Download biochem ch 44B [9-2

Intro
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In response to infection, leukocytes secrete cytokines (interleukins) that stimulate production of additional
leukocytes to fight infection
 Decreased supply of oxygen to tissues signals kidney to release erythropoietin (hormone that stimulates
production of RBCs)
 RBCs – glycolysis is main energy-generating pathway, with lactate production regenerating NAD+ for glycolysis to
continue; NADH produced in glycolysis used to reduce ferric form of Hgb (methemoglobin) to normal ferrous
state
o Glycolysis leads to side pathway in which 2,3-bisphosphoglycerate produced (major allosteric effector
for oxygen binding to Hgb
o Hexose monophosphate shunt pathway generates NADPH to protect RBC PM lipids and proteins from
oxidation through regeneration of reduced glutathione
o Heme synthesis occurs in precursors of RBCs and is complex pathway that originates from succinyl-CoA
and glycine – mutations in any of steps lead to group of diseases (porphyrias)
 RBC deformability because of cytoskeletal structure that consists of major proteins spectrin, ankyrin, and band 3
protein – mutations in proteins lead to improper formation of cytoskeleton, resulting in malformed spherocytes
in circulation (shortened life span leads to loss of RBCs)
 Mutations in genes that encode RBC metabolic enzymes, membrane structural proteins, and globins cause
hereditary anemias – some of most common genetic diseases known
 Hemoglobin switching – alteration of globin gene during development; switch between expression of one gene
to another regulated by transcription factor binding to promoter regions of genes
Cells of Blood
 RBCs lose all internal organelles during differentiation
 Thrombocytes contain cytoplasmic organelles but no nucleus
 Granulocytes (polymorphonuclear leukocytes) – neutrophils, eosinophils, and basophils; multilobed nuclei with
presence of secretory granules – when activated, granular vesicle membrnaes fuse with PM, resulting in release
of granule contents (degranulation)
o Granules contain cell-signaling molecules that mediate inflammatory processes
o Neutrophils stain light pink and eosinophils stain dark pink/red
o Neutrophils – phagocytic cells that migrate rapidly to areas of infection or tissue damage; engulf foreign
bodies and destroy them by initiating respiratory burst, which creates oxygen radicals that rapidly
destroy foreign material found at site of infection
o Eosinophils – fight viral infections (release RNase from granules) to remove fibring during inflammation
and protect against parasites such as worms; granules are lysosomes containing hydrolytic enzymes and
cationic proteins, which are toxic to parasitic worms; have been implicated in asthma and allergic
responses as well as antigen presenting to T cells
o Basophils – participate in hypersensitivity reactions such as allergic responses; histamine (produced by
decarboxylation of histidine) stored in secretory granules and stimulates smooth muscle cell contraction
and increase in vascular permeability; granules also contain enzymes such as proteases, βglucuronidase, and lysophospholipase, which degrade microbial structures and assist in remodeling
damaged tissue
 Mononuclear leukocytes – agranulocytes; lymphocytes and monocytes
o Lymphocytes – have high ratio of nuclear volume to cytoplasmic volume and are primary antigenrecognizing cells; differentiate into T cells, B cells, and NK cells; subclasses of T cells identified by
different surface membrane proteins correlating to function; NK cells target virally infected and
malignant cells for destruction
o Monocytes – precursors of tissue macrophages (phagocytic cells that enter inflammatory sites and
consume microorganisms and necrotic host cell debris left behind by granulocyte attack of foreign
material; macrophages in spleen play role in maintaining oxygen-delivering capabilities of blood by
removing damaged RBCs that have reduced oxygen-carrying capacity
 Thrombocytes – arise by budding of cytoplasm of megakaryocytes (multinucleated cells that reside in marrow)
Anemia
 MCV usually expressed in femtoliters (10-15 L), and MCHC usually expressed in g/L
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CBC performed by sending cells through automated analyzer based on flow cytometry (one cell at a time), and
laser shines light at cell and machine records predictable light scattering
Mature Erythrocyte Metabolism
 Metabolic enzymes limited to those found in cytoplasm; cytosol contains hemoglobin and enzymes necessary
for prevention and repair of damage done by reactive oxygen species and generation of energy
 RBCs only generate ATP by glycolysis – ATP used for ion transport across PM (Na+, K+, and Ca2+), phosphorylation
of membrane proteins, and priming reactions of glycolysis
o Glycolysis uses Rapoport-Luebering shunt to generate 2,3-bisphosphoglycerate (2,3-BPG) – RBCs have
much more than other cells (that require it for phosphoglycerate mutase reaction of glycolysis)
o As 2,3-BPG regenerated during each reaction cycle, it is required in catalytic amounts in non-RBCs
o 2,3-BPG is modulator of oxygen binding to Hgb that stabilizes deoxy form of Hgb, thereby facilitating
release of O2 to tissues
 To bind oxygen, iron of Hgb must be in ferrous state (2+)
 Some of NADH produced by glycolysis used to regenerate Hgb from methemoglobin by NADH-cytochrome b5
methemoglobin reductase system; cytochrome b5 reduces Fe3+ of methemoglobin, and oxidized cytochrome b5
then reduced by flavin-containing enzyme (cytochrome b5 reductase) using NADH as reducing agent
 Inherited deficiency in pyruvate kinase leads to hemolytic anemia – because amount of ATP formed from
glycolysis decreased by 50%, RBC ion transporters cannot function effectively
o RBCs tend to gain Ca2+ and lose K+ and H2O; H2O loss increases intracellular hemoglobin concentration,
which causes increased internal viscosity of cell to point that cell becomes rigid and therefore more
susceptible to damage by shear forces in circulation; once damaged, RBCs removed from circulation,
leading to anemia
o Effects of anemia moderated by 2-3x elevation in 2,3-BPG concentration that results from blockaged of
conversion of phosphoenolpyruvate to pyruvate
o Because 2,3-BPG binding to Hgb decreases affinity of Hgb for O2, RBCs remain in circulation highly
efficient in releasing bound oxygen to tissues
 5-10% of glucose metabolized by RBCs used to generate NADPH by hexose monophosphate shunt
o NADPH used to maintain glutathione in reduced state; glutathione cycle is RBC’s chief defense against
damage to proteins and lipids by reactive oxygen species
o Enzyme that catalyzes first step of hexose monophosphate shunt is glucose-6-phosphate dehydrogenase
(G6PD); lifetime of RBC correlates with G6PD activity – RBC cannot synthesize new G6PD, so as G6PD
activity decreases, oxidative damage accumulates, leading to lysis of erythrocyte
 Congenital methemoglobinemia – presence of excess methemoglobin; found in people with enzymatic
deficiency in cytochrome b5 reductase or in people who inherited hemoglobin M (single AA substitution in
heme-binding pocket stabilizes ferric oxygen)
o Individuals appear cyanotic but have few clinical problems
o Can be acquired by ingestion of nitrites, quinones, aniline, and sulfonamides – treated by administration
of reducing agents, such as ascorbic acid or methylene blue
 G6PD deficiency – most common enzyme deficiency because those with trait resistant to malaria; RBCs have
shorter life span and more likely to lyse under conditions of oxidative stress; gene for G6PD on X chromosome
o All known G6PD variant genes contain small in-frame deletions or missense mutations, so corresponding
proteins have decreased stability or lowered activity, leading to reduced lifespan for RBC
o No mutations found that result in complete absence of G6PD because this would result in embryonic
lethality
Erythrocyte Precursor Cells and Heme Synthesis
 Heme – consists of porphyrin ring coordinated with atom of iron; 4 pyrrole rings joined by methenyl bridges
(=CH-) to form porphyrin ring; 8 side chains serve as substituents on porphyrin ring (2 on each pyrrole) and may
be acetyl (A), propionyl (P), methyl (M), or vinyl (V) groups (in normal heme, it is MVMVMPPM clockwise,
characteristic of porphyrins of type III series, the most abundant type in nature)
o Most common porphyrin in body; complexed with proteins to form hemoglobin, myoglobin, and
cytochromes
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Heme synthesized from glycine and succinyl-CoA, which condense in initial reaction to form δ-aminolevulinic
acid (δ-ALA) – enzyme that catylzes reaction (δ-ALA synthase) requires participation of pyridoxal phosphate
(reaction is AA decarboxylation reaction; glycine is decarboxylated)
o Next reaction catalyzed by δ-ALA dehydratase (2 molecules of δ-ALA condense to form
porphobilinogen); 4 of these pyrrole rings condense to form linear chain and then series of
porphyrinogens; side chains initially contain A and P groups (A groups decarboxylated and oxidized to V
groups, forming protoporphyrin IX
o Final step of pathway – Fe2+ incorporated into protoporphyrin IX in reaction catalyzed by ferrochelatase
(heme synthase)
o δ-ALA dehydratase contains zinc; it and ferrochelatase inactivated by lead; in lead poisoning, δ-ALA and
protoporphyrin IX accumulate and production of heme is decreased, leading to anemia and decrease in
energy production because of lack of cytochromes for electron-transport chain
Heme is red and responsible for color of RBCs and muscles that contain large number of mitochondria
In B6 deficiency, rate of heme production slow because first reaction in heme synthesis requires pyroxidal
phosphate; less heme then synthesized, causing RBCs to be small and pale; iron stores elevated
Porphyrias – group of rare inherited disorders resulting from deficiencies of enzymes in pathway for heme
biosynthesis; intermediates of pathway accumulate and may have toxi effects on nervous system that cause
neuropsychiatric symptoms; when porphyrinogens accumulate, they may be converted by light to porphyrins,
which react with molecular oxygen to form oxygen radicals which may cause severe damage to skin; individuals
with excessive production of porphyrins photosensitive; scarring and increased growth of facial hair seen in
some porphyrias (werewolf syndrome)
Iron obtained from diet – heme from meat readily absorbed, and nonheme iron from plants not readily
absorbed because plants often contain oxalates, phytates, tannins, and other phenolic compounds that chelate
or form insoluble precipitates with iron preventing its absorption
o Vitamin c increases uptake of nonheme iron from digestive tract
o Uptake of iron increased in times of need by other mechanisms
o Iron absorbed in Fe2+ (ferrous) state but oxidized to ferric state by ferroxidase called ceruloplasmin
(copper-containing enzyme) for transport throughout body
Iron carried in blood as Fe3+ bound to apotransferrin (complex of iron and apotransferrin is transferrin);
transferrin usually only 1/3 saturated with iron; transferrin with bound iron binds to transferrin receptor on cell
surface and complex internalized into cell; internalized membrane develops into endosome with slightly acidic
pH; iron in ferrous form transported out of endosome into cytoplasm via divalent metal ion transporter 1 (DMT1); in cytoplasm, iron oxidized and binds to ferritin for long-term storage
o Storage of iron occurs in most cells, but especially in cells of liver, spleen, and bone marrow; in those
cells, storage protein (apoferritin) forms complex with Fe3+ to become ferritin
o Ferritin levels in blood increase as iron stores increase, so amount of ferritin in blood is most sensitive
indicator of amount of iron in body’s stores
o Iron can be drawn from ferritin stores, transported in blood as transferrin, and taken up via receptormediated endocytosis by cells that require iron like reticulocytes synthesizing Hgb
o When excess iron absorbed from diet, it is stored as hemosiderin (form of ferritin complexed with
additional iron that cannot be readily mobilized
Heme regulates its own synthesis by repressing synthesis of δ-ALA synthase and directly inhibits activity of δ-ALA
synthase via an allosteric modifier; heme synthesized when heme levels fall and rate of synthesis decreases as
heme levels rise
o Heme regulates synthesis of Hgb by stimulating synthesis of globin; heme maintains ribosomal initiation
complex for globin synthesis in active state
Heme degraded to form bilirubin, which is conjugated with glucuronic acid and excreted in bile; heme from
cytochromes and myoglobin also undergoes conversion to bilirubin, but major source is from Hgb
o After RBCs reach end of life span, they are phagocytosed by cells of reticuloendothelial system
o Globin cleaved to constituent AAs and iron returned to body’s iron stores
o Heme oxidized and cleaved to produce CO and biliverdin (biliverdin reduced to bilirubin, which is
transported to liver complexed with serum albumin)
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In liver, bilirubin converted to more water-soluble compound (bilirubin monoglucuronide) by reacting
with UDP-glucuronate – this is converted to diglucuronide and conjugated form excreted into bile
o In intestine, bacteria deconjugate bilirubin diglucuronide and convert bilirubin to urobilinogens; some
urobilinogen absorbed into blood and excreted in urine, but most oxidized to urobilins (like stercobilin)
and excreted in feces – this gives feces brown color
 Iron lost by desquamation of skin and in bile, feces, urine, and sweat; if man with normal diet has iron deficiency
anemia, his physician should suspect bleeding from GI tract as result of ulcers or colon cancer
 Drugs such as phenobarbital induce enzymes of drug-metabolizing systems of ER that contain cytochrome P450
o Because heme used for synthesis of cytochrome P450, free heme levels fall and δ-ALA synthase induced
to increase rate of heme synthesis
 Iron deficiency results in microcytic, hypochromic anemia – iron stores low
Red Blood Cell Membrane
 Biconcave disc shape serves to facilitate gas exchange across cell membrane
 Membrane proteins that maintain shape of RBC allow it to traverse capillaries with very small luminal diameters
to deliver oxygen to tissues
 When passing through kidney, RBCs traverse hypertonic areas and back again, causing RBC to shrink and expand
during its travels
 Erythrocytes pass through spleen 120x per day – must be highly deformable to make it through passageways
here; damaged RBCs become trapped here and are destroyed by macrophages
 Being not spherical (extra membrane) and cytoskeleton that supports it allows RBC to be stretched and
deformed by mechanical stresses as cell passes through narrow vascular beds
 On cytoplasmic side of membrane, proteins form 2D lattice that gives RBC flexibility – major proteins spectrin,
actin, band4.1, band 4.2, and ankyrin
o Spectrin – major protein of cytoskeleton; heterodimer composed of α- and β-subunits wound around
each other; dimers self-associated at heads
o At tails, actin and band 4.1 bind near to each other, and multiple spectrins can bind to each actin
filament, resulting in branched membrane cytoskeleton
o Spectrin cytoskeleton connected to membrane lipid bilayer by ankyrin, which interacts with β-spectrin
and integral membrane protein (band 3), and band 4.2 helps stabilize that connection
o Band 4.1 anchors spectrin skeleton with membrane by binding integral membrane protein (glycophorin
C) and actin complex, which has bound multiple spectrin dimers
o When RBC subjected to mechanical stress, spectrin network rearranges; some spectrin molecules
become uncoiled and extended while others become compressed, changing shape of cell but not SA
o Mature erythrocyte cannot synthesize new PM proteins or lipids, but PM lipids can be freely exchanged
with circulating lipoprotein lipids; glutathione system protects proteins and lipids from oxidative damage
o Band proteins got their names through study with PAGE (band 1, band 2, etc.); spectrin is band 1
 Defects in erythrocyte cytoskeletal proteins lead to hemolytic anemia; shear stresses in circulation result in loss
of pieces of RBC PM, resulting in RBC becoming more spherical and losing deformability (spherical cells more
likely to lyse in response to mechanical stresses or to be trapped and destroyed in spleen)
Agents that Affect Oxygen Binding
 2,3-BPG formed in RBCs from glycolytic intermediate 1,3-bisphosphoglycerate; 2,3-BPG binds to Hgb in central
cavity formed by 4 subunits, increasing energy required for conformational changes that facilitate binding of
oxygen; 2,3-BPG lowers affinity of Hgb for oxygen, so oxygen less readily bound (more readily released in
tissues) when Hgb contains 2,3-BPG
 Binding of protons by Hgb lowers its affinity for oxygen (Bohr effect) – pH of blood decreases as it enters tissues
because CO2 produced by metabolism converted to carbonic acid by reaction catalyzed by carbonic anhydrase in
RBCs; dissociation of carbonic acid produces protons that react with several amino acid residues in Hgb, causing
conformational changes that promote release of oxygen
o In lungs, oxygen binds to hemoglobin, causing release of protons, which combine with bicarbonate to
form carbonic acid, causing pH of blood to rise; carbonic anhydrase cleaves carbonic acid to H2O and
CO2, and CO2 is exhaled
 Carbon dioxide – most of CO2 produced by metabolism in tissues carried to lungs as bicarbonate, but some of
CO2 covalently binds to hemoglobin; in tissues, CO2 forms carbamate adducts with N-terminal amino groups of
deoxyhemoglobin and stabilizes deoxy conformation; in lungs where PO2 high, oxygen binds to Hgb and bound
CO2 is released
Hematopoiesis
 Hematopoietic stem cells renewable throughout life of host; population very low in bone marrow (not anywhere
else in body, though)
o In presence of appropriate signals, hematopoietic stem cells proliferate, differentiate, and mature into
any type of cell that makes up blood (differentiation hierarchical, and progenitors designated colonyforming unit-lineage or colony-forming unit-erythroid [CFU-E]; progenitors that form very large colonies
termed burst-forming units)
o Presence of hematopoietic cells enriched with stem cells can be isolated by fluorescence-activated cell
sorting, based on expression of specific cell-surface markers; increasing population of stem cells in cells
used for bone marrow transplantation increases chance of success of transportation
 Developing progenitor cells in marrow grow in proximity with marrow stromal cells (fibroblasts, endothelial
cells, adipocytes, and macrophages) – stromal cells form ECM and secrete growth factors that regulate
hematopoietic development
o Individual growth factor may stimulate proliferation, differentiation, and maturation of progenitor cells
and may also prevent apoptosis; factors may activate various functions within mature cell; some growth
factors act on multiple lineages, whereas others have more limited targets
o Leukemias arise when differentiating hematopoietic cell does not complete developmental program but
remains in immature proliferative state; leukemias found in every hematopoietic lineage
o Most hematopoietic growth factors recognized by receptors in cytokine receptor superfamily
 Binding of ligand to receptor results in receptor aggregation, which induces phosphorylation of
JAKs; activated JAKs then phosphorylate cytokine receptor, creating docking regions where
additional signal transduction molecules bind, including members of STAT family of transcription
factors; JAKs phosphorylate STATs, which dimerize and translocate to nucleus, where they
activate target genes
 Additional signal transduction proteins bind to phosphorylated cytokine receptor, leading to
activation of Ras/Raf/MAP kinase pathways; some other pathways also activated which lead to
inhibition of apoptosis
o Response to cytokine binding usually transient because cell contains multiple negative regulators of
cytokine signaling; family of silencer of cytokine signaling (SOCS) proteins induced by cytokine binding;
one member of SOCS family binds to phosphorylated receptor and prevents docking of signal
transduction proteins
 Other SOCS proteins bind to JAKs and inhibit them
o SHP-1 – tyrosine phosphatase found primarily in hematopoietic cells necessary for proper development
of myeloid and lymphoid lineages; function is to dephosphorylate JAK2
o Protein inhibitors of activated STAT (PIAS) family of proteins bind to phosphorylated STATs and prevent
dimerization or promote dissociation of STAT dimers
o STATs can also be inactivated by dephosphorylation or by targeting activated STATs for proteolytic
degradation
 In X-linked severe combined immunodeficiency (SCID) disease (most common form of SCID), circulating T
lymphocytes not formed, and B lymphocytes not active; affected gene encodes for γ-chain of IL-2 receptor;
mutant receptors unable to activate JAK3, and cells unresponsive to cytokines that stimulate growth and
differentiation
 Families identified with mutant erythropoietin (EPO) receptor unable to bind SHP-1; EPO is hematopoietic
cytokine that stimulates production of RBCs; individuals with mutant EPO receptor have higher than normal
percentage of RBCs in circulation because mutant EPO receptor cannot be deactivated by SHP-1; EPO causes
sustained activation of JAK2 and STAT 5
 Perturbed JAK/STAT signaling is associated with development of lymphoid and myeloid leukemias, severe
congenital neutropenia, and Fanconi anemia (characterized by bone marrow failure and increased susceptibility
to malignancy
 Complication of sickle cell disease is increased formation of gallstones; sickle cell crisis accompanied by
intravascular destruction of RBCs increases amount of unconjugated bilirubin transported to liver; if
concentration of unconjugated bilirubin exceeds capacity of hepatocytes to conjugate it to more soluble
diglucuronide through interaction with hepatic UDP-glucuronate, both total and unconjugated bilirubin levels in
blood increase; more unconjugated bilirubin then secreted by liver into bile, resulting in precipitation within
gallbladder lumen, leading to formation of calcium bilirubinate gallstones
 Production of RBCs regulated by demands of oxygen delivery to tissues; in response to reduced tissue
oxygenation, kidney releases hormone erythropoietin, which stimulates multiplication and maturation of
erythroid progenitors
o Progression along erythroid pathway begins with stem cell and passes through mixed myeloid
progenitor cell (CFU-GEMM, colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte),
burst-forming unit-erythroid (BFU-E), CFU-E, and to first recognizable RBC precursor (normoblast)
o Each normoblast undergoes 4 more cycles of cell division; during last cycle, nucleus becomes smaller
and more condensed; after last division, nucleus is extruded and RBC becomes known as reticulocyte
(still contains ribosomes and mRNA and is capable of synthesizing hemoglobin)
o Reticulocytes released from bone marrow and circulate for 1-2 days, then mature in spleen, where
ribosomes and mRNA lost
 Nutritional deficiencies in iron, B12, and folate prevent adequate RBC formation
o Iron deficiency – cells smaller and paler than normal; lack of iron results in decreased heme synthesis,
which affects globin synthesis; maturing RBCs following normal developmental program divide until Hgb
reached appropriate concentration; iron-deficient developing RBCs continue dividing past normal
stopping point, resulting in microcytic cells that are hypochromic because of lack of Hgb
o Deficiencies of folate or B12 can cause megaloblastic anemia; folate and B12 required for DNA synthesis,
so when vitamins deficient, DNA replication and nuclear division do not keep pace with maturation of
cytoplasm, and thus nucleus extruded before requisite number of divisions has taken place, and cell
volume greater than it should be and fewer cells produced
Hemoglobinopathies, Hereditary Persistence of Fetal Hemoglobin, and Hemoglobin Switching
 HbC found in high frequency in West Africa, in regions with high frequency of HbS; compound heterozygotes for
HbS and HbC not uncommon in some African regions and among some African Americans
o HbS/HbC individuals have significantly more hematopathology than individuals with sickle cell trait;
polymerization of deoxygenated HbS is dependent on HbS concentration within cell; presence of HbC in
compound heterozygote increases HbS concentration by stimulating K+ and H2O efflux from cell
o Because HbC globin tends precipitate, proportion of HbS tends to be higher in HbS/HbC cells than in
those with HbS/HbA
 More than 700 different mutant hemoglobins discovered, most arising from single base substitution, resulting in
single AA replacement; many not clinically significant
 HbS – most common Hgb mutation
 HbC – results from glu-to-lys replacement in same position as HbS mutation; promotes water loss from cell by
activating K+ transporter, resulting in higher than normal concentration of Hgb in cell
o Amino acid replacement substantially lowers Hgb solubility in homozygote, resulting in tendency of
mutant Hgb to precipitate in RBC, although cell does not deform
o Homozygotes have mild hemolytic anemia, and heterozygotes clinically unaffected
 Thalassemias – diseases characterized by one type of globin chain being produced more than other
o Mutations provide resistance to malaria in heterozygous state
o Caused by Hgb single AA replacement mutations that give rise to globin subunit of decreased stability,
but more commonly mutations that result in decreased synthesis of one subunit
o α-thalassemias usually caused by complete gene deletions, 2 copies per chromosome 16
 1 deletion is mostly normal (size and Hgb concentration may be minimally reduced)
 2 deletions have microcytic hypochromic cells, but individual not usually anemic
 3 deletions causes moderately severe microcytic hypochromic anemia with splenomegaly
 4 deletions usually fatal in utero
o β-thalassemias caused by deletions, promoter mutations, and splice-junction mutations
 Heterozygotes for β+ (some globin chain synthesis) or β0 (no globin chain synthesis) generally
asymptomatic, although they typically have microcytic hypochromic RBCs and may have mild
anemia
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Homozygotes of β+ have anemia of variable severity, heterozygotes of β+/β0 tend to be more
severely affected, and β0/β0 homozygotes have severe disease
 Diseases of β-chain deficiency more severe than diseases of α-chain deficiency because excess
β-chains form homotetramer (HbH) that is ineffective for delivering oxygen to tissues because of
its high oxygen affinity; as RBCs age, HbH precipitates in cells, forming inclusion bodies (RBCs
with inclusion bodies have shortened life spans because they are more likely to be trapped and
destroyed in spleen; excess α-chains unable to form stable tetramer
 Excess α-chains precipitate in erythrocytes at every developmental stage, resulting in
widespread destruction of precursors (ineffective erythropoiesis)
 Precipitated α-chains damage RBC membranes through heme-facilitated lipid oxidation by
reactive oxygen species; lipids and proteins (particularly band 4.1) damaged
 Difference in amino acid composition between β-chains of HbA and γ-chains of HbF result in structural changes
that cause HbF to have lower affinity for 2,3-BPG than HbA and thus greater affinity for oxygen; therefore
oxygen released from mother’s Hgb (HbA) readily bound by HbF in fetus, so transfer of oxygen from mother to
fetus facilitated by structural difference between Hgb molecule of mother and that of fetus
 Hemoglobin switching not 100%; most individuals continue to produce small amount of HbF throughout life
o Patients with hemoglobinopathies such as β-thalassemia or sickle cell anemia frequently have less
severe illnesses if levels of HbF are elevated
 Nondeletion forms of HPFH (hereditary persistence of fetal hemoglobin) derive point mutations in Aγ and Gγ
promoters; when these mutations found with sickle cell or β-thalassemia mutations have ameliorating effect on
disease because of increased production of γ-chains
 Deletion HPFH – both entire δ- and β-genes have been deleted one copy of chromosome 11 and only HbF can be
produced; in some individuals, fetal globins remain activated after birth and enough HbF produced that
individual is clinically normal; in others similar deletions that remove entire δ- and β-genes do not produce
enough HbF to compensate for deletion and have δ0β0-thalassemia
o Difference between asymptomatic and δ0β0-thalassemia patients is site at which deletions end within βglobin gene cluster
o Powerful enhancer sequences 3’ of β-globin gene resituated because of deletion so that they activate γpromoters
o In individuals with δ0β0-thalassemia, enhancer sequences have not been relocated so they don’t interact
with γ-promoters
 Embryonic megaloblasts (large embryonic RBCs with nucleus) first produced in yolk sac about day 15
o After 6 weeks, site of erythropoiesis shifts to liver (liver and to lesser extent spleen are major sites of
fetal erythropoiesis)
o In last few weeks before birth, bone marrow begins producing RBCs
o 8-10 weeks after birth, bone marrow is sole site of erythrocyte production
o Composition of Hgb changes with development because both α-globin locus and β-globin locus have
multiple genes differentially expressed during development
 α-globin locus on chromosome 16 contains embryonic ζ gene and 2 copies of α-gene (α1 and α2)
 β-globin locus on chromosome 11 contains embryonic ε-gene, 2 copies of fetal β-globin gene (Gγ and Aγ, which
differ by one AA), and 2 adult genes (β and δ)
 Embryonic Hgbs are ζ2ε2 (Gower 1), ζ2γ2 (Portland), and α2γ2 (Gower 2)
o Fetal Hgb predominantly α2Gγ2
o Fetal Hgb found in adult cells is α2Aγ2
 Timing of Hgb switching controlled by developmental clock not significantly altered by environmental conditions
and related to changes in expression of specific transcription factors (premature newborns convert HbF to HbA
on schedule with gestational ages)
Biochemical Comments
 HS40 – major regulatory element; nuclease-sensitive region of DNA that lies 5’ of the ζ-gene and acts as
erythroid-specific enhancer that interacts with upstream regulatory regions of ζ- and α-genes and stimulates
their transcription
 Region immediately 5’ of ζ-gene contains regulatory sequences responsible for silencing ζ-gene transcription
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Even after silencing, low levels of ζ-gene transcripts still produced after embryonic period but are not
translated because both ζ-globin and α-globin transcripts have regions that bind to mRNP (messenger
ribonucleoprotein) stability-determining complex, preventing mRNA from being degraded
5-25 kb upstream of ε-gene is locus control region (LCR), containing 5 DNAse hypersensitive sites; LCR necessary
for function of β-globin locus and maintains chromatin of entire locus in active configuration and acts as
enhancer and entry point for factors that transcribe genes of β-globin locus
o Each gene of β-globin locus has individual regulatory elements: promoter, silencers, or enhancers that
control developmental regulation
ε-globin gene has silencers in 5’-regulatory region; binding of proteins to these regions turns off ε-gene
Proximal region of γ-globin gene promoter has multiple transcription factor-binding sites; many HPFH mutations
map to these transcription factor-binding sites, either by destroying site or by creating new one
2 sites significant in control of hemoglobin switching: stage-selector protein-binding (SSP) site and CAAT box
region; when SSP complex bound to promoter, γ-globin gene has competitive advantage over β-globin promoter
for interaction with LCR
o Sp1 also binds at SSP-binding site, and competition between it and SSP complex helps determine activity
of γ-globin gene
o CP1 binds at CAAT box, and CAAT displacement protein (CDP) is repressor that binds at CAAT site and
displaces CP1
β-globin gene has binding sites for multiple transcription factors in regulatory regions; mutations that affect
binding of transcription factors can produce thalassemia by reducing activity of β-globin promoter
o Enhancer 3’ of poly A signal required for stage-specific activation of β-globin promoter
Transcription factor BCL11A strong repressor of γ-globin gene expression; BCL11A interacts with variety of other
transcription factors (GATA-1, FOG1, and NURD (nucleosome remodeling and histone deacetylase) repressor
complex) to repress γ-globin expression
o BCL11A expression regulated by transcription factor KLF1, which is essential for β-globin expression;
KLF1 increases BCL11A expression, which blocks γ-globin gene expression while KFL1 stimulates β-globin
gene expression