Page 748 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 45 Red Blood Cell Membrane Disorders 635

Pathobiology of Common Hereditary Elliptocytosis defects of protein 4.1R. Protein 4.1R is a multifunctional protein that
contains several important sites of protein interactions, including the
The possibility that the primary lesion of HE and HPP erythrocytes spectrin-binding domain, where 4.1R binds to the distal end of the
resides in the proteins of the RBC membrane skeleton was first raised spectrin αβ heterodimer, markedly increasing the binding of spectrin
by the findings of thermal instability of HPP spectrin, retention of to oligomeric actin, and the basic NH 2-terminal domain, where 4.1R
the elliptical shape in HE membrane skeletons, disintegration of interacts with GPC, phosphatidylinositol, and phosphatidylserine,
membrane skeletons after exposure to shear stress, defective self- facilitating the attachment of the distal end of spectrin to the
association of spectrin dimers to tetramers, altered susceptibility of membrane.
spectrin to tryptic digestion, and a deficiency of the membrane Studies of 4.1R mRNA from normal RBCs revealed 4.1R isoforms
skeleton proteins spectrin and protein 4.1R. Gene cloning and resulting from complex tissue- and developmental stage-specific pat-
determination of the primary structure of these proteins was soon terns of alternate mRNA splicing. Alternate translation initiation sites
followed by reports of mutations in the genes encoding erythrocyte are present in the protein 4.1R mRNA. When an upstream initiator
membrane proteins. methionine is used, isoforms greater than 80 kDa are synthesized.
During erythropoiesis, this upstream initiator methionine is spliced
out and a downstream initiator methionine is used, leading to the
Spectrin Mutations production of the 80-kDa mature erythroid protein 4.1R isoform.
On SDS-PAGE, protein 4.1R is resolved into two bands of different
The most common defects in HE, found in approximately two-thirds sizes: 4.1a and 4.1b. The larger band, 4.1a, is typically found in
to three-quarters of all patients, are mutations of α- or β-spectrin. normal RBCs, whereas the shorter one, 4.1b, represents the major
Both α- and β-spectrin are elongated flexible molecules consisting of isoform of reticulocytes. The 4.1b isoform is converted into the 4.1a
triple-helical repeats connected by nonhelical segments. These poly- isoform by deamidation of Asn 502.
peptides are associated side to side in an antiparallel position, forming A partial deficiency of protein 4.1R is associated with mild,
a flexible, rod-like αβ heterodimer in which the NH 2 -terminal of dominantly inherited HE, whereas a complete deficiency (a homo-
α-spectrin and the COOH-terminal of β-spectrin form the head zygous state) leads to a severe hemolytic disease. Homozygous protein
region of the heterodimer. Spectrin heterodimers associate head to 4.1R(−) erythrocytes fragment more rapidly than normal at moderate
head to form spectrin tetramers, the major structural subunits of the shear stresses, an indication of their intrinsic instability. Membrane
membrane skeleton. Spectrin tetramers in turn are interconnected mechanic stability can be restored by reconstituting the deficient
into a highly ordered two-dimensional lattice through binding, at RBCs with protein 4.1R or the protein 4.1R/spectrin/actin-binding
their distal ends, to actin oligomers with the aid of protein 4.1R. site. Homozygous protein 4.1R(−) erythrocytes also lack p55 and
The contact site between the α- and β-spectrin chains of the have only 30% of the normal content of GPC. These homozygous
opposed heterodimers is a combined “atypical” triple-helical repeti- protein 4.1R(−) erythrocytes, as well as GPC-deficient Leach erythro-
tive segment in which the first two helices are contributed by the cytes, demonstrate decreased invasion and growth of Plasmodium
COOH-terminal of β-spectrin, whereas helix 3 is the first helical falciparum in vitro.
segment of α-spectrin. Spectrin dimer-tetramer interconversion is Mutations associated with protein 4.1R deficiency have included
governed by a simple thermodynamic equilibrium that under physi- deletions that include the exon encoding the erythroid transcrip-
ologic conditions strongly favors spectrin tetramers. Most α-spectrin tion start size and mutations of the transcription initiation codon.
defects are at or near the NH 2 -terminal of α-spectrin, which is Qualitative defects of protein 4.1R protein include deletions and
involved in the heterodimer contact (the αI domain defined by duplications of the exons encoding the spectrin-binding domain,
limited tryptic peptide mapping; see the discussion under Laboratory leading either to truncated or elongated forms of protein 4.1R.
Manifestations), and impair the self-association of spectrin into tet- Electron microscopic studies of homozygous protein 4.1R(−) eryth-
ramers. Most α-spectrin mutations are point mutations. These rocyte membranes revealed a markedly disrupted skeletal network
mutations create abnormal proteolytic cleavage sites that typically with disruption of the intramembrane particles, suggesting that
reside in the third helix of a repetitive segment and give rise to protein 4.1R plays an important role in maintenance not only of the
abnormal tryptic peptides on two-dimensional tryptic peptide maps skeletal network but also of the integral proteins of the membrane
of spectrin. structure.
Elliptocytogenic β-spectrin mutations are COOH-terminal point
mutations or truncations that disrupt the formation of the combined
β triple-helical repetitive segment and consequently the self- Glycophorin C Deficiency
association of spectrin heterodimers to tetramers. All of these muta-
tions open a proteolytic cleavage site residing in the third helix of the GPC has been found absent because of a variety of molecular
combined repetitive segment, which gives rise to a 74-kDa αI peptide. defects. In contrast to other forms of HE, which are dominantly
Although most spectrin mutations reside in the vicinity of the inherited, heterozygous carriers are asymptomatic, with normal RBC
αβ-spectrin self-association site, a few mutations remote from the morphology, and homozygous patients have no anemia and only mild
self-association site have been described. These mutations are asymp- elliptocytosis apparent on the peripheral blood film.
tomatic in the simple heterozygous state but cause hemolytic anemia, GPC deficiency with elliptocytosis, the so-called Leach pheno-
which can be severe, in homozygous patients. Unlike mutations type, caused by reduced expression of GPC, should be distinguished
located in the self-association contact site, which are predicted to from the immunochemically defined phenotypes Gerbich and Yus,
disrupt the conformation of the local protein structure, mutations in which abnormal glycoproteins are formed that can functionally
outside this region are predicted to perturb long-range protein-protein substitute for normal GPC and preserve the normal RBC shape. The
interactions, disrupting the positively coupled, cooperative interac- Leach phenotype is usually caused by a large deletion of genomic
tions of αβ spectrin self-association, spectrin-ankyrin interactions, DNA (~7 kb) that removes exons 3 and 4 from the GPC/glycophorin
and ankyrin–band 3 interactions. One HE-associated mutation in a D locus. In one patient, the Leach phenotype was caused by a
linker region remote from the self-association contact site disrupted frameshift mutation.
the stability propagated from one spectrin repeat to the next. GPC-deficient patients are also partially deficient in protein 4.1R
and lack p55, presumably because these proteins form a complex
and recruit or stabilize each other on the membrane. It has been
Protein 4.1R Mutations speculated that the protein 4.1R deficiency in Leach erythrocytes
is the cause of the elliptocytic shape. In contrast, patients deficient
Another group of elliptocytogenic mutations, although much less in glycophorin A, the major transmembrane glycoprotein, are
common than spectrin mutations, are quantitative or qualitative asymptomatic.