Red Cell Membrane Defects

Lecture 5
BIOL4355 & BIOL6V29
Instructor: Betty Pace, MD
September 12, 2006
Objectives
 Understand the organization of proteins in the
red blood cell membrane

 Relate membrane structure to normal function

 Review progress made to identify molecular

causes of inherited red blood cell membrane
disorders
Red Blood Cell Function

Normal maturation in bone marrow

Function – oxygen transport
Specialized membrane structure
-Flexibility
-Gas exchange
Reticulocytes

 Immature erythrocyte
 1% of circulating erythrocytes
 Polyribosomes (RNA)

Methylene Blue Stain

Mature Erythrocytes

 Energy source glucose

 Mitochondria absent
 Protein synthesis absent
 Survival 120 days
 Senescent cell removed by the spleen
and bone marrow
 Smear of Peripheral Blood
Wright Stain

Red Blood Cell Indices – describe the size

and oxygen carry protein (hemoglobin) content
of cell
•MCV (mean corpuscular volume):
measure of size (normal value: 82-98 fl)
•MCHC (mean corpuscular hemoglobin
concentration): average hemoglobin
concentration in cells, related to color of
cells (normal value: 31-37 g/dl)
•RDW (red cell distribution width):
measures variation in red cell size, i.e.
anisocytosis (normal value: 11.5-14.5%)
 Red Blood Cells
Scanning Electron Microscopy

Biconcave disk
Membrane – provides resilience and flexibility!
 RBC Shapes and Sizes

Anisocytosis: variation in size

Poikilocytosis: increase in number
of abnormally shaped RBCs

Normocytic: normal size

Normochromic: normal hemoglobin
concentration
 Red Cell Membrane Structure

 Lipid Bilayer: polar heads (circles)

outward, apolar fatty acid chains face
inward
 Band 3 complex (A): tetramer
transmembrane protein with a tail
monomer for cytoplasmic domain
 Ankyrin-1 binds spectrin β-chain &
protein 4.2
 Glycophorin A receptor – erythroid
specific marker
 The Rh complex (B): Rh protein (Rh),
Rh-associated glycoprotein (RhAG),
CD47, and the Landsteiner-Wiener
glycoprotein (LW)
 CD47 interacts with protein 4.2 and Rh protein to contact ankyrin-1
 Junctional Complex (C): protein 4.1R, spectrin, actin, dematin (4.9), tropomyosin,
and β-adducin (not shown).
 Spectrin (D): α2β2 tetramer which forms a dense network lining the inner surface
of the lipid bilayer. The α- and β-chains are anti-parallel; dimers associate side-
by-side & head-to-head  N-terminal of α-chains to C-terminal of β-chains
 Red Blood Cell Membrane Skeleton

Spectrin forms a mesh-like pattern that is anchored to the membrane

by ankyrin (circles); the basic shape is hexagonal (shaded)
 Protein Structure
 Backbone
• Amide Group: -NH2
• Alpha Carbon (C)
• Carboxylic acid: -COOH
 R side chain amino acids:
• Hydrophobic: R=Cs, Hs; “water haters”
• Polar: R=O2, N2; “water lovers”
• Charged: R=basic or acidic moiety

backbone
 Protein Primary Structure
Polypeptide Bond

 Peptide: string of amino acids joined by

peptide bond
 Reaction between –COOH and NH2 to form
C-NH bond and H2O

PEPTIDE BOND
 Secondary Structure

 Helices from intra-molecular

hydrogen bonds
 -helix: 3.6 aa/360° turn

-helix
 Tertiary Structure

 Three dimensional shape, due to intra-molecular interactions

 Hydrogen & disulfide bonds (cross link cysteine residues)

twisted beta sheets four helical bundle

 Quaternary Structure

 Inter-molecular interactions that occur when multiple

polypeptides associate to form a functional protein
 Protein-protein contacts in multi-enzyme complexes

Tetramer Heterodimer
Methods to Detect of RBC Membrane Abnormalities
I. Rapid FACS: Eosin-5-Maleimide (EMA)  

1)  to determine the presence of abnormal red cells in blood

2)  to distinguish hemolytic anemia associated with membrane protein
abnormality from other types
3) A fluorescent-based probe
4) EMA binds to Band 3 and Rh-related proteins on intact red cells
5) Partial reductions in these proteins in hereditary spherocytosis (HS)
6) HS: a reduction of mean channel fluorescence (MCF) reading

II. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

Page)

1) RBC membrane proteins: spectrin, ankyrin, band 3, protein 4.1 and

protein 4.2, actin are separated according to their molecular mass (kDa)
2) Coomassie Blue dye stain
3) Quantitation of the bands by
densitometry 4) Each band is expressed
as a ratio to band 3 protein to detect an quantitative change in membrane
proteins.
RBC Membrane Proteins SDS-Page Method
SDS: anionic detergent used to denature proteins
SDS-PAGE: can be used to estimate relative molecular mass and
relative abundance of major proteins

Molecular mass (symbol m) is expressed in Daltons (Da); most

macromolecules are large enough to use kiloDalton (kDa) to describe
molecular mass

SDS

DTT

GEL ELECTROPHORESIS
COUMMASSIE STAIN
 RBC Membrane Protein Sizes

kDa spectrin

 -spectin = 260 kDa (240) 
 -spectrin = 225 kDa (240)
 Ankyrin = 215 kDa (2)
 Band 3 = 90-100 kDa (3)
 Band 4.1 = 78 kDa (4.1)
 Band 4.2 = 72 kDa (4.2)
 Actin = 43 kDa (5)
 Glycophorin C = 25 kDa (7)
 Globin = 16 kDa
 2D Gel Electrophoresis
 1st Dimension: isoelectric focusing (IEF) of proteins on
gradient strips
 pI value: pH where net charge is zero on protein
 2nd Dimension: run strips on SDS gel to separate by MW
 SDS detergent makes protein negative charged

IEF SDS
 Mass Spectrometry Approach

 Matrix Assisted Laser

Desorption Ionization
(MALDI)
 Mass Spectrometry
• Proteins bent by
magnetic fields
• Electric charge
generated and
recorded
• Peptide Mass
Fingerprint: series of
signal peak
 Database search to
identify peptides
Inherited Red Cell Membrane Disorders
 Type I: abnormal mechanical properties of membrane to
produce spherocytosis, elliptocytosis, or
pyropoikilocytosis

 Type II: abnormal passive flux of monovalent cations

across the membrane to produce stomatocytosis

 General Symptoms
• red cell hemolysis
• chronic hemolytic anemia
• elevated bilirubin – jaundice
• splenomegaly
• gallstones
• iron overload

 Red Cell Indices: elevated MCHC

 Hereditary Spherocytosis
 Most common in people of northern European ancestry
 Autosomal dominant and recessive inheritance
 Spontaneous mutations!
 Spherocytes: defects in vertical stabilization of the phospholipid bilayer 
separation of the spectrin from phospholipid bilayer
 Form vesicles: lost from the RBC surface resulting in decreased surface area
and spherocytes
 Spherocytes: reduced surface area relative to the RBC volume
 Spherocytes: less flexible and are destroyed in the spleen to produce
splenomegaly.
 Osmotic fragility test to diagnosis hereditary spherocytosis

Defects: 1) the actin - spectrin -

band 3 complex, 2) the spectrin -
4.1 -glycophorin complex or 3)
the connection between the
bilayer and spectrin.
Hereditary Spherocytosis – Band 3 Defects
 Band 3 gene (SLC4A1) mutations:
• DRTA : distal renal tubular acidosis
• Typical HS: Fukuoka & Coimbra
• CHC: Cryohydrocytosis
• SAO: Southeast Asian Ovalocytosis:
Hereditary Spherocytosis - Blood Smear

Normal Spherocytosis
vesicle formation
Osmotic Fragility Test
 Osmotic fragility is a test to detect abnormal
fragility of red blood cells
 Used to diagnose Hereditary Spherocytosis
 A negative test is normal

normal

abnormal
 Severity of Hereditary Spherocytosis
Mild Moderate Severe
Hemoglobin (g/l) 110–150 80–120 60–80
Reticulocyte count (%) 3–6 >6 >10
Bilirubin (µg/l) 17–34 >34 >51
Splenectomy not required school age delay to 6 yrs
before puberty if possible

Parvovirus B19
Parvovirus B19 is found in respiratory secretions
Fifth disease: mild illness; "slapped-cheek" rash on the face
Aplastic Crisis: may cause a serious illness in persons with hemolytic
anemia; acute severe anemia due to bone marrow suppression
No vaccine or medicine that prevents parvovirus B19 infection
Transfusions for severe anemia
 Hereditary Elliptocytosis
Hereditary elliptocytosis
Hereditary pyropoikilocytosis (HPP): more severe form
Caused by spectrin and protein 4.1 defect
RBC shape alterations i.e. elliptocytosis or ovalocytes
HPP: RBC fragmentation
Dominant inheritance
HE: symptomatic, without anemia or splenomegaly
Osmotic fragility is usually normal

HE HE HPP
 Hereditary Stomatocytosis

Autosomal dominant inheritance

Membrane abnormality leads to increased
permeability to sodium  water to move into
RBC  decreasing MCHC

Smear: RBC narrow mouth-like areas of

hypochromia i.e. stomatocytes.

Mild hemolytic anemia and splenomegaly

are typical clinical findings.