Biochimica et Biophysica Acta 1473 (1999) 4^8

www.elsevier.com/locate/bba

On the frequency of protein glycosylation, as deduced from analysis of

the SWISS-PROT database1
Rolf Apweiler
a

a;

*, Henning Hermjakob a , Nathan Sharon

EMBL Outstation Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK
b
Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel
Received 15 February 1999; accepted 19 May 1999

Abstract
The SWISS-PROT protein sequence data bank contains at present nearly 75 000 entries, almost two thirds of which
include the potential N-glycosylation consensus sequence, or sequon, NXS/T (where X can be any amino acid but proline)
and thus may be glycoproteins. The number of proteins filed as glycoproteins is however considerably smaller, 7942, of which
749 have been characterized with respect to the total number of their carbohydrate units and sites of attachment of the latter
to the protein, as well as the nature of the carbohydrate-peptide linking group. Of these well characterized glycoproteins,
about 90% carry either N-linked carbohydrate units alone or both N- and O-linked ones, attached at 1297 N-glycosylation
sites (1.9 per glycoprotein molecule) and the rest are O-glycosylated only. Since the total number of sequons in the well
characterized glycoproteins is 1968, their rate of occupancy is 2/3. Assuming that the same number of N-linked units and rate
of sequon occupancy occur in all sequon containing proteins and that the proportion of solely O-glycosylated proteins (ca.
10%) will also be the same as among the well characterized ones, we conclude that the majority of sequon containing proteins
will be found to be glycosylated and that more than half of all proteins are glycoproteins. 1999 Elsevier Science B.V. All
rights reserved.
Keywords: Glycosylation; Glycoprotein ; Database

Glycosylation is a common and highly diverse coand post-translational protein modication reaction.
Perhaps because almost all proteins of human serum
and of hen egg-white are glycosylated [1], as are
those of animal cell membranes [2], the sweeping
statement has been made that `most proteins are glycoproteins' [3,4]. The recent development of computerized protein sequence data banks allows us to put
this statement to a quantitative test. Here, we present

* Corresponding author..
1
Dedicated to Prof. Akira Kobata and Prof. Harry Schachter
on the occasion of their 65th birthdays.

the results of such an attempt, based on an analysis

of the SWISS-PROT database [5]. This data bank is
manually curated and strives to provide a high quality of annotation, with a minimal level of redundancy and a high level of integration with other biomolecular databases. It is thus more reliable than the
supplementary computer-annotated TrEMBL data
bank that contains translations of all protein coding
sequences in the EMBL nucleotide sequence database which are not yet in the SWISS-PROT and is
therefore much larger than the latter.
In almost all glycoproteins, the carbohydrate units
are attached to the protein backbone either by N- or
O-glycosidic bonds or by both types of linkage. The

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R. Apweiler et al. / Biochimica et Biophysica Acta 1473 (1999) 4^8

Fig. 1. Frequency of occurrence of sequons and carbohydrate units in the 749 well characterized glycoproteins listed in the SWISSPROT database by the end of 1998. Sequons per glycoprotein (A), and carbohydrate units in N-glycoproteins (B), O-glycoproteins
(C) and N-,O-glycoproteins (D).

N-glycosidic bond is always to the amide of an asparagine that is part of the consensus sequence NXS/
T, or `sequon', where X can be any amino acid except proline. The sequons are often referred to as
`potential glycosylation sites', since, for reasons that
are not understood, in not all of these, the asparagine is glycosylated. No consensus sequences for
O-glycosylation seem to exist.
The SWISS-PROT database contained by the end
of 1998 (release 36, including updates to 01/11/98)
74 988 entries. Potential N-glycosylation sites were
identied 151 993 times in 48 636 sequences, an average of 3.1 per protein. In 26 352 protein sequences,
such sites are absent, showing that about one third of
the proteins cannot be N-glycosylated. Examination
of the TrEMBL entries leads to similar conclusions
(Table 1).
The number of proteins in SWISS-PROT that
have been led as glycoproteins is relatively small,

namely 7942 (10.6% of the total). In this database,

a protein is labelled with the keyword `GLYCOPROTEIN' only when it is beyond reasonable doubt
that it is really glycosylated, even if information on
the nature of its carbohydrate units and their linkage
to the protein is lacking, as is the case for most of
these glycoproteins. Detailed annotation on the glycosylation of these substances is done by the following format: `FT CARBOHYD 6 position s 6 poTable 1
Protein entries and sequons in SWISS-PROT and TrEMBL databases by the end of 1998
Database

SWISS-PROT

TrEMBL

Number of entries
Entries containing NXS/T sequon

74 988
48 636
64.9%
151 933
3.12

156 187
107 551
68.9%
394 483
3.66

Number of sequons
Sequon/sequon containing entry

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R. Apweiler et al. / Biochimica et Biophysica Acta 1473 (1999) 4^8

Fig. 2. Amino acid residues per sequon in all well characterized glycoproteins (A) and per real glycosylation site in the N-, O- and
N-,O-glycoproteins of the same group (B, C and D, respectively).

sition s 6 description s '. For example, a glycoprotein with a N-acetylgalactosamine at position 34 will
be annotated as `FT CARBOHYD 34 34 N-ACETYLGALACTOSAMINE'.
As a thorough biochemical characterization of

most of the glycoproteins is lacking, numerous 6 description s tags contain the strings `BY SIMILARITY', `PROBABLE' or `POTENTIAL'. These denote that no biochemical characterization of the
glycosylation site(s) is available. For the purpose of

Table 2
Potential and real glycosylation sites in the 749 well characterized glycoproteins listed in the SWISS-PROT database by the end of
1998

Potential N-glycosylation
sites (sequons)
Real glycosylation sites
Real N-glycosylation sites
Real O-glycosylation sites

Glycoproteins with at
least one biochemically
characterized (`real')
glycosylation site

Glycoproteins with
at least one real
N-glycosylation site
and at least one real
O-glycosylation site

Glycoproteins with
at least one real
N-glycosylation site
and no real
O-glycosylation site

Glycoproteins with
at least one real
O-glycosylation site
and no real
N-glycosylation site

Sites

Entries

Sites

Entries

Sites

Entries

Sites

2 066

697

289

80

1 679

582

98

35

1 965
1 279
686

749
662
167

556
238
318

80
80
80

1 041
1 041
0

582
582
0

368
0
368

87
0
87

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Entries

R. Apweiler et al. / Biochimica et Biophysica Acta 1473 (1999) 4^8

Fig. 3. Origin of the 749 well characterized glycoproteins in

SWISS-PROT.

the present study, we denote as `real' glycosylation

sites only those sites where the 6 description s tag
does not contain any of the above strings. We consider the glycosylation annotation of a SWISSPROT entry to be based on biochemical characterization if it contains at least one `real' (or occupied)
glycosylation site.
It should be noted that at present, at least one
third of all proteins in SWISS-PROT lack any biochemical characterization and 12 921 are hypothetical
proteins, not yet proven to exist.
Furthermore, the proportion of biochemically
characterized proteins is getting smaller due to the

increasing number of predicted proteins derived

mainly from the fully sequenced genomes of dierent
organisms.
Of the 7942 listed glycoproteins, 1295 (16%) are
devoid of N-glycosylation sites, while 6647 (84%)
are N- (or N-,O-)glycoproteins. The latter contain a
total of 33 550 potential N-glycosylation sites, on
average 4.6 per molecule, which is more than that
found for all proteins containing such sites. Only a
small fraction of these, namely 749, have been thoroughly characterized with respect to their glycosylation patterns and complete information is available
as to the number and types of carbohydrate units per
molecule (Table 2 and Fig. 1). The majority of these
glycoproteins (83%) is from animals, while the proportion of those isolated from other sources is relatively small, 12% from plants, including fungi, 3%
from viruses and 2% from microorganisms (bacteria
and protozoa) (Fig. 2).
Of the 749 well characterized glycoproteins, 662
are N- or N-,O-linked, containing in total 1968 sequons. This is an average of three potential N-glycosylation sites per molecule of sequon containing protein, which is slightly lower than found for all sequon
containing proteins (Table 1). Of the sequons in the
well characterized glycoproteins, on average 1.9 per
molecule (i.e. about 2/3) are occupied. Typically, one
or two N-glycosidic carbohydrates are found in a
glycoprotein (Fig. 1B,D). They average 1.8 U per
N-linked glycoprotein and 3.0 U per N-,O-linked glycoprotein (Table 2), but their number may be as high
as 24 per molecule, as in gp120 of HIV [6]. The
number of O-linked units in the glycoproteins is
larger (Fig. 1C,D), averaging four per glycoprotein
(Table 2), with as many as 96 carbohydrate units
found in polysialoglycoproteins of Salmon sh eggs

Table 3
Spacing of sequons and real glycosylation sites in the 749 well characterized glycoproteins
Amino acid per
Minimum
Maximum
Mean
Median
Range
S.D.
a

Sequon

Real N-site

Real O-site

Real N+O-sitea

18.00
1 669.00
159.04
121
1 651.00
164.07

23.11
3 412.00
249.92
144
3 388.89
348.09

3.10
2 813.00
269.90
167
2 809.90
338.83

3.10
3 412.00
231.66
133
3 408.90
335.80

In N-,O-glycoproteins.

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R. Apweiler et al. / Biochimica et Biophysica Acta 1473 (1999) 4^8

[7]. The range of spacing of the sequons and the real

glycosylation sites is extremely wide (Fig. 3 and Table 3).
Assuming that the well characterized glycoproteins
are a representative sample of the glycoproteins
present in nature, some three quarters of all glycoproteins should be N-linked, about one tenth N-,Olinked and about one eighth just O-linked. Based on
the rate of occupancy of the sequons in the well
characterized glycoproteins, the 48 636 sequon containing proteins of SWISS-PROT should each carry
on average two N-linked carbohydrates. This is close
to the average number of such units per molecule of
well characterized glycoprotein. It is thus highly
likely that most of the sequon containing proteins
will prove to be glycosylated. To this estimate should
be added the 10% proteins expected to be O-glycosylated. We conclude therefore that more than half
of all proteins in nature will eventually be found to
be glycoproteins.

[2]

[3]

[4]

[5]

[6]

[7]

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