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Chapter 33 Pathobiology of the Human Erythrocyte and Its Hemoglobins 455

Ontogeny of Hemoglobin Hb biosynthesis and Hb switching. However, posttranscriptional
mechanisms contribute to the final distribution of globin and non-
The Hb composition of the erythrocyte depends on when in gestation globin mRNAs and to the balance of α-globins and non–α-globins
or postnatal development it is measured. This is a result of sequential within the erythroblasts. When compared with many other mRNAs,
activation and inactivation (i.e., switching) among genes within the such as cytokine mRNAs, globin mRNAs are extraordinarily stable.
α-globin and non–α-globin gene clusters (see Fig. 33.8). What con- Their half-lives have been estimated at 30–50 hours. Most other
trols these switches in globin gene transcription is not understood. mRNAs have turnover rates, or half-lives, measured within the range
The two early embryonic Hbs consist of ζ- and ε-globin chains (Hb of a few minutes to 5 or 6 hours. The increase in the percentage of
Gower-1) and α- and ε-globin chains (Hb Gower-2). The ζ-globin total mRNA that is globin mRNA is greatly accentuated because
gene is akin to the α-globin genes but is expressed only during early the newly transcribed globin mRNAs accumulate and remain quite
embryogenesis. The ε-embryonic globin chain is a β-like element. stable in the cell, but nonglobin mRNAs, which are no longer being
The combination of ζ- and γ-globin chains forms Hb Portland. produced, are also disappearing at a faster rate. Consequently, the
These early Hbs are made primarily in yolk-sac erythroblasts and mRNA content of the reticulocytes consists of 90% to 95% globin
are detectable only during the very earliest stages of embryogenesis mRNA.
except in certain pathologic states, in which they may persist until The transcription rates of the α-globin and non–α-globin genes
gestation is complete. The major Hb of intrauterine life is HbF, which are not precisely equal. (This phenomenon has been studied in detail
consists of two α- and two γ-globin chains. Expression of the γ-globin only in adult erythroid cells expressing the α- and β-globin genes) A
gene begins early in embryogenesis, peaks during midgestation, and slight, but reproducibly detectable, excess of α-globin mRNA is
begins a rapid decline just before birth. By 6 months of age in normal present in erythroblasts. However, β-globin mRNA is translated
infants, only a remnant of prior γ-globin gene expression remains. somewhat more efficiently than α-globin mRNA. These counterbal-
The level of HbF in the blood declines rapidly thereafter to less than ancing forces result in almost equal syntheses of α- and β-globin
1% of the total. Expression of the α-globin gene starts early in the polypeptide chains. There is a very slight excess of α-globin produc-
first trimester, peaks quickly, and is sustained for life. Expression of tion, resulting in a small pool of free α-globin chains.
the β-globin gene also commences early in gestation and reaches its Alpha Hb-stabilizing protein (AHSP), a small protein present at
zenith within a few months after birth. The combination of α-globin high concentrations in RBCs, binds specifically to the α-globin
with β-globin chains forms HbA the predominant Hb of postnatal polypeptide, protecting the unstable free α-globin chain by inhibiting
life. Adult cells also contain HbA 2 . The δ-globin gene, which directs heme loss and oxidant-mediated chain precipitation. It remains
synthesis of the non–α-globin chain of HbA 2 , is very inefficiently unclear whether mutations of this protein can modify the phenotype
expressed. Only low levels of HbA 2 are present; defects in the δ-globin of β-thalassemia by increasing the imbalance in globin chain synthe-
gene are of no clinical consequence. In adult blood, HbF is not evenly sis. Some α-globin chain variants, because the mutations alter AHSP
distributed among erythrocytes and is present in only a very small binding, are associated with mild thalassemia-like features.
number of RBCs, called F cells. HbA 2 is present in all RBCs, albeit Newly synthesized β-globin chains are rapidly and completely
at levels less than 3.5% of the total Hb in adult life. incorporated into αβ dimers that spontaneously associate as tetra-
mers. Hb tetramers are remarkably stable throughout the life span of
the circulating RBC by virtue of their long half-lives. Only small
Hemoglobin Biosynthesis and Its Regulation amounts sustain oxidative or proteolytic damage.
Hb molecules are exposed for prolonged periods to chemically
Throughout development, genes coding for α-globin, non–α-globin, active compounds in the milieu of the bloodstream. They often
and heme exhibit coordinated expression. Almost equal amounts of become nonenzymatically modified by such processes as glycosyl-
each of the moieties that ultimately constitute the Hb tetramer are ation, acetylation, and sulfation. Glycosylation occurs more exten-
made. Excess unpaired globin chains and mutant globins are removed sively during periods of hyperglycemia and leads to elevated levels of
from the cell by ATP-dependent proteases, ensuring a balance the glycosylated form of HbA, HbA 1c . This phenomenon is the basis
between accumulation of α-globin and non–α-globin chains. Bal- of a useful test for control of the blood sugar in diabetes. Other
anced chain synthesis and coordination of globin chain production posttranslational modifications are of little clinical importance except
with synthesis of heme are important because Hb tetramers are highly as already noted for 2,3-BPG, carbon dioxide, and NO.
soluble, but the components of Hb (i.e., unpaired chains, protopor-
phyrin, and iron) are not. Precipitation of any of these is deleterious
to cell survival. Erythroblast proteases are not efficient enough to Transcriptional Regulation of Globin
eliminate the substantial excesses of unpaired chains that accumulate Gene Expression
when an α-gene or non–α-gene is selectively impaired by severe
thalassemia mutations. The mechanisms regulating heme production Precise regulation of the globin gene clusters involves a complex
and some of the interactions between heme and globin synthesis are interplay between trans-acting proteins, such as transcription factors,
discussed in Chapter 35. and cis-acting sequences that act as promoters, enhancers, and silenc-
The proper production of the individual globin chains within ers of gene activity. DNA-binding proteins interact with sequences
erythroid tissues at the appropriate states of differentiation and in regulatory regions of the globin gene cluster and with other pro-
development is predominantly ensured by regulation at the level of teins through specific protein–protein interactions, forming DNA–
transcription. The onset of phenotypic maturation at the proeryth- protein complexes that regulate gene transcription. Trans-acting
roblast stage is marked by the onset of globin mRNA biosynthesis in factors mediate the remodeling of chromatin structure, influencing
dramatically increasing quantities. Expression of α-globin and non–α- gene expression for the entire globin gene clusters. Mutations in the
globin genes begins at essentially the same time, although some cis-acting sequences or trans-acting proteins cause dysregulated
studies suggest a slightly earlier onset for α-globin gene expression. expression of globin genes, resulting in thalassemia-like syndromes
Transcription persists at a high level throughout most of the remain- although most thalassemia-causing mutations are in the cognate
der of erythropoiesis, declines as the nucleus condenses, and is globin gene clusters. Elucidating the full extent of sequences required
eventually lost in late erythroblasts. Even as the absolute rates of for appropriate expression of globin genes will inform the develop-
globin gene transcription begin to decrease, however, the relative ment of constructs for gene therapy.
percentage of total transcriptional activity devoted to globin gene The nuclei of erythroid cells contain numerous proteins that have
expression continues to increase; this reflects the silencing of tran- been identified as transcription factors, including GATA1, NFE2,
scription of almost every other gene in the erythroblast. LRF, and EKLF. GATA1 is named on the basis of the DNA sequence
The transcriptional activation of the globin genes is the major event motif (T/A) GATA (A/G), the GATA motif that it recognizes and
that must be understood to define and manipulate the regulation of binds. It is a zinc finger class DNA-binding protein (see Chapters 22