A basic overview of a complex concept

2. THE RED CELL
MEMBRANE
JG Nel
June 2009

3.  Dutch biologist and
microscopist
 First observed and
described red cells
in 1668,
observations were
recorded in his
notebooks
Jan Swammerdam

4.  Published in the
“Philosophical
transactions of the
Royal Society”
 Description of the
unique features of red
cells
 “in a healthy body they
should be soft and
flexible, that they may
be capable of passing
through the cappiliary
veins ”
Anton van Leewenhoek

5.  Published the primary
features of red cell
membranes in “Blood of
the vertebrata” in 1862
 “…it is really composed of
two different parts. One of
these is membranous,
colourless and insoluble in
water; the other is semi
fluid or viscid, containing
the colour and very soluble
in water”
A drawing by George Gulliver George Gulliver

7. Overview of this lecture
1. The organization of a normal red cell
membrane as we see it at present.
2. Implications of the structural organization of
various membrane components for red cell
function.
3. Mechanistic basis for altered membrane and
red cell function in inherited red cell
disorders.

8. The organization of the red cell
membrane
• Highly elastic
• Responds rapidly to
applied stress
• Capable of
undergoing large
membrane extensions
(250%)
• Stronger than steel
ito. structural
resistance
A red cell traversing from the
splenic cords to the splenic
sinus
Material properties of the
membrane

9. THE ORGANIZATION
OF THE RED CELL
MEMBRANE
How it is seen at present

10. The organization of the red cell
membrane
 Membrane comprises
3 layers
 External carbohydrate
rich later
 Lipid bilayer
containing numerous
transmembrane
proteins
 Inner network of
proteins that forms the
red cell membrane
cytoskeleton

11. Membrane carbohydrates
 Glycocalix
 Contains some blood group antigens
 Protect the cell against pathogen invasion and
mechanical damage
 Plays a role in red cell senescence
 Exact function not understood

12. Membrane lipids
 Charged
phosphatidyl groups
of PL
 Hydrophillic
 Form outer and
inner surfaces of
bilayer
 Interior of bilayer
 Hydrophobic binding
of the acyl chains
with cholesterol

13. Membrane lipids
 Equal proportions cholesterol and
phospholipids
 Cholesterol
 Enters the membrane via diffusion from plasma
 Distributed equally between the 2 leaflets
 Phospholipids
 4 major
 Asymmetrically disposed
 PC and SM outer monolayer
 PE and PS +minor phophpinositide=inner

14. Membrane lipids:
Phospholipid asymmetry
 Energy dependant and energy independent
phospholipid transport proteins maintain
asymmetry
 Flippases
 Move PL from outer monolayer to inner monolayer
 Floppases
 Opposite, against [],energy dependant
 Scramblase
 Move PL bi-directionally, down [], energy indep
manner
 ?molecular identity –lipid rafts enriched in cholesterol and
sphingolipids in association with specific membrane

15. Membrane lipids:
Phospholipid asymmetry
 Maintenance of asymmetry several functional
implications
 Especially PS and PIP to inner monolayer
 Macrophages recognize and phagocytose red cells
that expose PS at their outer surface
 Inhibits red cell adhesion to vascular endothelial cells
 Binding of spectrin to PS enhances membrane
mechanical stability
 Binding of 4.1 complex to PIP enhaces it’s interaction
with Band 3 and glycophorin C

16. Membrane proteins
 Cell surface Proteins
 Linker Proteins
 Cytoskeletal proteins

17. CELL SURFACE
PROTEINS

18. Cell surface proteins
 Cell surface proteins are not necessarily
discrete units within the cell membrane
 They may
 congregate together in lipid rafts
 Be part of complexes of proteins, contributing to the
function of the complex as a whole

19. Cell surface proteins
 More than 100 have been characterized
 Large fraction define the various blood group
antigens
 Some are multifunctional
 Diverse functional heterogeneity
 Transport proteins and channels
 Receptors and Adhesion molecules
 Enzymes
 Structural proteins

20. Membrane transporters and
channels
 Facilitate the transfer of biologically important
molecules in and out of cells
 Typically serpentine , crossing the membrane
several times
 Examples
1. Urea transporter
2. Aquaporins
3. Band3/Rh Protein complex
4. Glucose transporter
5. Na/K ATPase pump
6. Ca/Mg ATPase pump

21. Membrane transporters and
channels
Urea transporter
 Kidd blood group glycoprotein
 Urea has a low permeability across lipid
bilayers
 Increased in the red cell by the presence of
UT-B
 Speeds up the transfer rate of urea as the cells
pass through the descending and ascending
vasa recta

22. Membrane transporters and
channels
Water and glycerol channels
 Aquaporins
 Two groups
 AQP1: permeated mostly by water
 AQP3: permeated by water and other small
solutes such as glycerol
 Colton and Gill blood groups

23. Membrane transporters and
channels
Band3/Rh macrocomplex
 Red cell anion exchanger
 1 000 000 copies per cell
 Three domains
1. Cytoplasmic N-terminal
 Interacts with
 ankyrin and protein 4.2
 glycolytic enzymes
 Deoxihemoglobin
 hemichromes
2. Membrane domain
 Spans the membrane 14
times
3. Cytoplasmic C-terminal
domain
 Binds CAH

24. Membrane transporters and
channels
Band3/Rh macrocomplex
 Plays an important role in the efficient
transport of respiratory gasses in blood
CO2 + H2O H+ + HCO 3
-
 Transports HCO3
- out of the cell in exchange
for Cl-
 The proton then interacts with Hb release of
O2

25. Membrane transporters and
channels
Rh proteins
 Exact function of Rh proteins in red cells not
known
 Some sequence corr with NH4
+ transporters in
bacteria
 Part of macrocomplex with band3 tetramers
and ICAM4,CD47 and glycophorins A&B
 Adhesion molecules might facilitate transient
adhesive interactions between the red cell and
vascular endothelium to maximize gas transfer

26. Band3/Rh macrocomplex

27. Receptors and adhesion
molecules
 Duffy glycoprotein
 Immunoglobulin superfamily
 Complement control proteins

28. Receptors and adhesion
molecules
Duffy glycoprotein
 G protein-coupled superfamily for receptors
 But lacks the motifs required for G protein
coupling
 Binds many different ligands especially
chemokines
 Functions as a sink –binding excess
chemokines to prevent inappropriate activation
of neutrophils
 Exploited as a receptor by P.vivax for the
invasion of red cells

29. Receptors and adhesion
molecules
Immunoglobulin superfamily
 Large family of receptors with extracellular
domains containing different numbers of repeating
domains with sequence homology to
immunoglobulin domains
 Mostly function as receptors and adhesion
molecules
 Include:
 ICAM4
 ERMAP
 CD47
 Lutheran glycoproteins

30. Receptors and adhesion
molecules
Complement control proteins
1. DAF(CD55)
 Inhibits C3 convertases
2. MIRL(CD59)
 Prevents assembly of MAC
 1 and 2 anchored to the membrane via a GPI anchor
3. CR1
 Binds and processes immune complexes and
transport the complexes to the liver and spleen
 Involved with the rosseting of cells infected with
P.falciparum and uninfected cells

31. Enzymes
 Three glycoproteiens have structures
suggestive of enzymatic activity
1. Acetylcholinesterase
2. Kell glycoprotein
 Endopeptidase
 Activates endothelin 3 via cleavage of it’s inactive
progenitor
3. ART4 glycoprotein

32. Structural proteins
Enables effective attachment of the cell
membrane to it’s cytoskeleton
Transmembrane proteins
 Band3
 Glycophorin C&D
 CD44
 RhAG
Connected by linker proteins to cytoskeleton

33. Linker proteins
 attachment of skeletal proteins to
transmembrane proteins
 Ankyrin-1: spectrin to band3 (Prot 4.2)
 4.1R : spectrin to glycophorin C/D

34. CYTOSKELETAL
PROTEINS

35. Cytoskeletal proteins
 Principal proteins
 Spectrin a+b
 Actin
 Protein4.1R
 Other proteins
involved
 Adducin
 Dematin
 Tropomyosin
 tropomodulin

36. Cytoskeletal proteins
Spectrin
 Large number of triple
helical repeats of 106
amino acids
 20 in a-spectrin
 16 in b-spectrin
 These triple helical
bundles define the
spectrin family
 Repeats are
structurally
heterogenous ito
thermal stability

37.  Connected to one
another at 2 sites:
 2 or more spectrin
dimers articulate at
the spectrin self
association site
 The ends of several
tetramers converge
to a junctional
complex

38. Cytoskeletal proteins
Spectrin
lateral association
a-spectrin + b-
spectrin
Dimer
Head to head assoc
tetramer
End to end
association
Junctional complex

39. Cytoskeletal proteins
Junctional complex

40. Cytoskeletal proteins
 Basic mesh of
skeleton comprized
of inter-penetrating
hexagons
 Spectrin forms the
sides and radi
 Middle –spectrin self
association site
 Points of
convergence-
junctional
complexes

41. Horizontal and vertical stability
 Vertical stability
 Spectrins
 Band 3
 Ankyrin
 Protein4.2
 Horizontal stability
 Contribute to red cell
shape
 Spectrins
 Actin
 Protein 4.1

42. IMPLICATIONS OF
ORGANIZATION FOR
FUNCTION
Where does it all fit in?

43. Implications for function
In performing it’s primary function of oxygen
delivery the red cell must absorb mechanical
punishment throughout it’s lifetime –without
structural deterioration.
Three primary regulators of the ability of the cell
to undergo the neccesary deformations
 Cell geometry
 Cytoplasmic viscosity and cell volume
regulation
 Membrane deformability

44. Cell geometry
 Biconcave
 Volume 90fL
 Surface area 140 µm2
 Excess surface area of
40% when compared to
a sphere with the same
volume
 Without excess surface
area to volume ratio the
cell cannot deform
 Any deviation from the
spherical state at a
constant volume implies
an increase in surface
area

45. Maintenance of Cell geometry
Maintenance of membrane surface area
 Mediated by
 strong cohesion between the bilayer and the
membrane skeleton prevents vesiculation
 Mechanically stable spectrin based membrane
skeleton that prevents membrane breakup
Maintenance of cell volume
 Mediated by various membrane-associated ion
transporters

46. The concept of “vertical” vs
“horizontal” stability

47. Maintenance of cell geometry
 Membrane cohesion
 “vertical” linkages between bilayer and
membrane skeleton

48. Maintenance of cell geometry
Membrane mechanical stability
 Major determinant of structural stability of cell
 Dependant on
 Avidity of the interaction of spectrin dimers
 Interactions that define the juctional complex at
the distal ends of the spectrin tetramers

49. Maintenance of cell geometry
Avidity of the
interaction between
spectrin dimers
 Adjacent triple helical
repeats needed for
effective interaction
between the dimers
 Dimer-dimer
interaction not static
–opens up reversibly
irt tensile forces
imposed by
deformation

50. Maintenance of cell geometry
The role of lipids
 Certain of the triple helical repeats of b&a-
spectrin bind to PS increases
membrane mech stab
 PIP2 binds to N-terminus of b-spectrin
the propensity of b-spectrin to
form
ternary complexes with
 Actin
 Protein4.1R

51. Cytoplasmic viscosity and cell
volume regulation
 Ability of the red cell to rapidly change it’s
shape in response to fluid shear stress
Cytoplasmic
viscosity
Intracellular
[Hb]

52. Cytoplasmic viscosity and cell
volume regulation
 Distribution of [Hb] in normal cells vary
between 27-37g/dl
 Viscosity of Hb rises steeply starting at 37g/dl
 By tightly regulating [Hb] within a narrow range
red cells minimize the cytoplasmic viscous
dissipation during cell deformation
 Increased [Hb] >37g/dl rate at which the
cell recovers it’s initial shape after extensional
and bending deformations

53. Cytoplasmic viscosity and cell
volume regulation
 The ability of the cell to regulate it’s [Hb] within
narrow limits is critically dependant on it’s
ability to control it’s volume
 Volume determined by total cation content

54. Membrane deformability
 A unique feature of the normal red cell
membrane is it’s high elasticity
 Enables the cell to rapidly respond to fluid shear
stresses
 Precise structural basis for this ability remains
uncertain
 Unfolding and refolding of distinct spectrin
repeats make a major contribution to the
elasticity of the red cell membrane

55. RED CELL MEMBRANE
DISORDERS
Mechanistically explained

56. HS
Non-haemolytic HE
Haemolytic HE

57. The two classes
 Altered function due to mutations in various
membrane or skeletal proteins
 Hereditary spherocytosis
 Heriditary eliptocytosis
 Heriditary ovalocytosis
 Heriditary stomatocytosis
 Altered function due to secondary effects on the
membranes resulting from mutations in the globin
genes
 Sickle cell disease, HbSC,HbCC, unstable Hb and
thalassemias

58. Altered cell geometry
 cell surface: volume ratio cell sphericity
 Distinguishing feature of red cells in
 HS
 HE
 OHS

59. Altered cell geometry
Heriditary spherocytosis
 Common feature of all
cases of HS
 Loss of membrane
surface area
 Cells with decreased
membrane surface area
unable to safely
traverse the spleen
 Severity of the disease
related to the extent of
decrease of the
membrane surface area

60. Altered cell geometry
Heriditary spherocytosis: decreased vertical
stability
 The mechanismDeficiencies in proteins that
anchor bilayer to cytoskeleton
cohesion
anchoring
Band 3
RhAG
Ankyrin
Protein 4.2

61. Altered cell geomtery
Heriditary eliptocytosis
 10% cases moderate to
severe anaemia
 Heterozygotes asymptomatic
 Homozygotes
 Mild to severe anaemia
 Heriditary pyropoikilocytosis
 Common feature
 Mechanically unstable
membrane
 progressive transformation
from: discocyte eliptocyte
 Severity dependant on the
extent of degree of
membrane instability and
resultant loss of membrane
surface area

62. Altered cell geometry
HE: decreased horizontal stability
 The mechanism Mutations in the genes
encoding for a&b
spectrin, protein 4.1R
Defective spectrin
dimer-dimer or spectrin-
protein4.1 or interaction
Weakened horizontal
stability
Protein 4.1R
adduci
n
spectrin

63. Altered cell geometry
Overhydrated heriditary
stomatocytosis
 Rare disorder
 Large numbers of
stomatocytes on peripheral
blood associated with
moderately severe to
severe anaemia
 Autosomal dominant
 Distinctive feature of red
cells is their sphericity due
to increased cell volume
without an increase in
membrane surface area

64. Increased cytoplasmic viscosity
Dehydrated heriditary stomatocytosis
 Red cell dehydration
 Increased MCHC due to decreased total
cation content and concom. Water loss
 Dehydration does not compromise cell survival
as severely as Overhydration
 Well compensated anaemia

65. Altered membrane deformability
 Decreased
membrane
deformability is the
distinguishing feature
of Heriditary
ovalocytosis
 Homozygosity is
embrionic lethal
 Membranes of
ovalocytes: 4-8times
less elastic
 Mutation in band3
?decreased elasticity

66. The future
 Studies on the red cell membrane have shed
much new, often unexpected light on the
structure and function of plasma membranes
 Great potential in continued red cell research
 ?nature and function of macromolecular
complexes in the membrane
 Dynamics of assembly and function of membrane
microdomains
 Molecular basis for cell volume regulation
 Etc.

67. Sources
 Red cell membrane: past, present and future.
Mohandas, Gallager blood 2008 112:3939-3948
 Disorders of the red cell membrane. Xuili, Mohandas
bjh 2008 141:367-375
 Functions of red cell surface proteins. Daniels Vox
Sangiunis 2007 93: 331-340
 Blood Groups and Diseases Associated with Inherited
Abnormalities of the Red Blood Cell
Membrane.Yadanbakhsh, Lomas-Francis Transfusion
Medicine reviews 2000 14:364-374
 Mechanisms in Haematology
 Post Graduate Haematology