phenotype that has been analyzed using a combination of mass spectrometry, gene sequencing and PCR analysis. This combined analyses revealed the HbQ variant to be associated with a beta chain mutation, IVS 1-1 [G>T]. Though HbQ has earlier been reported with thalassemic trait using different techniques, this is the first report of a compound α and ß chain Hb heterozygous mutant involving HbQ and IVS1-1 being validated using Mass Spectrometry and Reverse dot blot hybridization.

Structural analysis of glycated human hemoglobin using native mass spectrometry.

Glycated hemoglobin (GHb) is the indicator of the long-term glycemic index of an individual. GHb is formed by the irreversible modification of N-terminal α-amino group of ß globin chain with glucose via Amadori rearrangement. Cation exchange chromatography exploits the difference in surface charges between GHb and native hemoglobin (HbA0 ) for their separation and quantification. However, glucose condensation is specific to primary amino groups. Therefore, structural characterization of GHb synthesized in vivo is essential as multiple glycation may interfere with GHb assessment. The stoichiometric composition of different glycated hemoglobin from a 19% GHb sample was deduced using native mass spectrometry. We observed a comparable population of α and ß glycated tetramers for mono-glycated HbA0 . Surprisingly, doubly and triply glycated HbA0 also showed mono-glycated α and ß globins. Thus, we propose that glycation of hemoglobin (HbA) occurs symmetrically across α and ß globins with preference to unmodified globin first. Correlation between conventional and mass spectrometry-based quantification of GHb showed a reliable estimation of the glycemic index of individuals carrying HbA0 . Mutant HbAs have different retention time than HbA0 due to the differences in their surface charge. Thus, their glycated analog may elute at different retention time compared to GHb. Consequently, our method would be ideal for assessing the glycemic index of an individual carrying mutant HbA.

Y-27632 enhances differentiation of blastocyst like cystic human embryoid bodies to endocrinologically active trophoblast cells on a biomimetic platform.

Trophoblast differentiation and formation of the placenta are important events linked to post-implantation embryonic development. Models mimicking the biology of trophoblast differentiation in a post-implantation maternal microenvironment are needed for understanding disorders like placental-ischemia or for applications in drug-screening, and would help in overcoming the ethical impasse on using human embryos for such research. Here we attempt to create such a model by using embryoid bodies (EBs) and a biomimetic platform composed of a bilayer of fibronectin and gelatin on top of low-melting agarose. Using this model we test the hypothesis that cystic-EBs (day 30) that resemble blastocysts morphologically, are better sources as compared to noncytic EBs (day 10), for functional trophoblast differentiation; and that the Rho kinases inhibitor Y27632 can enhance this differentiation. Non/cytic EBs with/out Y27632 were grown on this platform for 28 days, and screened from secretion and expression of trophoblast and other lineage markers using ECLIA, RT-PCR, and Immunofluorescence. All EBs attached on this surface and rapidly proliferated into hCG and progesterone (P2) secreting functional trophoblast cells. However, the cells derived from cytic-EBs and cytic-EBs+ Y27632 showed the maximum secretion of these hormones and expressed IGF2, supporting our hypothesis. Also Y27632 reduced extraembryonic endoderm and trophoblast lineage differentiation from early noncystic-EBs, whereas, it specifically enhanced the induction of trophoblast and multinucleated syncitiotrophoblast differentiation from late cystic-EBs. In vivo trophoblast differentiation can be replicated in fibronectin based biomaterials, using cytic-EBs and by maneuvering the Rho-ROCK pathways. Response of EBs to a compound may vary temporally, and determination of their right stage is crucial for applications in directed-differentiation or drug-screening.