2018 Biochemistry 110 California Institute of Technology Lecture 7: Molecular Disease: Sickle-Cell Anemia

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1 2018 Biochemistry 110 California Institute of Technology Lecture 7: Molecular Disease: Sickle-Cell Anemia James Herrick ( ) Phase-Contrast microscopy image of Sickle Cells intermingled with erythrocytes. In Chicago in 1904, Herrick first observed the sickle-shaped red blood cells from patients that suffered from anemia, irregular heartbeat and a series of physical ailments. His findings went unexplained until 1949 when Linus Pauling examined the physical properties of hemoglobin from those afflicted with the disease and identified a single amino acid substitution as the cause of the disease. Today, thousand Americans live into their 40s and 50s with the disease that previously had a life expectancy of no more than 30 years. The study and treatment of this disease, which affects 100,000 African Americans in the U.S., continues to be an active endeavor in research and medicine.!review of Hb: Cooperativity, Allosteric Effect, Bohr Effect ( ).!Sickle Cell Anemia: Effects of Single Nucleotide Changes to Hb ( ).

2 Features of Hemoglobin that allow 88% O2 transport from Lungs to Tissues 1. Cooperativity β1 β1 α1 15 x eli FH α1 x eli FH x eli FH x eli FH α2 Tetrameric Hb structure can switch from Low Affinity State (T State) to High Affinity State (R state). This allows a Sigmoidal-Shaped binding curve (S-Curve). α2 β2 T State (favors deoxyhemoglobin) β2 R State (favors oxyhemoglobin) 2. Allosteric Effect 2,3-Bisphosphoglycerate in red blood cells binds at a single site between the two beta chains and stabilizes the Low Affinity State (T State). 3. Bohr Effect Affinity of Hemoglobin for O2 decreases as ph decreases and also as CO2 levels increase in Tissues. The effect is a combination of carbonic acid (H2CO3) lowering ph which protonates key Histidines and also carbamate formation from CO2 reacting with N-terminal amino group.

3 Erythrocytes are Designed to Move O 2 Efficiently Through Capillaries Bi-concave shape maximizes surface area for O 2 uptake/release while maintaining smooth profile and flexibility mammalian erythrocytes are anuclear - made in bone marrow x erythrocytes (1/4 of total cell count) in the human body» SCD is associated with red blood cells that are crescent-shaped, sticky and suffer rapid hemolysis.

4 Pauling s Research on Hemoglobin In 1935 the Rockefeller Foundation had been supporting my work on the crystal structure of the sulphide minerals and they said to me, You know, we re really not interested in the sulphide minerals. We re interested in biological substances - The Pauling Blog, paulingblog.wordpress.com measured magnetic susceptibility of blood - venous blood was paramagnetic - arterial blood was diamagnetic In 1945, Pauling attended a presentation by physician William Castle, who noted that for sickle-cell patients: - venous blood showed predominance of sickle cells - arterial blood showed most cells normally shaped Pauling s notebook Pauling s Laboratory in Gates (1935) Pauling Observed Altered Electrophoretic Mobility of Hemoglobin S Science 1949, 110, Subsequent 2D electrophoretic paper analysis of tryptic peptides of the β-hb chain and comparison with those of β-hb S by Baglioni and Ingram: single tryptic peptide: Val-His-Leu-Thr-Pro-Glu-Glu-Lys in β-hb S is changed to: > Val-His-Leu-Thr-Pro-Val-Glu-Lys apply sample HbA HbS _ apply electric field + then elute then stain vertically with ninhydrin with AcOH/BuOH (cathode) - + (anode) (cathode) - + (anode)

5 A Single Nucleotide Difference Leads to Sickle Cell Behavior We call it β-e6v. (E in the 6th position of chain β changes to V) DeoxyHemoglobin S forms Long Helical Fibers The Hemoglobin S tetramer with the Val-6 associates with the hydrophobic patch on another Hemoglobin β-chain in the deoxygenated state.

6 The Aggregation of Deoxy-Hemoglobin Makes SCD Particularly Dangerous Depletion of oxygen due to blood vessel blockage drives the deoxygenation of the tetramer. SCD increases opportunity for infections that can initate more aggregation.» Children growing up with SCD require special medical attention. The Sickle Cell Heterozygous Trait Confers Malaria Resistance

7 Sickle-Cell Phenotype Develops in Infants as Fetal Hemoglobin is Replaced by Adult Hemoglobin α -VLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFPTTKTYFPHF-DLSH-----GSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHK β VHLTPEEKSAVTALWGKVNV--DEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDK γ GHFTEEDKATITSLWGKVNV--EDAGGETLGRLLVVYPWTQRFFDSFGNLSSASAIMGNPKVKAHGKKVLTSLGDATKHLDDLKGTFAQLSELHCDK δ VHLTPEEKTAVNALWGKVNV--DAVGGEALGRLLVVYPWTQRFFESFGDLSSPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFSQLSELHCDK ε VHFTAEEKAAVTSLWSKMNV--EEAGGEALGRLLVVYPWTQRFFDSFGNLSSPSAILGNPKVKAHGKKVLTSFGDAIKMNDNLKPAFAKLSELHCDK LRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR 141 LHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH 146 (Adult Hemoglobin (HbA) - α 2 β 2 ) LHVDPENFKLLGNVLVTVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYH 146 (Fetal Hemoglobin (HbF) - α 2 γ 2 ) LHVDPENFRLLGNVLVCVLARNFGKEFTPQMQAAYQKVVAGVANALAHKYH 147 (Adult Hemoglobin A 2 (HbA 2 ) - α 2 δ 2 ) LHVDPENFKLLGNVMVIILATHFGKEFTPEVQAAWQKLVSAVAIALAHKYH 147 (Embryonic Hemoglobin Gower II α 2 ε 2,1A9W) To date, the best treatment for SCD is hydroxyurea. - antineoplastic drug increases the proportion of HbF for sickle-cell patients. - carriers of the HbS trait from Saudia Arabia were known to produce an unusual proportion of HbF in their blood. - HbF was known to be a very potent inhibitor of the polymerization of deoxy-hbs. Vernon Ingram, Genetics 2004, 167, 1-7.

8 The Bohr Effect CO 2 generated in the tissues is converted to H + - and HCO 3 in RBC s by Carbonic Anhydrase. Deoxy-Hb has increased affinity for H + (pka s of certain groups are raised). The groups that have pka s near ph are His and the peptide terminal NH 2 group. In addition, CO 2 carbamoylates the α-chain terminal amino group:

9 Early Treatment for SCD with Cyanate : The Bohr effect (deoxy-hb transporting protons and CO 2 ) is reduced when the terminal amino groups of the α-chains are carbamoylated. O R NH 2 + -NCO + H + R N NH2 Terminal Amino Group Cyanate H Carbamoylated derivative Cyanate enters red blood cells and carbamoylates the terminal amino groups of hemoglobin increasing the affinity of Hb for oxygen. (Thus decreasing the propensity to aggregate deoxy-hbs.) Unfortunately, cyanate was found to have several side effects and is no longer used. Treatment of SCD is still an active area of medicinal research.

10 There are Over 100 Examples of Site-Specific Changes in Hemoglobin α-chain I G Honolulu Norfolk M Boston M Iwate G Philadelphia O Indonesia K16E E30Q G57D H58Y H87Y N68K E116K β-chain C S G SanJose E M Saskatoon M Zurich M HydePark M Milwaukee D Punjab E6K E6V E7G E26K H63Y H63R H92Y V67E E121Q Tyrosine F8 CH 2 Heme Proximal N Histidine F8 N Fe2+ O 2 HN CH 2 Hemoglobin A Heme H O Fe 3+ O H Hemoglobin M Iwate H O H Heme Fe 3+ M signifies the propensity for the Methemoglobin form (Fe3+). N Distal Histidine E7 CH 2 O Tyrosine E7 CH 2 Hemoglobin M Boston The frequency of any non-s mutant allele is <10-4. These other mutations do not confer any (known) benefit. 1. Altered Exterior: Nearly all substitutions on the surface of the hemoglobin tetramer are harmless. Hb-S is a striking exception. 2. Altered Active Site: The defective subunit can no longer bind O Altered Tertiary Structure: The polypeptide chain can no longer fold. 4. Altered Quaternary Structure: Mutations at the subunit interfaces result in loss of allosteric properties.