Early analyses by SDS-PAGE of the polypeptides present in red blood cells revealed 10 major proteins (Figure 53–5). These proteins were initially designated based upon their migration on SDS-PAGE, with the slowest migrating (highest molecular mass) designated band 1 protein, also known as spectrin (Table 53–4). As illustrated by Figure 53–6, certain of these proteins are glycosylated and several span the membrane bilayer (integral membrane proteins), while others associate with its surface, generally via protein-protein interactions (peripheral membrane proteins).
Major membrane proteins of the human red blood cell. Proteins separated by SDS-PAGE were detected by staining with Coomassie blue dye. (Reproduced, with permission, from Beck WS, Tepper RI: Hemolytic anemias III: membrane disorders. In: Hematology, 5th ed. Beck WS (editor). The MIT Press, 1991.)
TABLE 53–4Principal Proteins of the Red Cell Membrane ||Download (.pdf) TABLE 53–4 Principal Proteins of the Red Cell Membrane
|Band Numbera ||Protein ||Integral (I) or Peripheral (P) ||Approximate Molecular Mass (kDa) |
|1 ||Spectrin (α) ||P ||240 |
|2 ||Spectrin (β) ||P ||220 |
|2.1 ||Ankyrin ||P ||210 |
|2.2 ||Ankyrin ||P ||195 |
|2.3 ||Ankyrin ||P ||175 |
|2.6 ||Ankyrin ||P ||145 |
|3 ||Anion exchange protein ||I ||100 |
|4.1 ||Unnamed ||P || 80 |
|5 ||Actin ||P || 43 |
|6 ||Glyceraldehyde-3-phosphate dehydrogenase ||P || 35 |
|7 ||Tropomyosin ||P || 29 |
|8 ||Unnamed ||P || 23 |
| ||Glycophorins A, B, and C ||I ||31, 23, and 28 |
Interactions of cytoskeletal proteins with each other and with certain integral proteins of the membrane of the red blood cell. (Reproduced, with permission, from Beck WS, Tepper RI: Hemolytic anemias III: membrane disorders. In: Hematology, 5th ed. Beck WS (editor). The MIT Press, 1991.)
The Red Blood Cell Membrane Contains Anion Exchange Protein & the Glycophorins
Band 3 protein is a transmembrane glycoprotein oriented with its carboxyl terminal end projecting from the external surface of the erythrocyte membrane and its amino terminal end from the cytosolic face. Thought to exist as a dimer, band 3 protein is a multipass membrane protein whose polypeptide chain crosses the bilayer 14 times. The principle function of this anion exchange protein is to provide a channel within the membrane through which chloride and bicarbonate anions can be exchanged. At the tissues, bicarbonate generated from the hydration of CO2 is exchanged for chloride. At the lungs, where carbon dioxide is exhaled, this process is reversed. The amino terminal end serves as an anchoring point for several red blood cell proteins, including band 4.1 and 4.2 proteins, ankyrin, hemoglobin, and several glycolytic enzymes.
Glycophorins A, B, and C are single-pass transmembrane proteins (the polypeptide chain crosses the membrane only once). The 23 amino acid transmembrane segment is α-helical in configuration. Glycophorin A, the predominant form, is heavily glycosylated. The amino terminal end of this 131-amino acid polypeptide is modified by 16 oligosaccharide chains, 15 of them O-linked, that account for roughly 60% of its mass. The oligosaccharide chains of glycophorin A account for nearly 90% of the sialic acid residues bound to the red cell membrane. The carboxyl terminal end extends into the cytosol and binds to band 4.1 protein, which in turn binds to spectrin. Polymorphism of glycophorin A provides the basis of the MN blood group system (see below). Some viral and bacterial pathogens, such as influenza virus and Plasmodium falciparum, target erythrocytes by recognizing and binding to glycophorin A. Intriguingly, individuals whose red cells lack glycophorin A exhibit no adverse effects.
Spectrin, Ankyrin, & Other Peripheral Membrane Proteins Help Determine the Shape & Flexibility of the Red Blood Cell
In order to maximize the efficiency of gas exchange, red blood cells must possess the structural strength to maintain their biconcave shape, yet remain sufficiently flexible to squeeze through peripheral capillaries and the sinusoids of the spleen. The red blood cell membrane’s lipid bilayer, which is inherently fluid, contributes significantly to the deformability of the erythrocyte membrane. This flexible bilayer is pulled into the biconcave shape by a strong but flexible network of cytoskeletal proteins (Figure 53–6).
Spectrin is the most abundant protein of the erythrocyte cytoskeleton. It is composed of two polypeptides more than 2100 residues in length: spectrin 1 (α chain) and spectrin 2 (b chain). The α and β chains of each spectrin dimer intertwine in an antiparallel orientation to form a highly extended structural unit ≈ 100 nm in length. Normally, two spectrin dimers self-associate head-to-head to form an approximately 200 nm long tetramer that is linked to the inner surface of the plasma membrane (and is bridged to other spectrin tetramers) via ankyrin, actin, and band 4.1 protein. The result is an internal mesh, the cytoskeleton, that is strong enough to maintain cell shape and resist swelling due to osmotic pressure, yet flexible enough to allow the erythrocyte to fold when needed.
Ankyrin is a pyramid-shaped protein that binds spectrin. In turn, ankyrin binds tightly to band 3, securing attachment of spectrin to the membrane. Ankyrin is sensitive to proteolysis, accounting for the appearance of bands 2.2, 2.3, and 2.6, all of which are derived from band 2.1.
Actin (band 5) exists in red blood cells as short, double-helical filaments of F-actin. The tail end of spectrin dimers binds to actin. Actin also binds to protein 4.1.
Protein 4.1, a globular protein, binds tightly to the tail end of spectrin, near the actin-binding site of the latter, and thus is part of a protein 4.1-spectrin-actin ternary complex. Protein 4.1 also binds to the integral proteins glycophorin A and glycophorin C, thereby attaching the ternary complex to the membrane. In addition, protein 4.1 may interact with certain membrane phospholipids, thus connecting the lipid bilayer to the cytoskeleton.
Certain other less quantitatively prominent proteins, such as band 4.9, adducin, and tropomyosin, also participate in cytoskeletal assembly.
Abnormalities in the Amount or Structure of Spectrin Cause Hereditary Spherocytosis & Elliptocytosis
Hereditary spherocytosis, a genetic disease transmitted as an autosomal dominant, affects about 1:5000 persons of Northern European ancestry. It is characterized by the presence of spherocytes (spherical red blood cells, with a low surface-to-volume ratio) in the peripheral blood, by a hemolytic anemia, and by splenomegaly. Spherocytes are more vulnerable to lysis when exposed to lower than normal osmotic pressure, since their spherical shape offers little capacity to accommodate additional water. Their abnormal shape also renders them less deformable and more prone to destruction in the spleen, thus greatly shortening their life in the circulation.
Hereditary spherocytosis is caused by a deficiency in the amount of spectrin or abnormalities of its structure that undermine its capacity to associate with other cytoskeletal components. The consequent weakening of the links that anchor the erythrocyte membrane to the cytoskeleton leads to the adoption of the spherocytic shape. Hereditary spherocytosis also can results from mutations that produce abnormalities in ankyrin or in bands 3, 4.1, or 4.2. The anemia associated with hereditary spherocytosis is generally relieved by surgical removal of the patient’s spleen (splenectomy).
Hereditary elliptocytosis also results from genetic disorders that generate abnormalities in spectrin or, less frequently, in band 4.1 protein or in glycophorin C. It can readily distinguished from hereditary spherocytosis by virtue of the fact that the affected red blood cells assume an elliptic, disk-like shape.