63

BIOCHEMISTRY AND ROLES OF GLYCOPHORIN A

SAAD TAYYAB* and MOHAMMAD ABUL QASIM
D e p a r t m e n t of Biochemistry, Faculty of Medicine
Aligarh Muslim University
Aligarh-202001, India

Introduction Membranes play important roles in many cellular phenomena. The transmembrane
proteins contain carbohydrates, and are usually implicated in such important biological
functions of the cell as virus binding, cell-surface antigenicity, cell-cell recognition,
cell-cell communication, cellular transformation, transport, energy transduction, etc.
These proteins are embedded in the lipid milieu of the membrane which provides the
suitable environment for their action. The erythrocyte membrane is the most thoroughly
studied. 1-3 About six proteins and four sialoglycoproteins may be observed by SDS-
polyacrylamide gel electrophoresis, but the actual number of polypeptides is in fact much
higher. 4
The glycoproteins of the membrane have been classified into asialo and sialoglyco-
proteins depending upon the presence of sialic acid. All the sialic acid of the membrane
appears to be bound to proteins. 5 These glycoproteins comprise about 10% of the total
protein of human erythrocyte membrane and their sialic acid residues are responsible for
most of the negative charge on the cell surface. Cell-surface carbohydrates may be
involved in the stability of the cell and also contribute to the specificity of blood group
substances. 6 In human erythrocytes, there are about 20 million sialic acid molecules per
cell. 7,s
The human erythrocyte membrane, after solubilization in 1% SDS, gives four PAS-
stainable bands, PAS-1, PAS-2, PAS-3 and PAS-4, on SDS-polyacrylamide gel electro-
phoresis. 9 These sialoglycoproteins were called glycophorins (glyco - - carbohydrate and
phorin - - to bear) by Marchesi and his group. 10 The major sialoglycoprotein constituting
about 75% of total was termed glycophorin A. 1°

Orientation The orientation of glycophorin A in the red cell membrane has been studied by several
workers. Its N-terminus was found to be exposed to the exterior by labelling the
membranes with phytohemagglutinin conjugated to ferritin. Lactoperoxidase-catalyzed
iodination of glycoproteins in intact cells and resealed ghosts clearly demonstrated the
transmembrane orientation of glycophorin A. Studies based on fingerprinting of the
cytoplasmic segment and C-terminal CNBr fragment of glycophorin A showed the C-
terminal fragment to be cytoplasmic. 11 All of these results strongly support a transbilayer
orientation of glycophorin A (Fig 1). A segment which passes through the bilayer has also
been recognized owing to its unusual sequence and by the use of photosensitive apolar
reagents. An insoluble tryptic peptide composed of 35 amino acids, of which 22 are
uncharged and form a unique sequence, appears to constitute the bilayer-embedded
domain of glycophorin A. ~2 Reincorporated glycophorin A in phospholipid vesicles
assumes the same orientation as found in intact cells in the sense that N- and C-terminal
portions are exposed to the aqueous environment and with the hydrophobic region
embedded in the lipid bilayer. The orientation, however, does not seem to be asymmetric
as is found in cell membranes, as only 50% of the sialic acid residues could be removed by
neuraminidase treatment.

Extraction and Chemical Extraction of glycophorin A from membranes requires the disruption of the lipid bilayer
Composition either by the use of detergents or organic solvents.~3 Affinity chromatography using sialic
acid-binding lectins such as lima bean lectin and wheat-germ agglutinin has also been used
for the purification of this protein.
The chemical composition of glycophorin A from various mammalian sources has been
studied. Similarities in all glycophorins are a high carbohydrate content (40-80%) which is
localized exterior to the N-terminal segment of protein, and a large amount of sialic acid.
This constant level of sialic acid at the cell surface is suggestive of some generalized
function. According to one view, during senescence, 10-15% loss in sialic acid acts as a
trigger for removal of these cells from circulation. 14 Carbohydrates are linked to the

BIOCHEMICAL EDUCATION 16(2) 1988

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Figure 1
polypeptide chain through O-glycosidic linkages either to serine or threonine residues and
N-glycosidic linkage to asparagine residues of the protein. Human glycophorin A contains
15 O-glycosidic and 1 complex N-glycosidic linkages. Both carbohydrates and protein
moiety contribute to the antigenicity of the protein and the differences in these
components account for differences in antigenic behaviour.
Amino acid compositions of glycophorins from different mammalian sources are not
identical. Marked variations in aromatic as well as basic amino acid residues are found. A
higher content of acidic amino acids as compared to basic amino acids explains the acidic
nature of glycophorin A. The composition does not show any unusual behaviour except
that the content of serine and threonine residues is higher than is typically found in
proteins. The content of hydrophobic amino acids is more or less the same ( - 3 0 % ) as is
found in various globular proteins.

Amino Acid Sequence The amino acid sequence of glycophorin A from various sources has been determined.
Human glycophorin A is made up of 131 amino acid residues with no disulfide bonds.
Leucine and glutamic acid residues at positions 1 and 5 in the M-form of glycophorin A are
replaced by serine and glycine in the N-form. An interesting feature of the primary
structure of glycophorin A is that the molecule can be divided into three distinct segments.
The first 62 amino acid residues form the N-segment which is exposed to the cell surface
and contains all the carbohydrate present in glycophorin. Abnormally high contents of
serine and threonine residues are found in this segment to which carbohydrates are linked
through O-glycosidic bonds. The C-terminal one-third of the molecule extends into
cytoplasm. This segment is rich in acidic amino acid residues with proline residues at
regular intervals. The intermediate segment, which connects N-terminal and C-terminal
segments, is predominantly made up of hydrophobic amino acid residues (Fig 2). This
region passes through the lipid bilayer. The exact length of the segment which traverses
the lipid bilayer seems to be uncertain and results obtained by different workers do not
agree. Probably this is due to the inherent uncertainty in the different techniques used in
identifying this segment. The numbers of amino acid residues present in this segment have
been reported to be in the range 20-34. This hydrophobic rich segment is suitable for
interaction with lipids and with other transmembrane proteins and glycophorins as well. It
also contains a significant number of hydrophilic amino acid residues which may be
involved in formation of salt bridges or hydrogen bonds.
Comparison of the amino acid sequence of glycophorin A from different mammalian
sources shows a striking homology in the amino acid sequence of membrane embedded
domain while there is no homology in the N-terminal region. ~5These observations suggest
that probably the hydrophobic domain of the protein has been conserved during evolution
while the N-terminal region which acts as a receptor for several ligands, has undergone
considerable changes.

BIOCHEMICAL EDUCATION 16(2) 1988

 65

• l l m - 0

+ + + + + +
I0 ~0 30 40 50 60

lm mm m m m m I

+ + +
70 80 90

+ + + +
IOO I10 120 130
Figure 2
Secondary Structure Glycophorin A exists in a mostly unordered form in dilute aqueous solutions. Schulte and
Marchesi a6 found that glycophorin A contained about 27% a-helix, 10% 13 structure and
63% unordered structure. SDS or Ammonyx-LO had little effect on the a-helical content
of glycophorin A. N-terminal and C-terminal segments are predominantly unstructured.
However, 10% 13 structure was reported in N-terminal region. Hydrophobic segment
exists exclusively in c~-helical form both in 1% SDS and ammonyx-LO. The helix is
remarkably resistant to conventional denaturing conditions. However, it undergoes
reversible unfolding in trifluoroacetic acid. 17 Infrared spectroscopic studies on glyco-
phorin integrated with liposomes revealed the major structure as either random coil or a-
helix. 18

Molecular Weight The molecular weight of glycophorin A has been determined by various techniques but
these have yielded conflicting results. By SDS-polyacrylamide gel electrophoresis, the
molecular weight was reported to be in the range 50 000-83 000 which decreased to 30 000
on heating at 100°C, representing the molecular weight of glycophorin A monomer. A
similar value (29 000) was also obtained in 1% SDS by sedimentation equilibrium. These
values are remarkably close to the value calculated from the amino acid sequence data.

Biosynthesis In normal human bone marrow basophilic normoblasts and erythroid cell stages are the
sites for glycophorin expression. In this intermediate form it contains neither galactose nor
sialic acid, is readily incorporated into microsomes and then glycosylated completely
within the cell. mRNA for glycophorin A has also been used for its synthesis.

Subunit Association Glycophorin's strong tendency to aggregate leads to the uncertainty in identifying PAS-
stainable bands under different conditions. Several factors such as protein concentration,
ionic strength, buffer composition, temperature, etc, make significant contributions to the
aggregation-disaggregation equilibrium and thus marginal differences in experimental
conditions can produce totally different patterns on SDS-polyacrylamide gels. The
aggregation of glycophorin A in aqueous media is well known 17 and persists even in
presence of detergents and denaturants. It has been shown that PAS-1 (an apparently
homogeneous protein) could be split into two protein bands when its solution was heated
to 100°C for 3 minutes in the presence of SDS before electrophoresis. PAS-1 was
proclaimed to be the dimer which was stable to SDS at ordinary temperature and comes
into equilibrium with its monomer only at high temperature. The relative concentrations
of PAS-1 and PAS-2 appear to depend on protein and SDS concentrations, and time and
temperature of incubation. Modification of one of the two methionine residues causes the
dissociation of dimer into monomer. However, when the ionic strength was increased
more than 0.25, both dimer and monomer converted into a third form, designated as PAS-
4. This shows that intermolecular aggregation occurs through hydrophobic domains of
glycophorin A. 19

Interaction with Other Several lines of evidence suggested the involvement of the hydrophobic domain of
Membrane Components glycophorin A in its interaction with lipids. However, these results are based on indirect
observations. In addition to its interactions with lipids, glycophorin A can also interact
with itself and with other proteins in the membrane which can be observed in freeze-

BIOCHEMICAL EDUCATION 16(2) 1988

66

fractured membranes. Association of glycophorin A with another transmembrane protein,

namely, band 3, was deduced from observations based on reduced rotational mobility of
band 3 in the presence of specific antibody against glycophorin A. 2°

Biological Functions At present, the exact functional role of glycophorin A is uncertain. Its structural features
likely to contribute to its biological functions include a high content of carbohydrate and
its transmembrane orientation. Due to the presence of carbohydrates, it interacts with a
number of lectins. 21 The biological importance of these interactions, if any, is obscure.
The carbohydrate moieties of glycophorin A have been implicated in MN-antigenic
activity of the protein. 22 However, according to some workers the protein part also plays
some role. 23
Glycophorin A acts as a major receptor for encephalomyocarditis virus, influenza
virus, 24 Sendai virus, M pneumoniae 25 and for Portuguese man-of-war toxin. It has also
been implicated in several red blood cell diseases such as leukopenia and thrombopenia. 26
Additionally, Tn antigen, which is a marker in human carcinoma cells is also exposed when
some of the carbohydrate residues are removed from glycophorin A. It may play a role in
the aging mechanism of the red cell as well as in binding of some important metal ions such
as Mg ++ and Ca ++ to the red cell membrane. 6 Binding of these metal ions is important
since glycophorin A is involved in cytoplasmic Ca++-induced morphological changes in
red blood cells. It is also a receptor for enterotoxigenic E c o l i . 26 An interesting finding is
that erythrocytes deficient in glycophorin A resist invasion by the malarial parasite. 27 In
fact, a protein from malarial parasite has been isolated which combines specifically with
glycophorin A, 2s proving the receptor function of this glycoprotein for malarial parasite
and in this interaction sialic acid residues of glycophorin A are involved since this
interaction is inhibited by sialic acid. z9

References IBranton, D, Cohen, C M and Tyler, J (1981) Cell 24, 24-32

2Gratzer, W B (1981) Biochem J 198, 1-8
3Lux, S E and Glader, B E (1981) in 'Hematology of Infancy and Childhood' (Nathan, D G and Oski, F A
editors) second edition, pp 456-565, Philadelphia
4Thompson, S, Rennie, C M and Maddy, A H (1980) Biochim Biophys Acta 600, 756-768
5Abraham, G and Low, P S (1980) Biochim Biophys Acta 597, 285-291
6Viitala, J and Jarnefelt, J (1985) Trends Biochem Sci 10, 392-395
7Schauer, R (1982) Adv Carbohydrate Chem Biochem 40, 131-234
8Schauer, R (1985) Trends Biochem Sci 10, 357-360
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l°Furthmayr, H, Tomita, M and Marchesi, V T (1975) Biochem Biophys Res Commun 65, 113-121
1~Bretscher, M S (1975)J Mol Biol 98, 831-833
12Tomita, M, Furthmayr, H and Marchesi, V T (1978) Biochemistry 17, 4756-4770
t3Furthmayr, H and Marchesi, V T (1983) Methods Enzymol 96, 268-280
~4Lutz, H U and Fehr, J (1979) J Biol Chem 254, 11177-11180
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l~Schulte, T H and Marchesi, V T (1979) Biochemistry 18,275-280
17Cramer, J A, Marchesi, V T and Armitage, I M (1980) Biochim Biophys Acta 595, 235-243
lSRichard, M, Richard, D A, Terry, G and Henry, M H (1984) Biochemistry 23, 1498-1504
~Daman, M E, Batstone-Cunningham, R L, Hardy, R E and Dill, K (1983) Int J Biol Macromol 5,371-373
2°Nigg, E A, Bron, C, Girardit, M and Cherry, R J (1980) Biochemistry 19, 1887-1893
21Marchesi, V T, Furthmayr, H and Tomita, M (19761 Ann Rev Biochem 45,667-698
22Batstone-Cunningham, R L, Hardy, R E, Daman, M E and Dill, K (19831 Biochim Biophys Acta 746. 1-7
23Furthmayr, H, Metaxas, M and Metaxas, B M (1981) Proc Natl Acad Sci USA 78, 631-635
24Burness, A T H and Pardoe, I U (1983) J Gen Virol 64, 1137-1148
25Kahane, I, Bahai, M, Razin, S and Bredt, W (1980) Infect lmmun 30, 628-634
2OLindahl, M and Wadstroem, T (1984) Vet Microbiol 9,249-257
27pasvol, G, Wainscat, J S and Weatherall, D J (1981) Nature 297, 64-66
2SJungery, M, Boyle, D, Patel, T, Pasvol, G and Weatherall, D J (1983) Nature 301,704-705
2'~Vanderberg, J P, Gupta, S K, Schulman. S, Oppenheim, J D and Furthmayr, H (1985) Infect lmmun 47,
201-210

* Author's present address: Department of Biochemistry, University of Kashmir, Srinagar 190006, India.

BIOCHEMICAL EDUCATION 16(2) 1988