REVIEWS

37
38
39
40

(1995) Biechem. Biophys. Res. Commun. 214,

576-581
Muscatelli, F. et al. (1994) Nature 372,
672-676
Bardoni, B. et al. (1994) Nat. Genet. 7,497-501
King, V. et al. (1995) Curr. Biol. 5, 37-39
Ryner, L. C. and Swain, A. (1995) Cell 81,
483-493

TIBS 2 1 - AUGUST1996
41 Sockanathan, S., Cohen-Tannoudji,M.,
Colignon, J. and Lovell-Badge,R. (1993) Genet.
Res. 61, 149
42 Kamachi, Y. et al. (1995) EMBO J. 14,
3510-3519
43 Denny, P. et al. (1992) EMBO J. 11, 3705-3712
44 van de Wetering, M., Oosterwegel, M.,
van Norren, K. and Clevers, H. (1993) EMBO J.

12, 3847-3854
45 Affara, N. A. et al. (1993) Nucleic Acids Res. 2,

785-789
46 Harley,V. R. et al. (1992) Science 255,453-456
47 Braun, A. et al. (1993) Am. J. Hum. Genet. 52,

578-585
48 Zeng, Y. et al. (1993) J. Med. Genet. 30, 655--657
49 Pou~at,F. et al. (1994) Hum. Mutat. 3, 200-204

Thus, this protein seemed to be an

exception to the glycosylation rule.
However, more recent work has
shown that this is not the case. There is
now evidence that the Rh-polypeptides
form part of a large glycopolypeptide
complex, including among others the
Rh50 glycoproteins and the LandsteinerWiener (LW) blood group glycoprotein
(intercellular adhesion molecule 4)
(Refs 13, 14). This situation is similar to
that of ~2-microglobulin in class I transMost proteins presented at the external surface of mammalian cells conplantation antigens, where the unglycotain carbohydrate. The reason for this is not fully understood, but recent
sylated protein associates with the
work has shown that such carbohydrate has two major functions. Inside
heavy chains of the transplantation
the cell, it helps proteins fold and assemble correctly in the endoplasmic
antigens. The importance of the associreticulum, and it might also act as a signal for the correct migration of
ation of the Rh-polypeptide with other
glycoproteins. Outside the cell, it provides specific recognition structures
glycosylated proteins is underscored by
for interaction with a variety of external ligands.
the fact that it has not yet been possible to express the Rh cDNA in any
mammalian cell expression system.
EARLY WORK IN the 1960s on the mor- encoding them have been characterized,
In a recent survey of the SWlSS-PROT
phology of mammalian cells showed cloned and sequenced, this proposal database (release 33.0, April 1996) we
that the external surface of the plasma has turned out to be largely correct.
found 1823 complete animal protein
membrane is rich in carbohydrate,
Most glycoproteins are N-glycosylated, entries with reported extracellular feawhereas the inner side is devoid of con- i.e. they contain asparagine-linked oligo- tures, of which 1671 (91.7%) were
ventional-type oligosaccharides 1. Until saccharides located at the peptide se- described as 'glycoproteins' in the keyrecently, it was unclear whether the quence(s) NxS/T (where x stands for word field; 1630 of these 1671 contained
carbohydrate was confined to few or any amino acid except for proline) at the the N-glycosylation peptide sequence
many different cell surface glycoconju- external aspect of the membrane. Some NxS/T. The remaining 8.3%, representing
gates 2, and the development of radio- membrane proteins are O-glycosylated, 152 potentially non-glycosylated plasmaactive techniques in particular has al- with the carbohydrate chains attached membrane proteins, contained 116 prolowed the carbohydrate portions of to serine or threonine residues, which are teins with multiple transmembrane reexposed cell surface glycoproteins and often clustered in distinct regions of the gions, 15 proteins that are known to
glycolipids to be labeled3,4. It was then polypeptides. Most O-glycosylated pro- associate with glycosylated subunits in
possible to study the larger number of teins also contain one or more N-glyco- a complex such as CD3 chains and the
glycoconjugates specifically presented sidic oligosaccharides 7. The importance Rhesus D-polypeptide, and seven that
at the surface of various cells. Also, it and requirements for O-glycosylation contained 5-38 potential N-glycosylation
sites, i.e. polypeptides highly likely to be
became apparent that cell membranes have yet to be elucidated.
glycosylated, yet not marked as glycocontain a multitude of glycoproteins,
proteins. This leaves only 14 sequences
many more than previously thought 5,6.
Exceptions to the rule
Early searches of the literature for un- (0.7%) that are candidates for nonIn 1976, after studying human erythrocytes and other cells, it was proposed glycosylated surface proteins in mam- glycosylated, non-complexed plasma
that cell surface proteins are always malian cells met with little success. How- membrane proteins with a single transglycoproteins s. A similar proposal was ever, in 1982, the human red cell Rh(D) membrane domain.
In another survey, we assessed
made independently by Bretscher and (Rhesus) protein, with an apparent molRaff2. As more and more mammalian ecular weight of 30-32 kDa, was identi- whether this high representation of the
cell membrane proteins and the genes fied s,9. Importantly, no evidence for the N-glycosylation tripeptide sequence is
presence of carbohydrate was found 1~ more than should be found by chance
and subsequent cloning and sequencing alone*. To do this, we extracted all sec. G. Gahmberg and M, Tolvanen
of its cDNA and that of other polypep- quence features marked as extracelluare at the Departmentof Biosciences,
tides belonging to the Rh-blood group lar domains from animal proteins in
Division of Biochemistry, P.O.Box 56,
system, showed that they do indeed SWlSS-PROT 33.0, which resulted in
Viikinkaari 5, FIN-O0014,Universityof
lack N-glycosylation sequences u32. 4259 stretches of sequence from 1933
Helsinki, Finland.

Why mammalian cell surface

proteins are glycoproteins

Carl G. Gahmberg and Martti Tolvanen

308

9 1996, Elsevier Science Ltd

PII: S0968-0004(96) 10034-7

REVIEWS

TIBS 2 1 - AUGUST1996

proteins. By chance alone, this material

is expected to contain 3343 potential
N-glycosylation sites, but it actually contains 6725 sites, an over-representation of
more than twofold. This high frequency
of potential N-glycosylation sites in extracellular domains might reflect recent
gene duplication and shuffling events as
well as a possible evolutionary pressure
to enrich for glycosylation sites.
For comparison we analysed all reported c~oplasmic sequences in animal proteins in the same way. This material contained 1656 occurrences of
the N-glycosylation sequence versus
1727 expected occurrences.

Glycosylation is essential in the

endoplasmic reticulum
It has been difficult to understand why
the glycosylation machinery, especially
that of N-glycosylation, is so remarkably
complex is. Is Nature wasteful? Briefly,
dolichol-containing glycolipids are used
to donate the initial glucose3-mannose 9N-acetylglucosamine 2 oligosaccharide to
asparagine residues in the lumen of the
endoplasmic reticulum ~R) ~ig. 1). The
peripheral glucose residues are subsequently removed by cr
I
and II, followed by c~-mannosidase removing e-mannosyl residues; the final
oligosaccharides ~ig. 2) are formed by
the action of various glycosyltransferases. If the protein is still unfolded after
removal of the glucose residues, reglucosylation takes place by the ER enzyme UDPglucose: glycoprotein glucosyl
transferase 16 ~ig. 1). Importantly, this
enzyme acts only on denatured or unfolded substrates. The subsequent
glycosylation reactions take place during the transport of the glycoproteins
through the ER and Golgi apparatus en
route to the cell surface.
Using glycosylation mutants, Stanley
and co-workers have convincingly shown
that, whereas N-glycosylation is essential for the viability of cells, hybrid and
complex oligosaccharides are not an
absolute necessity ~7. They are, however, required for the whole organism
to develop normally ]7. Thus, mice with
inactivated N-acetylglucosamine transferase ] die at mid-gestational age. This
*If the probability of a given tripeptide being an
N-glycosylation sequence (N-gs) is Prigs, a sequence
of n residues is considered as n - 2 tripeptides and
a binomial distribution is assumed for the number
of glycosylation sites in this population of tripeptides, the expectation value of the number of glycosylation sites, p. = P~s
for N sequences of
total length L. The assumption of a binomial distribution is fair when P~gs is small, but it gives a slight
overestimation for the number of sites.

transferase is the key enzyme

GIc
in the initiation of complexGlucosidase I ~
I c~1-2
and hybrid-type N-linked
GIc
UDP-GIc:glycoprotein
oligosaccharide biosynthesis.
I c~1-3 / glucosyltransferase
It is still poorly underGlucosidase II
G]c ~-/
stood how the biosynthesis
i o~1-3
of membrane oligosacchaMan
Man
Man
rides is regulated. Evidently,
{zl-2 I
~1-2l
I {x1-2
the activity of the glycosyl
Man
Man
Man
~1-3\
/ (zl-6
~1-2l
transferases and also the
availability of nucleotide sugMan
Man
/ c~1-6
~1 - 3 \
ars are of key importance.
Man
The following example might
I 131-4
illustrate this: human En(a-)
GIcNAc
red cells lack the gene that
Ipl-4
encodes for glycophorin A,
GIcNAc
the major er~hroc~e sialoIP
glycoprotein 18, but the cell
compensates for this loss by
making a larger-than-normal
Band 3 (anion transport proFigure 1
Structure of untrimmed N-glycosidic oligosaccharide.
tein) oligosaccharide of the
This oligosaccharide is later modified in the endoplaspolylactosamine type. Indimic reticulum (ER) by removal of the peripheral gluviduals heterozygous for
cose
by glucosidase I and of the other two glucose
the glycophorin A defect
residues by glucosidase I1. The innermost glucose
(containing 50% the normal
residue is essential for the interaction with calnexin
amount of glycophorin A)
and calreticulin. UDPglucose: glycoprotein glucosylsynthesize a Band 3 oligotransferase, which acts on unfolded proteins, can
restore it. Abbreviations used: GIc, D-glucose; GIcNAc,
saccharide with a size beN-acetyl-o-glucosamine; Man, D-mannose.
tween that of normal and
En(a-) cells 18. Although the
carbohydrate chains in this example carbohydrate portions (specifically to
are profoundly different, the findings in- the innermost glucose residue) of newly
dicate that there is competition for acti- synthesized glycoproteins is of fundavated sugars, and the lack of one major mental importance ~9-23. Whereas the
polypeptide acceptor gives others the biosynthesis of the polypeptides is relatively quick, their subsequent folding
opportunity to get more carbohydrate.
and the association of subunits are
Calnexin and calreticulin
much slower processes. By binding to
The realization that calnexin and cal- newly synthesized glycopolypeptides,
reticu]in, both ER proteins and molecular calnexin anchors the polypeptides in
chaperones, recognize and bind to the the ER until they have achieved their
SA
(z2-3 I

Gal
#1-41
GIcNAc
p1-2 I
Man
o~1-3 ~

Gal
pl-41
GIcNAc
pl-21
Man

(Man)
o~1-2 I

(Man)
(z.1-2 I

c~1-3~

Man

/o~1-6

c~1-3

Man
1#1-4
GIcNAc
1~1-4
Fuc c~1-6 GIcNAc

(a)

(Man)
c~1-21
Man

(Man)
1~1-2
Man
/'od-S

Man
/cr

Man
I P1-4
GIcNAc
IPl-4
GIcNAc

(b)
Figure 2

(a) Complex-typeand (b) high-mannose-type N-glycosidic oligosaccharides. Many complex-type

oligosaccharides are important for cell-ligand interactions, but also high-mannose-type
structures can function as lectin and microbial ligands. Abbreviations used: Fuc, L-fucose; Gal,
D-galactose; GIc, D-glucose;GIcNAc, NacetyI-D-glucosamine; Man, D-mannose; SA, sialic acid.

309

REVIEWS

TIBS 2 1 - AUGUST1996

SA
1.2-6
SA ~_~3Gal ~1-3 GIcNAc ~

SA.2-3 Gal ~1-4GIcNAc

Ipl-6
SA ~ 3 Gel pl-3 GIcNAc ~

Figure 3
Structures of Oglycosidic oligosaccharides.
O-glycosidic oligosaccharides are found
in mucin-type glycoproteins, often in large
numbers. The multi-valency is probably
important in increasing their avidity for
various interactions. A large number of
sugar chains might also have protective
functions against proteolysis. Abbreviations used: Gal, D-galactose; GalNAc,
N-acetyl-D-galactosamine; SA, sialic acid.

correct folding conformation and, where

appropriate, associated with other polypeptides into supramolecular complexes.
However, if deglycosylation is prevented by glucosidase inhibitors like
castanospermine and deox3mojirimycin,
the proteins remain bound to calnexin
and calreticulin, their transport is delayed in the ER and the proteins are
subsequently degraded. Treatment with
tunicamycin, which completely inhibits
the N-glycosylation machinery, blocks
calnexin and calreticulin interactions
with their substrates, resulting in incorrectly folded polypeptides, protein
degradation and mislocalization of
newly synthesized proteins. These findings do not exclude the fact that other
molecular chaperones such as BiP
Oinding protein) are also important.
In addition to these glycoprotein
chaperones, a few proteins have been
identified that can act as intracellular

TA SI"ING
310

lectins (chaperones?) later in the bioHowever, one could also interpret the
synthetic pathway. These include the results to suggest that the oligosacERGIC-53 protein and VIP36, which are charide of the N-glycosylated tripeptide
homologous to plant lectins and can bind in fact gives the molecule a positive
high-mannose type oligosaccharides 24.
migration signal. Support for such a
Little is known about the importance function of oligosaccharides has been
of O-glycosylation, but because those cell obtained using a recombinant chimeric
surface proteins that are O-glycosylated membrane protein based on rat growth
Gig. 3) are often heavily so, they in- hormone. The soluble protein is norfluence strongly the three-dimensional mally not glycosylated, and when a
structure of these mucin-type membrane membrane-anchored form was engiglycoproteins. Furthermore, O-glycosyl- neered it remained intracellular 27. Howation efficiently protects such proteins ever, the introduction of N-glycosylation
from proteolysis. Olinked oligosaccha- sites resulted in its glycosylation, and
rides might extend surface proteins into presentation at the cell surface. Interrod-like structures, which could be estingly, the same glycosylated protein
important in mediating or preventing showed apical sorting in Madin-Darby
cell-cell interactions. Moreover, carbo- canine kidney cells, whereas the
hydrate--carbohydrate interactions 25can non-glycosylated growth hormone was
be important during various stages of secreted from both the apical and
glycoconjugate biosynthesis, but little basolateral membranes 28.
is lmown about their significance.
Oligosaccharide function at the cell surface
Exit from the cell
A number of carbohydrate-specific
It has been argued that plasma mem- functions occur at the cell surface, many
brane glycoproteins do not need any of which involve recognition events.
'exit signal', but migrate by bulk flow These include cell adhesion, interactions
without specific retention. This would between cells and soluble ligands, and
mean that glycoproteins remain at the between cells and various microbes.
same concentration in the transport Such interactions often involve carbovesicles during intracellular migration. hydrate-binding lectins.
Wieland et al. 26 used an 1251-labeled,
Although the presence of mammalian
formylated N-glycosylation consensus se- cell lectins has been known for a numquence tripeptide (NYS), which was taken ber of years, their importance was iniup by intact cells, glycosylated and tially largely neglected. This was owing
then secreted in 5-10 min. Whether this to the fact that rather few specificities
sequence employed the physiological were found, and the lectins were conmigration route through the Golgi appa- sidered important only in a few special
ratus is not known. The fast, intracellu- cases. Only a few examples of specific
lar migration by this simple molecule carbohydrate-plasma membrane glycowas interpreted to mean that secretion protein interactions can be mentioned
involves neither any specific retention here, and the reader is referred to more
signals, nor any signals that would extensive recent reviews for additional
reading29,30.
target the tripeptide to the cell surface.
The finding of a hepatoc~e lectin for
de-sialylated serum glycoproteins opened
the field 31. This lectin, presented at the
surface of liver epithelial cells, binds to
serum glycoproteins that have lost terminal sialic acids, resulting in exposure
of galactosyl/N-acetylgalactosaminyl residues. A corresponding macrophage activity involving binding of mannosecontaining proteins was subsequently
described. But more widespread interest in the importance of glycoprotein
oligosaccharides arose when leukoc~e
adhesion was found to involve initial
carbohydrate-ligand interactions. Several studies have shown that 'rolling' of
neutrophils and monocytes along capillary endothelia results from reversible
interactions between selectins presented
on endothelial cells and leukocytes, and

byDr
nWnsfHra;aBuSx

TIBS 2 1 -

REVIEWS

AUGUST1996

specific carbohydrate structures on the

counter-ligand cells. These selectins 32,33
bind to sialyl Lex, sialyl Lea (Fig. 4), sulfatides and related carbohydrate structures. L-selectin is found on nucleated
blood cells, whereas E- and P-selectins
are mainly found on endothelial cells
(P-selectins are also found on platelets).
Interestingly, many selectin receptors
are cell surface proteins rich in O-glycosidic oligosaccharides. These include
glycosylation-dependent cell adhesion
molecule 1 (GlyCAM-1), CD34, mucosal
vascular addressin cell adhesion molecule 1 (MAdCAM-1) and P-selectin
glycoprotein ligand 1 (PSGL-1) (Ref. 34).
The selectin-mediated 'rolling' phenomenon is essential for subsequent
stronger binding, and for tissue migration of leukocytes involving integrins
and ligands of the immunoglobulin
superfamily [intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM)].
Additional leukocyte carbohydratebinding proteins have recently been described; among them is the B lymphocyte surface protein CD22, which binds
~2-6 sialic acid-galactosyl structures,
and the macrophage sialo-adhesin with
specificity for u2-3 sialic acid-containing
glycoconjugates35. Of particular interest
is the binding of sperm to zona pellucida
glycoproteins through cr
residues 33, although this field is still controversial and is probably more complex
than currently thought.
However, it is anticipated that this list
of important surface glycoproteins will
grow over the next few years, along with
the elucidation of their binding specificities. In fact, the carbohydrate structures of only a few cell surface glycoproteins are currently known. This is, of
course, largely owing to the fact that it
is difficult to purify native glycoproteins
from mammalian cell membranes in sufficient quantities. However, it is already
evident that cell surface proteins originating from the same cell can have very
different oligosaccharide compositions.
On one hand, CD45, a major cell surface
glycoprotein of leukocytes, which contains tyrosine phosphatase activity on
the inner aspect of the membrane, is
enriched in ~2-6 sialic acid-galactosyl
residues. On the other hand, the leukocyte CD11/CD18 integrins contain ct2-3
sialic acid-galactose structures, but no
~2-6 sialic acid. These facts are reflected
in their binding specificities. Thus, CD22
binds to CD45 (and to some other proteins), but not to the integrins, whereas
the opposite is true for E-selectin36.

Currently, a large number

of binding specificities of
cell surface carbohydrate
are known for unphysiological ligands such as bacteria,
viruses, toxins etc. Although
clinically important, these
activities are obviously not
physiological, but reflect
evolutionary adaptations of
various microbes.

Concluding remarks

(a)

SA

] q.2.--3

(b)

Gal

Fuc

I~1--4

Fuc ~-3 GIcNAc

I c~1--4

SA ~2-3 Gal ~-3 GIcNAc

Sialyl Le x

Sialyl Lea

Rgure 4
Structures of the selectin ligands (a) sialyl Lex and
(b) sialyl Lea. These oligosaccharides are important
ligands for selectins and are found as terminal
structures in several different oligosaccharides, both
in glycoproteins and glycolipids. Abbreviations used:
Fuc, L-fucose; Gal, D-galactose; GIcNAc, N-acetyl-Dglucosamine; SA, sialic acid.

The main purpose of this

short review is to point out
the requirement of cell surface proteins for carbohydrate. For successful biosynthesis, folding and intracellular migration, cell
surface proteins (and most secreted
proteins) need to be glycosylated or, we
postulate, linked to a glycosylated
protein. The few exceptions to this rule
include cell surface proteins that span
the membrane several times. Intracellular lectins such as calnexin and calreticulin are responsible for retaining
glycoproteins on the ER until the time is
right for their migration to the membrane, but carbohydrates might also be
important as positive plasma membrane
signals. However, many of the specific
functions of mature cell surface glycoprotein oligosaccharides are physiologically important, but not always
essential for the protein function.
Although this research area is still
largely in its infancy, it is rapidly developing. Whether most (or all) carbohydrate structures present at the cell
surfaces eventually will turn out to
be important in interactions with surrounding cells, soluble ligands and
infecting microbes remains to be seen.

Acknowledgements
The original research from the authors' laboratory was supported by the
Academy of Finland, the Sigrid Jus61ius
Foundation and the Finnish Cancer
Society. We thank K. Simons (European
Molecular Biology Laboratory) for useful comments on the manuscript, and
Yvonne Heinil~ for secretarial assistance.

References
1 Rambourg, A., Neutra, M. and LeBIond, C. P.
(1966) Anat. Rec. 154, 41-71
2 Bretscher, M. S. and Raft, M. C. (1976) Nature
258, 43-49
3 Gahmberg, C. G. and Hakomori, S. (1973)
J. Biol. Chem. 248, 4311-4317
4 Gahmberg, C. G. and Andersson, L. C. (1977)
J. Biol. Chem. 252, 5888-5894
5 Gahmberg, C. G. (1976) J. Biol. Chem. 251,
510-515
6 Gahmberg, C. G., H~yry, P. and Andersson, L. C.

(1976) J. Cell Biol. 68, 642-653

7 Kornfeld, R. and Kornfeld, S. (1976) Annu. Rev.
Biochem. 45, 217-237
8 Gahmberg, C. G. (1982) FEBS Lett. 140, 93-97
9 Moore, S., Woodrow,C. F. and McClelland,D. B. L.
(1982) Nature 295, 529-531
10 Gahmberg, C. G. (1983) EMBO J. 2, 223-227
11 Cherif-Zahar, B. et al. (1990) Proc. Natl. Acad.
Sci. U. S. A. 87, 6243-6247
12 Avent, N. D., Ridg~vell,K., Tanner, M. J. A. and
Anstee, D. J. (1990) Biochem. J. 271, 821-825
13 Moore, S. and Green, C. (1987) Biochem. J.
244, 735-741
14 Agre, P. and Cartron, J-P. (1992) In Protein
Blood Group Antigens of the Human Red Cell

15
16

17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33

34
35
36

(Agre, P. and Cartron, J-P., eds), pp. 20-52,

Johns Hopkins University Press
Kornfeld, R. and Kornfeld, S. (1985) Annu. Rev.
Biochem. 54, 631-664
Parodi, A. J., Mendelzon,D. H., Lederkremer,G. Z.
and Martin-Barrientos, J. (1984) J. Biol. Chem.
259, 6351-6357
Stanley, P. and Ioffe, E. (1995) FASEB J. 9,
1436-1444
Gahmberg, C. G. et al. (1976) J. Biol. Chem.
251, 6108-6116
Degen, E. and Williams, D. B. (1991) J. Cell
Biol. 112, 1099-1115
Ou, W-J., Cameron, P. H., Thomas, D. Y. and
Bergeron, J. J. M. (1993) Nature 364, 771-776
Hammond, C., Braakman, I. and Helenius, A.
(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 913-917
Hebert, D. N., Foellmer, B. and Helenius, A.
(1995) Cell 81, 425-433
Fiedler, K. and Simons, K. (1995) Cell 81,
309-312
Fiedler, K. and Simons, K. (1994) Cell 77,
625-626
Eggens, I. et al. (1989) J. Biol. Chem. 264,
9476-9484
Wieland, F. T., Gleason, M. L., Serafini, T. A. and
Rothman, J. E. (1987) Cell 50, 289-300
Guan, J-L., Machamer, C. E. and Rose, J. K.
(1985) Cell 42, 489-496
Scheiffele, P., Per~nen, J. and Simons, K.
(1995) Nature 378, 96-98
Paulson, J. C. (1989) Trends Biochem. Sci. 14,
272-276
Varki, A. (1993) Glycobiology 3, 97-130
Ashwell, G. and Harford, J. (1982) Annu. Rev.
Biochem. 51, 531-554
Bevilacqua, M. P. and Nelson, R. M. (1993)
J. Clin. Invest. 91, 379-387
Gahmberg, C. G., Kotovuori, P. and Tontti, E.
(1992) Acta Pathol. Microbiol. Immunol. Scand.
100, 39-52
McEver, R. P., Moore, K. L. and Cummings, R. D.
(1995) J. Biol. Chem. 270, 11025-11028
Kelm, S. et al. (1994) Curr. Biol. 4, 965-972
Kotovuori, P. et al. (1993) Glycobiology 3,
131-136

311