Proteomics 2003, 3, 363–379 363

Review
Raj K. Upreti Bacterial glycoproteins: Functions, biosynthesis
Manoj Kumar
V. Shankar and applications
Biomembrane Laboratory, Although widely distributed in eukaryotic cells glycoproteins appear to be rare in pro-
Industrial Toxicology karyotic organisms. The prevalence of the misconception that bacteria do not glyco-
Research Centre, sylate their proteins has been a subject matter of discussion for a long time. Glycocon-
Lucknow, India
jugates that are linked to proteins or peptides, generated by the ribosomal translational
mechanism have been reported only in the last two to three decades in a few prokar-
yotic organisms. Most studied prokaryotic glycoproteins are the S-layer glycoproteins
of Archeabacteria. Apart from these, membrane-associated, surface-associated,
secreted glycoproteins and exoenzymes glycoproteins are also well documented in
both, Archea and Eubacteria. From the recent literature, it is now clear that prokaryotes
are capable of glycosylating proteins. In general, prokaryotes are deprived of the cel-
lular organelles required for glycosylation. In prokaryotes many different glycoprotein
structures have been observed that display much more variation than that observed in
eukaryotes. Besides following similar mechanisms in the process of glycosylation, pro-
karyotes have also been shown to use mechanisms that are different from those found
in eukaryotes. The knowledge pertaining to the functional aspects of prokaryotic gly-
coproteins is rather scarce. This review summarizes developments and understanding
relating to characteristics, synthesis, and functions of prokaryotic glycoproteins. An
extensive summary of glycosylation that has been reported to occur in bacteria has
also been tabulated. Various possible applications of these diverse biomolecules
in biotechnology, vaccine development, pharmaceutics and diagnostics are also
touched upon.

Keywords: Bacteria / Biosynthesis / Glycoprotein / Membrane / Prokaryote / Review PRO 0371

Contents 6 Industrial potential of bacterial

glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . 376
1 Introduction and perspectives . . . . . . . . . . . . 363 7 Concluding remarks . . . . . . . . . . . . . . . . . . . . 376
2 Occurrences, location and structure 8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
of bacterial glycoproteins . . . . . . . . . . . . . . . . 364
3 Functional role of bacterial glycoproteins . . . 364
4 Glycoprotein biosynthesis . . . . . . . . . . . . . . . 372 1 Introduction and perspectives
4.1 Enzymatic glycosylation . . . . . . . . . . . . . . . . . 372
4.1.1 Role of glycosyl transferase . . . . . . . . . . . . . . 372 Prokaryotes possess an extreme variety of carbohydrates
4.1.2 Lipid-linked oligosaccharides . . . . . . . . . . . . . 373 as constituents of their cell structure. These carbohy-
4.1.3 Transient modifications . . . . . . . . . . . . . . . . . . 373 drates are either attached to the lipids or occur as a part
4.1.4 Transport across the membrane . . . . . . . . . . 373 of the elongated glycan chains. Glycoconjugates that are
4.1.5 Glycosylation in bacterial flagellins . . . . . . . . 374 linked to proteins or peptides generated by ribosomal
4.2 Nonenzymatic glycosylation . . . . . . . . . . . . . . 375 translational mechanism have long thought to be absent
5 Escherichia coli membrane glycoproteins . . . 375 from prokaryotes. Previously, glycosylation was thought
to be a phenomenon exhibited exclusively by eukaryotes
and not prokaryotes, mainly because of the short life
Correspondence: Dr. R. K. Upreti, Head, Biomembrane Divi- span of prokaryotes and the lack, in prokaryotes, of the
sion, Industrial Toxicology Research Centre, P.O. Box No. 80,
cellular organelles shown to be required in eukaryotic
M. G. Marg, Lucknow-226 001, India
E-mail: [email protected] glycoprotein biosynthesis. Following the first report on a
Fax: +91-522-2228227 glycoprotein in halobacteria [1], subsequently, in the last

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0173-0835/03/0404–363 $17.501.50/0
364 R. K. Upreti et al. Proteomics 2003, 3, 363–379

few years, there has been an overwhelming response in sidic linkage, whereas, Archaea bacteria may have either
relation to the discovery of bacterial glycoproteins, and an N- or O-glycan or both, with the predominant type
around 70 bacterial glycoproteins have been reported so being the N-glycosidic linkage.
far [2–5]. Most of the bacteria reported to possess glyco-
The glycoprotein structure is expected to be the same in
proteins belong to the Archaebacteria. More recently,
eukaryotes and prokaryotes but from the available data
evidence has also been provided for the occurrence of
it is obvious that prokaryotic glycoproteins are far more
glycoproteins in eubacteria. Experimental evidence for
diverse than the structures found in eukaryotes. However,
the glycoprotein nature is not always very convincing
they do share some characteristics. In prokaryotic and
and in some cases, already has been proved to be incor-
eukaryotic glycoproteins, sugar chains are attached to
rect [6]. Due to the abundance of glycans in most prokar-
the protein core either via the amide nitrogen of an Asn
yotic cell walls, lectin binding and periodic acid Schiff
residue (N-glycosylation) [9–11] or via the hydroxyl groups
staining, even after SDS-PAGE, might be caused by firmly
of mainly Ser or Thr (sometimes Tyr) residues (O-glycosyla-
associated but noncovalently linked saccharides. The
tion) [12–14]. However, in some bacteria distinct consensus
final proof for the glycosylation of a protein is the identifi-
sequences have also been reported, for example Asp-Ser
cation of the amino acid-attached sugar linkage. In some
and Asp-Thr in Chryseobacterium meningosepticum and
cases this claim still needs further evidence [7, 8].
Val-Tyr in Thermoanaerobacter kivui [15, 16]. In both pro-
The chemistry of glycoprotein structure and biosynthesis karyotes and eukaryotes, the glycans are hetereogenous
is expected to be basically the same in eukaryotes and where either a protein carries more than one type of glycan
prokaryotes, but prokaryotes are deprived of the cellular or some glycans remain incomplete or modified.
organelles required for glycosylation. Hence, there is a
Some prokaryotic glycans such as O-antigens of Gram
possibility that the biosynthetic pathway of prokaryotic
negative bacteria resemble surface polysaccharides and
glycoproteins follows routes different from those de-
display a repetitive structure such as the S-layer pro-
scribed for eukaryotes. However, similar mechanisms
teins of Paenibacillus alvei, Clostridium symbiosum and
such as the utilization of nucleotide-activated sugar resi-
Thermoanaerobacter thermohydrosulfuricus L-77-66 and
dues for the assembly of the oligosaccharide chain, the
L-111-69 [17–20]. In prokaryotes, variations are also
occurrence of trimming reactions, and the existence of
found in sugar constituents and linkage units, for exam-
lipid-bound intermediates, were also observed in the
ple, a 2,4,diacetamido-2,4,6, trideoxyhexose on Neisseria
process of prokaryotic glycosylation [5]. Conversely, the
meningitides pillin and asparaginyl-rahmanose for S-layer
occurrence of nucleotide diphosphate-linked oligosac-
protein of Bacillus stearothermophilus [8, 21]. Even indi-
charide, differentiates between prokaryotic and eukaryo-
vidual strains of a species can exhibit a remarkable de-
tic glycosylation mechanisms. Despite discovering a
gree of heterogeneity regarding the structural organiza-
wide variety of functions of the eukaryotic glycoprotein,
tion and chemical composition of a glycoprotein. All the
knowledge pertaining to the functional aspects of prokar-
reported prokaryotic glycoproteins, depending upon their
yotic glycoproteins still remains unexplored. The present
localization, can be classified into five major types. These
review focuses on various aspects of bacterial glyco-
include the surface-layer glycoproteins (glycoprotein sub-
proteins including location, structural features, biosynthe-
units, that are present in the outermost macromolecular
sis and their functional roles. In addition, recent coverage
monolayer of a bacterial cell envelope), membrane–asso-
of Escherichia coli membrane glycoproteins including a
ciated glycoproteins (glycoprotein units that are distrib-
novel glycoprotein (Gp45) recently isolated and purified
uted in the outer/inner membrane and in the periplasmic
in our laboratory is discussed.
space of the bacteria), cell-surface glycoproteins (sub-
units associated with the pili or flagelli), secreted glyco-
proteins and exo-enzymes, and cellular glycoproteins
2 Occurrences, location and structure of (whose location in the cell, is either uncertain or nonspe-
bacterial glycoproteins cific). Details are summarized in Table 1.

In eukaryotes, the crucial and characteristic covalent

bond(s) which join the carbohydrate and the peptide/ 3 Functional role of bacterial glycoproteins
protein form five different types of glycosidic linkages:
N-glycosides, O-glycosides, phosphorus-linked glyco- Glycosylation has been found to alter the function of
sides, S-glycosides and C-glycosides. Of these only both eukaryotic and prokaryotic proteins [22, 23]. Much
N- and O-glycosylation have been reported in bacterial of the precise information on the effects of glycosylation
glycoproteins. Eubacteria may possess either an O- or on protein functions is derived from studies with eukaryo-
N-glycan with the predominant type being the O-glyco- tic glycoproteins. The glycan moieties of eukaryotic gly-
Proteomics 2003, 3, 363–379 Bacterial glycoproteins 365

Table 1. Various bacterial glycoproteins

Classification Organism Nomenclature Proposed linkage/sugars Reference(s)

Surface-layer glycoproteins:
Eubacteria Gram-positive Aneurinibacillus thermo-
aerophilius DSM 10155 Glycopeptides A and B b-GalNAc-O-Ser [115, 116]
b-GalNac-O-Thr
Bacillus stearothermophilius Glycopeptide GP I Rha-N-Asn [8, 115]
NRS 2004/3a
Bacillus alvei CCM2051 Trisaccharide repeating b-Gal–O-Tyr [18, 76]
glycan-containing protein
Bacillus sphaericus 133–155 kDa acidic glyco- 0.69–1.81% [117]
NTCC9602 proteins carbohydrates
Clostridium difficile HMW S-layer subunit Not known [118]
Clostridium symbiosum 15 kDa-phosphate contain- ManNAc, GalNAc, BacNAc [17]
HB25 ing glycoprotein
Clostrium thermohydro- 140 kDa glycoptrotein b-GlC-O-Tyr [17]
sulfuricum S102-70.
Clostridium thermohydro- HMW, Tetrasaccaride- 14% total carbohydrate [19]
sulfuricum L77-66 repeating units-contain- content
ing glycoprotein
Clostridium thermo- Amino-sugar rich glyco- 3-amino- 3,6- dideoxy- [119, 120]
saccarolyticum E207-71 peptide D-glucose-O-Tyr
b-GlC-O-Tyr
b-Gal-O-Tyr
Desulfotomaculum S-layer protein Not known [121]
nigrificans
Dienococcus radiodurans HPI-layer glycoproteins 6 tightly bound sugar [122]
residues per polypeptide
chain
Lactobacillus buchneri S-layer protein a-Glc-O-Ser [123]
Paenibacillus alvei S-layer protein b-Gal-O-Tyr [20]
Thermanaerobacter thermo- – a-Gal-O-Tyr [20, 124, 125]
hydrosulfuricus b-Glc-O-Tyr
L111-69/L110-69
Thermoanerobacterium Xylanase (S-layer protein) Not known [126]
Thermosulfurigenes EM1
Thermanaerobacter thermo- Tyrosine-bound S-layer b-Glc-O-Tyr [127]
saccharolyticum glycan
D120-70
Archaebacteria Gram-positive Acetogenium kivui 80 kDa protein GalNAc-?-Tyr [128]
Glc-?-Tyr
?-?-Tyr
Gram-negative Halobacterium halobium HMW CSG b-GalNAc-N-Asn [88]
b-Glc-N-Asn
Gal-O-Thr
Halofeax volcanii 52, 56, 105, 109 kDa b-GalNAc-N-Asn [80, 129]
Cell-surface glycoproteins b-Glc-N-Asn
Gal-O-Thr
Halobacter salinarium 194 kDa CSG b-GalNAc-N-Asn [130]
Gal-O-Thr
Haloarcula japonica TR-1 HMW CSG Not known [131]
Methanolobus tindarius S-layer glycoprotein Not known [132]
Methanothermus fervidus SlgA glycoprotein b-GalNAc-N-Asn [86]
366 R. K. Upreti et al. Proteomics 2003, 3, 363–379

Table 1. Continued

Classification Organism Nomenclature Proposed linkage/sugars Reference(s)

Methanothermus sociabilis SlgA glycoprotein b-GalNAc-N-Asn [11]

Sulfolobus acidacaldarius 140–170 kDa glycoprotein Not known [133]
Sulfolobus solfactaricus 140–170 kDa glycoprotein Not known [134]

Membrane-associated glycoproteins:
Eubacteria Gram-positive Bacillus megaterium 52 kDa PG I Not known [49]
Clostridium thermocellum 210 kDa S1 subunit a-Gal-O-Thr [56, 135]
(cellulosome)
Steptococcus Mutans IDG-60 Sialic acid,mannose, [47]
galactose, and N-acetyl
glucosamine
Streptococcus pyogenes M-protein/SAGP Not known [114]
Streptococcus salivarius Protein Antigen C Glucose, Galactose, [136]
Rhamnose (6: 1.5: 1)
Gram-negative Azinetobacter species 20 kDa ComP protein Not known [137]
BD413 (OMP)
Bacteroides cellulosolvens 230 kDa subunit a-Gal-O-Thr [56]
(cellulosome) a-Gal-O-Ser
Bacteroides succinogenes 75 kDa enzyme cello- 8–16% carbohydrates [138]
S85 biosidase (mainly cellohexaose)
Borellia burgdorferi OspA and OspB b-GlcNAc-N-Asn [139]
(31 and 34 kDa) (OMP)
Campylobacter (Vibrio) fetus Antigen [a] (OMP) 4% carbohydrates [140]
(hexose, pentose,
methyl pentose)
Chlamydia trachomatis MOMP (40 kDa) b-GlcNAc-N-Asn [123]
Erwinia herbicola Ice-nucleating protein Man-N-Asn [141]
Gal-O-Ser
Gal-O-Thr
Escherichia coli 104 kDa TibA (OMP) O-glycosidic linkage [42, 101]
Myxococcus xanthus 74 kDa VGP (OMP) 13.5% carbohydrates- [142]
neutral sugars, hexo-
samines, uronic acids
Pseudomonas syringae Ice-nucleating protein Man-N-Asn [141]
Gal-O-Ser
Gal-O-Thr
Treponema pallidum 4 protein antigens (30.5, Not known [33]
33, 35.59 kDa) (OMP)
Treponema phagedensis 2 protein antigens Not known [33]
(33, 34 kDa) (OMP)

Archaebacteria Gram-positive Methanosarcina mazei Membrane-bound Not known [143]

S-6 glycoprotein
Gram-negative Chloroflexus auranticus Aurocyanin B Glc-N-Asn [144]
(Periplasmic protein) Glc-N-Thr
Haloferax volcanii Four (150, 98, 58, 54 kDa) Not known [89]
glycoproteins
Thermoplasma acidophilum Plasma membrane GlcNAc-N-Ser [48]
glycoprotein
Methanoplanus limicola Cell-envelope glycoprotein Not known [145]
Proteomics 2003, 3, 363–379 Bacterial glycoproteins 367

Table 1. Continued

Classification Organism Nomenclature Proposed linkage/sugars Reference(s)

Flagelli/pili-associated glycoproteins:
Eubacteria Gram-positive Streptococcus sanguis 300 kDa LFP Low levels of basic amino- [146]
(Long Fibril Protein) acids, high levels of
nonpolar aminoacids,
and carbohydrates
Gram-negative Azospirillum brasilense Sp 7 flagellin Not known [65]
Campylobacter coli VC167 T 1 and T 2 flagellins O-glycosidic linkage [35, 147]
Campylobacter jejuni T 1 and T 2 flagellins O-glycosidic linkage [35]
Escherichia coli Col B2 and EDP208 pilin One covalently bound glucose [99]
molecule, per pilin subunit
Myxococcus xanthus 8 kDa fimbriae 0.8% carbohydrate content [148]
Neisseria gonorrhoeae Pilin High levels of galactose [40]
Neisseria meningitidids Pilin 2,4-diacetamido- [37]
2,4,6-trideoxy hexose-
O-Ser
Pseudomonas aeruginosa 50.8 kDa pilin Not known [41]
1244
Pseudomonas aeruginosa a-type flagellins Not known [34]
type A
Spirochaeta aurantia Periplasmic flagellar protein Glycosylation proposed. [62]
Not established
Archaebacteria Gram-negative Halobacterium halobium Sulfated flagellins Glc-N-Asn [77]
Halobacter saccharavorum Flagellin Not known [149]
Methanococcus deltae Flagellin Not known [150]
Methanogenium marsnigri Flagellin Not known [151]
Methanospirillum hungatei GP 1 (24 kDa) Not known [61, 152]
JF 1 (25 and 35 kDa)
Flagellins
Natranobacterium Flagellin Not known [153]
pharanois 12
Natranobacterium magadii Flagellin Not known [154]
Pyrodictum Occultum Flagellin Not known [155]
Sulfolobus shibate 31 and 33 kDa flagellins Not known [152]
Thermococcus stetteri flagellin Not known [152]
Thermoplasma volcanicum 41 kDa flagellin Not known [152]
Cellular glycoproteins:
Eubacteria Gram-positive Bacillus thurigiensis 250 kDa and 72 kDa Rha-O-Ser [54]
glycoprotein (in sporangia) GlcNAc-O-Ser
?-O-Ser
Bacillus thuringiensis subsp. 27.3 kDa crystal protein GlcNAc-N-Asn [156]
israelensis
Bacillus thuringiensis subsp. 120 kDa parasporal 5–6% carbohydrate content, [157]
kurstaki crystalline prototoxin with 20 glucose and
mannose, per subunit
Streptococcus sanguis PAAP GlcNAc-N-Asn [67]
Secreted glycoproteins:
Eubacteria Gram-positive Bacillus amyloliquefaciens H1-b-glucanase GlcNAc-N-Asn [55]
Bacillus macerans (1,3 ? 1,4 ) b-glucanase GlcNAc-N-Asn [55]
Cellulomonas fimi CenA, CenX (cellulases) Man-O-Thr Gal-O-Thr [13]
368 R. K. Upreti et al. Proteomics 2003, 3, 363–379

Table 1. Continued

Classification Organism Nomenclature Proposed linkage/sugars Reference(s)

Clostridium acetobutylicum 28 kDa Autolysin Not known [158]

Corynebacterium Phytotoxin Man-?-Thr [159]
sepedonicum
Erysipelothrix rhusiopathiae Glycoprotein antigen Not known [160]
Streptococcus faecium Autolytic enzyme O-glycosidic linkage [53]
muramidase involving glucose/methyl
pentose
Streptomyces lividans 66 Xylanase B Gal-?-Thr [58]
Man-?-Thr
Gram-negative Flavobacterium meningo- 1.2 kDa endoglucosidase Man-O-Ser [16, 161]
septicum Man-O-Thr
Mycobacteria Mycobacterium tuberculosis 45 kDa Antigen complex a-Man-O-Thr [14, 32]
Mycobacterium para- 35–45 kDa glycopeptide Not known [162]
tuberculosis
Mycobacterium smegmatis 19 kDa Antigen Not known [27]
Mycobacterium bovis Antigens Xylose (22b) Mannose, [163]
(22b, 19, 15, 12 kDa) Arabinose (19, 15, 12 kDa)
Archaebacteria Gram-positive Methanobacterium bryantii Crx Proteins One or both of a-D-Man or [164]
BKYH a-D-Glc residues
Thermonospora fusca YX 46 kDa E2 protein Monomeric enzyme, contain- [165, 166]
(b-1,4-endoglucanase) ing 25% carbohydrates,
by weight

coproteins are thought to play important roles in: the matis [26, 27]. The covalent modifications of protein with
maintenance of protein conformation and stability; sur- acyl or glucosyl moieties are known to alter immunoge-
face or intracellular recognition and cell adhesion; modu- nicity and pathogenicity [28]. For example, the triacylated
lation of many physicochemical properties like solubility, protein of Gram-negative bacteria are found to possess
viscosity, surface charge etc.; proteolytic processing and various activities including the ability to prime cytotoxic
solubilization of the polypeptide against uncontrolled pro- T-cells [29], activate B-lymphocytes [30] and stimulate
teolysis; mediation of biological activity; intracellular sort- phagocytosis and cytokine production by macrophages
ing; externalization of glycoprotein and embryonic devel- [31]. T-lymphocytes recognize short antigenic peptides
opment and differentiation [24, 25]. Despite discovering a bound to either MHC class I/II molecules. Glycosylation
wide variety of functions of the eukaryotic glycoprotein, of such peptide molecules modulates the interaction of
the knowledge pertaining to the functional aspects of the T-lymphocytes with the modified peptide that is pre-
prokaryotic glycoproteins, believed to follow similar func- sented by the MHC molecule. Both the glycan and the pep-
tional roles, requires further exploration. The known and tide make contact with the T-lymphocyte’s receptor-bind-
speculative functional roles of bacterial glycoproteins ing site. This has been found to occur in the case of the
have been summarized in Fig. 1. antigen complex of Mycobacterium tuberculosis [32], where
demannosylation of the glycoprotein reduces significantly,
The functional role and importance of glycosylation of the potency of the peptide to stimulate T-lymphocytes and
prokaryotes may be analyzed by comparison between regulates the cell-mediated immune response. In contrast,
glycan deficient mutants and wild-type strains or by com- the glycoproteins of Treponema sps. were shown to in-
parison between experimental conditions with or without duce and maintain the primary humoral response [33].
competing sugars, or by introduction or deletion of nutri-
ents in culture medium, or by the use of recombinant DNA Glycoproteins on the surface of bacterial cells may be
technology. Several reports have demonstrated that pro- involved in cell-cell interactions between the bacteria
tein glycosylation alters immunogenicity by inhibiting the and eukaryotic cells. It is possible that glycans on
major histocompatibility complex (MHC) presentation of enzymes facilitate interaction with the substrate. How-
individual peptides to T-cells. Evidence for this has been ever, removal of the glycan from meningococcal pili,
obtained from the glycoproteins of Mycobacterium smeg- did not affect adhesion to endothelial or epithelial cells.
Proteomics 2003, 3, 363–379 Bacterial glycoproteins 369

Figure 1. Functional role of bacterial glycoproteins.

The glycans (specifically, the terminal galactose residues pathogenesis by facilitating adhesion, followed by inva-
of the glycan moiety) of Neisseria meningitidis pili cause sion of the host cell [38, 39]. The glycoproteins localized
the pili to become sialylated [21], thus increasing its resis- on the membrane or in the cell surface structures (pili/
tance to complement-mediated killing. Similar sialylation flagelli) usually participate in such adhesive functions,
in the lipopolysaccharides of N. gonorrhoea also results in thus mediating close contact between the host and the
increased complement resistance [34]. The sialic acid mol- bacterium [21, 40, 41]. Similar host-related adhesion has
ecule (which is also the surface component of host erythro- also been observed in the TibA glycoprotein of entero-
cytes) of the organism, is identified as the self-antigen, toxigenic E. coli [42]. The larvicidal glycoprotein toxin of
thus camouflaging the bacterium from the immune system. Bacillus thuringiensis subsp. israelensis contains cova-
Alternatively, the glycosyl groups could also play a role in lently-associated amino sugars, which are recognized
the antigenicity of flagellin protein, constituting an immuno- by the lectin receptors in the larval mosquito gut, thus
dominant epitope, as in the flic genes of Pseudomonas exerting its entomotoxic activity [43].
aeruginosa [34]. The involvement of glycosylated flagellin
Surface/intracellular recognition and cell-adhesion phe-
monomers in antigenicity has also been established in
nomena are properties shared by almost all glycan moi-
Campylobacter species and Mycobacteria [14, 35].
eties [17]. The glycosylation of proteins has been thought
Several studies have confirmed that glycosylated pro- to enhance the hydrophilic character along the mem-
teins are key to the aspects of immunogenicity and brane to aid in permeability and phage-mediated infec-
pathogenicity of the host organism [26, 36, 37]. In terms tion [44]. Modification of cell-surface structures may
of the association of glycoproteins to virulence, the most stabilize the attachment of the pathogen to host cells,
notable pathogens are from a variety of bacterial genera, containing the proper receptors. The absence of glyco-
including Treponema, Campylobacter, Streptococcus, sylation has been found to have no effect on either the
Neisseria etc. (Table 2). While organisms like Campylo- formation or extension and retraction of pilus fibres in
bacter fetus produce antiphagocytic components, thus the pilin subunits of P. aeruginosa 1244 [41]. Instead, the
conferring virulence to the bacterium, others exhibit glycoproteins are believed to contribute to overall cell-
370 R. K. Upreti et al. Proteomics 2003, 3, 363–379

Table 2. Bacterial glycoproteins responsible for pathogenicity

Organism Category Glycoprotein Location Mechanism Clinical syndromes Reference

ETEC E. coli Entero- Tib A OMPa) TibA mediates close contact Diarrhoea [39, 42]
toxigenic (Adhesin/ between the bacterium and
Invasin) host cell, and thus invades
host
Campylobacter Enterotoxic – Flagella Identical to other adhesins Campylobacteriosus [35]
jejuni (Adhesin) Acute colitis; Guillan-
Barre syndrome
Campylobacter Entero- Antigen [ a ] OMP Cells possessing this antigen Septicaemia, spontane- [167]
fetus pathogenic become refractory to ingestion ous abortion and other
by macrophages systemic infections
Treponema Pathogenic Protein OMP Glycoproteins mediate host Syphilis [33]
pallidum Antigens cell adhesion
Neisseria Pathogenic Gonococcal Pili Terminal galactose residue of Gonorrhoea, pelvic [40]
gonorrhoea pili the pilin mediates adherence inflammatory disease
to the host cell (PID); Disseminated
gonococcal infection;
Proctitis; Septic
arthritis etc.
Neisseria Pathogenic Pilin Pili Identical to other adhesins Meningitis; Meningoc- [21]
meningitidis occemia; Waterhouse-
Friedrichson syndrome
Borrelia Pathogenic OspA OspB OMP Adherence to cell-surface Lyme disease [168]
burgdorferi in both vertebrate and
invertebrate hosts
Pseudomonas Pathogenic Pilin Pili Identical to other adhesins Cystitis; Bronchitis [41]
aeruginosa Pneumonia; Keratitis;
Otitis externa; Endo-
carditis
Clostridium Pathogenic HMW glyco- S-layer May promote adherence and Acute colitis; Antibiotic- [169]
difficile protein colonization or contribute to induced diarrhoea
subunit virulence by evading immune
system
Streptococcus Pathogenic M-Protein Cell-wall Colonization and resistance to Purpureal fever; Scarlet [114]
pyogenes phagocytosis; Major cause fever; Pharyngitis;
of antigenic shift, and anti- Erysipelas; Glomerulon-
genic drift in Group A ephritis etc.
streptococci
Streptococcus Oral pathogen Protein Membrane Glycoprotein mediates host- Dental caries [136]
salivarius antigen-C related adhesion functions,
such as binding of epithelial
cells or erythrocytes and
aggregation with salivary
components
Corynebacterium Phytotoxic Phytotoxin Secreted Infects the seeds and the Ring-rot of potato [159]
sepedonicum enzyme affected tubers fade and
rot to give access to
microbes that cause
secondary soft rot
Bacillus thuringiensis Entomocidal Parasporal Cellular Amino sugars of the glyco- Larvicidal activity [43, 170]
subsp. israelensis crystalline localiza- protein bind to the lectin
protoxin tion receptor in the mosquito gut
and infects the host

a) OMP, outer membrane proteins

Proteomics 2003, 3, 363–379 Bacterial glycoproteins 371

surface negativity. Similar to this, are the glycans of conformational alterations in the protein structure and
Clostridium thermohydrosulfuricum L-77-66, which add increase their sensitivity to proteases [9, 24]. This reflects
greatly to the cell surface density [19]. The glycoproteins the steric hindrance of the protease by the glycan. This
of Halobacteria have been found to be involved in main- protective mechanism (glycosylation), whereby the pro-
taining the structural integrity of the rod-shaped organ- teins achieve resistance against proteases, is character-
ism. These glycoproteins have been shown to aggregate istic of flagellins of P. aeruginosa [52], M. tuberculosis [14],
strongly in the presence of divalent cations, suggesting in the secreted glycoprotein of Cellulomonas fimi and
that protein-protein interactions via salt-bridges may be Streptococcus faecium [53], and in the sporangia of
involved [45]. A high concentration of monovalent cation B. thuringiensis [54].
has been proved to be necessary to shield the negative
charges and prevent repulsion within a protein or between Various bacterial enzymes are glycosylated, but glyco-
adjacent proteins [1]. sylation does not appear to directly play any role in enzy-
matic activity, for example, as in the case of glucanase of
The cell surface glycoproteins (CSG) are important con- Bacillus sps. and F. meningosepticum in which removal of
stituents contributing to the form-giving or shape-main- the glycan does not affect activity [16, 55]. However, the
taining function of the bacteria. When the incorporation presence of carbohydrates in muramidase of S. faecium
of the sulfated glycosaminoglycan into the polypeptide has been shown to have some recognition function,
chain is inhibited by the inclusion of an antibiotic (bacitra- which enables the enzyme to bind to the substrate with
cin), the rod-shaped halobacterial cells are converted high affinity [53]. Similarly, the O-glycans of C. fimi and
to spherical cells [46], proving that glycosaminoglycans Clostridium thermocellum have been shown to enhance
might be involved in maintaining the structural integrity of the substrate-binding property which in turn increases
the rod-shaped organism. Similar observations have also the possibility of cellulose (substrate) degradation [13,
been identified in the 60 kDa glycoprotein of Strepto- 56]. On the other hand, the nonglycosylated cellulase of
coccus mutans [47]. The carbohydrate coat, formed by C. fimi retains the enzyme activity but loses its capacity
the membrane glycoproteins of Thermoplasma acido- to bind with the substrate [57]. Xylanase A and B isolated
philum has been found to trap water molecules, immobi- from their respective clones Streptomyces lividans IAF18
lize them and form a mesh-like network, thus imparting and IAF42 were found to be glycosylated [58]. Xylanase A
rigidity to the limiting plasma membrane [48]. These hydrolysed efficiently the water soluble short chain oli-
complexes may further contribute to the stability of the goxylosides, but had no or little action on the longer,
membrane. While the glycoproteins have been observed water insoluble, fragments. Its reaction products were
to govern the shape and rigidity of the bacterial cells in xylobiose and xylotriose, the latter being hydrolyzed to
some organisms, the polypeptide factor PG I of Bacillus xylobiose and xylose upon further incubation. Xylanase
megaterium, has been found to assist in the synthesis B acted on both substrate fractions, but appeared to
of peptidoglycans by stimulating the incorporation of di- cleave preferentially the long-chain oligoxylosides. Al-
aminopimelate [49]. though glycosylation does not always directly affect enzy-
matic activity, it has been shown to enhance some
Important biological functions have been attributed to enzyme’s thermostability. The glycosylated forms exhib-
O-glycosylation. This modification has been proposed ited a long half-life compared to their unglycosylated
to play a direct role in the structure of the S-layer and in counterparts at higher temperature in Bacillus amyloli-
the ability of organisms to survive in hostile environments quefaciens and Bacillus macerans [55]. Another unique
[10, 28]. The glycans are clustered in heavily glycosylated property exhibited by the glycoproteins of Erwinia and
domains, and protein folding within these domains is some species of Pseudomonas is to induce ice-nuclea-
restricted, leading to an extended conformation [50]. tion in super-cooled water [59].
Thus, a combination of glycosyl units in the vicinity of
proline residues have been proposed to result in a stiff, The function of glycosylation in archaeal flagella has not
extended conformation of the protein [14]. Similar fea- been clearly elucidated. It has been hypothesized to play
tures have been observed in mycobacteria, C. symbio- a role in subunit interaction in the assembled product.
sum HB25 [17] and Flavobacterium meningosepticum Methanococcus deltae grown in the presence of bacitra-
[16]. It has been proposed some glycans are added at cin does not appear to possess fully glycosylated flagel-
the b-turns of proteins, masking the turn conformation, lins, suggesting inhibition of the glycosylation machinery,
and thereby protecting the protein from proteolysis [51]. and the minimum amount of glycosylation of flagellins
The enzymatic removal of bulky oligosaccharide moieties required for the proper assembly of the flagellum was not
of these glycoproteins have been believed to expose new produced. As a result, flagella were not observed in those
protease cleavage sites directly or indirectly by inducing species [60]. The function of glycosylation in the flagellins
372 R. K. Upreti et al. Proteomics 2003, 3, 363–379

of Methanospirillum hungatei does not comply with this 4.1 Enzymatic glycosylation
[61]. In fact, the glycosylation of this organism is unusual,
as glycosylation in flagella occurs only in low phosphate 4.1.1 Role of glycosyl transferase
media. Similar phenomenona have been reported in Halo-
Glycosyl transferases, that are located externally at the
bacter halobium and minor periplasmic components of
cell-surface, catalyze the stepwise synthesis of both
Spirochaeta aurantia [62]. Glycosylation may not play a
N- and O-linked oligosaccharides by carrying out the
role in the transport through archaebacterial cell envel-
transfer of a sugar residue from an activated donor sub-
opes, as in the case of H. halobium, as it occurs at the
strate (which can be either nucleotide sugar derivatives-
cell-surface [63]. It has also been suggested that glyco-
like UDP-Gal, UDP-GalNAc, UDP-GlcNAc etc, or the lipid-
sylation of the halobacterial flagellins helps in the smooth
linked sugars, such as dolicholphosphoryl glucose) to the
sliding of the individual filaments (of flagellar bundle)
hydroxyl group of an acceptor substrate on the growing
against one another. As a result, reversal of the direction
oligosaccharide chain [63]. These enzymes are grouped
of flagellar rotation does not disrupt the flagellar bundle
into many families, including sialyl transferase, galactosyl
[64]. Similar functions have been served by the peri-
transferase, fucosyl transferase etc, depending upon the
plasmic flagellins of S. aurantia.
type of sugar it transfers. According to the one-linkage-
An additional function has been identified in the polar fla- one-enzyme hypothesis, a different enzyme for each glyco-
gellum of Azospirillum brasilense, in which glycosylation sidic linkage is involved in the biosynthetic process.
was responsible for increased adsorption to plant roots.
In a eukaryotic cell, the biosynthesis of oligosaccharides
A plant root lectin could either bind to the glycan chains
O-glycosidically linked to Thr (Ser) in the polypeptide
of the flagella or the flagellin protein itself may act as a
chain, appears to occur almost entirely by sequential gly-
lectin and can recognize the plant sugars [65]. Further-
cosylation, where the product of one glycosyl transferase
more, O-glycosylation has also been thought to play an
is utilized as an acceptor substrate for another glycosyl
important role in enhancing the protein export out of cells
transferase [9]. The bacterial glycoproteins may also fol-
in Trichoderma reesei [66]. Thus, glycosylation is a most
low a similar pathway, as seen in the case of Clostridium
diverse post-translational modification as displayed by
thermocellum [68]. In this context Gerwig et al. [68] have
the proteins of both prokaryotes and eukaryotes. The
shown additional O-linked carbohydrate chains in the cel-
exact role of the carbohydrate units glycosidically linked
lulosome of eubacteria C. thermocellum and suggested
to the polypeptide chain in various bacteria needs further
that the initiation could take place by transfer of a Galp
chemical characterization of the individual glycoproteins.
or Galf residue to the protein core, followed by elongation
through addition of the individual monosaccharides
4 Glycoprotein biosynthesis from their nucleotide-activated derivatives. In contrast,
in eukaryotes the N-linked oligosaccharides are syn-
Glycosylation of proteins by eukaryotic organisms is an thesized by the sequential addition of sugars to the lipid
established phenomenon. Eukaryotic glycoproteins are moiety, dolichol monophosphate. The reaction is found to
glycosylated in intracellular membrane bound processes progress slowly and once the lipid-linked oligosaccharide
with nucleotide and lipid linked saccharides as sugar precursor is complete, the oligosaccharide may be trans-
donors. A wide variety of polysaccharides are present in ferred ‘en bloc’ to the newly synthesized protein releasing
prokaryotic cell walls, usually attached to lipids or pepti- diphosphoryl dolichol, which then undergoes dephos-
doglycans. Such covalently associated carbohydrates phorylation, to remove one phosphate, thus completing
may bind to the inner surface of the bacterial membrane, the dolichol cycle. The transfer of oligosaccharide from
facilitating the secretion and subsequent incorporation of dolichol phosphate to the amide nitrogen is observed
the protein into the cell wall [67]. Biosynthesis of bacterial only when the motif, Asn-Xxx-Ser/Thr is present [69–73].
glycoproteins has been the focus of investigation since Here, Xxx can be any amino acid, although proline is
the last decade. Prokaryotes do not possess intracellular not favoured. A trisaccharide of L-rhamnose is known to
membrane; hence, they may follow different mechan- be attached to the Asn residue in the glycopeptide-1 of
isms. The whole process of glycosylation in bacteria is B. stearothermophilus [8]. It has also been postulated
not yet clearly understood. Based on the present data, that two different N-glycosyl transferases exist in H. halo-
glycoprotein biosynthesis in prokaryotic organisms could bium, one of which does not use the typical consensus
either involve glycosyl transferases and/or follow non- Asn-Xxx-Ser(Thr), necessary for all other N-glycosyl
enzymatic glycosylation. On the basis of earlier descrip- transferases described so far. In fact, the exchange of
tions and recent literature, a proposed mechanism for the Ser-4 for Val, Leu, or Asn in the sequon, Asn-Ala-Ser,
biosynthesis of bacterial cell-surface glycoproteins is does not prevent N-glycosylation of the cell surface gly-
depicted in Fig. 2. coproteins [74].
Proteomics 2003, 3, 363–379 Bacterial glycoproteins 373

Intracellularly, all prokaryotes, including Archaea and transfer of monomeric sugars from nucleotide precursors
Eubacteria, appear to follow a similar pathway for carbo- or by the ‘en bloc’ transfer of carbohydrate repeating
hydrate synthesis. Evidence favouring this, has been units from other lipid carriers [67].
obtained from characterizing the cell-surface glycopro-
teins of Halobacteria [10], Thermoanerobacter species
[75], Bacillus alvei [76], PAAP protein of Streptococcus 4.1.3 Transient modifications
sanguis [67] and many archaeal flagellins [35, 61, 65, 77].
Investigations on halobacterial cell surface glycoproteins In some bacterial species, the lipid-linked saccharides
revealed the involvement of lipid-linked intermediates in contain some distinct chemical modifications that do not
the biosynthesis of the amino sugar containing saccha- exist in the protein-linked stage. This represents the tran-
ride. This finding suggested that glycoprotein synthesis sient modification of lipid oligosaccharides. The methyla-
is a membrane-mediated process [78–80]. If transfer of tion of position 3 of the oligosaccharide’s peripheral, non-
the saccharide to the protein occurs in a compartment reducing end glucose residue has been reported to be
equivalent to the luminal site of the eukaryotic rough transient in the case of halobacteria. The study shows
endoplasmic reticulum, bacterial glycosylation could occur that this methylation helps in translocation of lipid oligo-
at the extracellular surface of the cell membrane [63]. The saccharides through the membrane [81]. However, such
whole glycosylation process begins with the synthesis of modifications at position 3 are stable and found as a
polysaccharides on a lipid carrier. A unique carbohydrate stoichiometric component in M. fervidus glycoprotein
core is established by the sequential addition of nucleo- [86]. In M. fervidus and H. halobium the transient sugar
tide or lipid monophosphate-activated sugars to the lipid residues are found [9, 87].
carrier [81], and then these sugars are assembled by po-
lymerization of preformed pentasaccharides in a mechan-
ism similar to the one described for the biosynthesis of the 4.1.4 Transport across the membrane
salmonella O-antigen [82]. There is evidence for common
Although the biosynthetic pathway of intracellular carbo-
mechanisms for the synthesis of bacterial glycoproteins
hydrates in the bacterial cells is known, the mechanism
and other cell surface polysaccharides. In N. meningitides,
for incorporation of polymers into the cell wall of these
a galE mutation (the galE gene product is a UDP-galactose
organisms is not yet experimentally determined. In eukar-
4’-epimerase) results both in lipopolysaccharide truncation
yotes, the glycoconjugates are covalently modified at the
and in a reduction of the molecular mass of the pilin [21]. In
protein-linked level in the Golgi apparatus, where trimming,
A. brasilense, an exoC mutation (exoC encodes a phos-
further glycosylation, sulfation or epimerization occurs and
phomannomutase) results in both altered exopolysaccha-
hence follow a different biosynthetic pathway of synthesis
rides and a reduced molecular mass of the flagellin [65].
of their glycoproteins. The bacterial oligosaccharides are
completed and sulfated, while still attached to the lipid car-
rier on the cytosolic side of the cell membrane [10]. There-
4.1.2 Lipid-linked oligosaccharides after, the whole complex of lipid-linked oligosaccharides
(sulfated in the case of halobacteria) is transported to the
The lipid carrier is a C60-polyprenol of the eukaryotic cell-surface, where transfer of the carbohydrate to the
dolichol, rather than of the bacterial undecaprenyl type. core-protein occurs [88]. This was considered to be func-
Bacitracin, an inhibitor of peptidoglycan synthesis, is tionally analogous to the lumen of the rough endoplasmic
known to form a complex with prenyl diphosphate com- reticulum in animal cells. However, it has also been claimed
pounds and inhibit the hydrolytic regeneration of the cor- that these post-translational modifications occur following
responding monophosphate [83]. Furthermore, studies translocation across the plasma membrane [89].
carried out by Zhu et al. [80] using glycosylation inhibitors
Amphomycin [84] and sugar nucleotide analogs, pp36 For polysaccharide polymers to be incorporated into the
and pp55 [85] have indicated that high-energy poly- external face of the cell-wall, nucleotide linked precursor
prenyl-phospho glucose intermediates and not dolichyl must transfer monomeric carbohydrate units to the grow-
phosphate, are involved in Heloferax volcanii protein and ing polysaccharide as a lipid-linked intermediate on either
lipid glycosylation and that glucose was found to be the the inner face of the cytoplasmic membrane or external
major constituent of lipid and protein fractions of the cell to the membrane [90]. Exogenous nucleotide precursors
envelope. In the case of halobacteria, N-acetyl glucosa- may become incorporated into a growing carbohydrate
mine (Gal/Glc in other organisms) is first added to the intermediate on the external surface of the cytoplasmic
lipid carrier to form isoprenyl pyrophospho-N-acetyl membrane, but these are not capable of re-entering the
glucosamine. Other sugars are then attached via glyco- cell through the membrane. If these were not translocated
sidic bonds to the growing saccharide by enzymatic back through the membrane, nucleotide phosphate by-
374 R. K. Upreti et al. Proteomics 2003, 3, 363–379

products would not be recovered for future use [67]. On of glycosylation outside the cytoplasmic membrane as in
the other hand, polysaccharides could be incorporated the case of halobacteria; and the homology of the primary
into the cell wall by an ‘en bloc’ transfer, the mechanism sequences of archeal flagellins [60, 62, 63, 92].
of which is not clearly defined. The glycosylation machin-
The assembly of extracellular structures of bacterial
ery in Streptococcus sanguis involves the cotransport
flagella-filament and hook involves transport of the com-
of carbohydrate through the membrane covalently asso-
ponent proteins (filament and hook proteins, respectively)
ciated with an exported PAAP precursor, a secretory pro-
through a central channel in the flagellar base. These
tein, which may cotransport the covalently linked poly-
component proteins are able to distinguish between the
saccharides [91]. Moreover, transient methylation may
signal sequences of flagellar proteins, which are to be
represent an obligatory step for the lipid oligosacharides
exported with other cellular proteins. In addition, there
through the membrane [81]. Once, the lipid-linked oligo-
may be another barrier in the basal body in order to main-
saccharide moiety reaches the cell-surface, polysaccha-
tain the proton-gradient across the cell membrane. This
rides are cleaved from the protein backbone and trans-
barrier may be the site for ‘export apparatus’ and ‘glyco-
ferred by glycosyl transferase to other cell wall compo-
sylation machinery’ [65]. In eubacteria, filament elonga-
nents, such as lipids and peptidoglycan (Fig. 2).
tion follows the same route, by the passage of the fila-
ment monomers across both the inner and outer cell
4.1.5 Glycosylation in bacterial flagellins membranes, up through the axial pore within the filament
followed by integration into the distal end of the growing
Limited data are available on flagella of the archae- filament [93, 94]. This is observed in the case of campylo-
bacteria. The flagella of this kingdom differ from those bacter flagellins, in which glycosylation presumably
found in eubacteria in many key features including the occurs in the cytoplasm prior to flagellin secretion [35],
presence of multiple flagellins (which may be glyco- whereas, in archaebacteria, flagellin is bound to cyto-
sylated); the presence of leader peptides in the archaea, plasmic chaperones that prevent the nonspecific aggre-
which are not found in eubacterial flagellins (with the gation of the flagellins. The flagellin leader peptide helps
exception of presumptive signal sequences on the flagel- in the transfer of the chaperone-flagellin complex to the
lins of the internal flagella of S. aurantia), the occurrence vicinity of the polar cap structure. The leader peptidase

Figure 2. Proposed mechanism for the biosynthesis

of bacterial cell surface glycoproteins. A lipid (DOI-P) or
nucleotide (GDP, UDP) molecule serves as sugar carrier.
GT, glycosyl transferase; UDP, uridine diphosphate; GDP,
guanidine diphosphate.
Proteomics 2003, 3, 363–379 Bacterial glycoproteins 375

cleaves the flagellin molecules from the chaperones, and 5 E. Coli membrane glycoproteins
the subunits in the cytoplasmic membrane are incorpo-
rated into the growing filament at the base of the cyto- Most of the inner and outer membrane proteins of E. coli
plasmic membrane situated above the polar cap [60]. have been characterized. E. coli outer membrane pro-
As glycosylation of flagellin has been proposed to occur teins, except for those with host-related adhesion proper-
outside the cytoplasm [63], the process of glycosylation ties, have been identified as nonpathogenic. The exact
and then incorporation are thought to occur simulta- physiological role for a few of them has been established.
neously. However, it is not clear if glycosylation takes The pathogenic E. coli strains impart pathogenesis to
place before or after flagellin incorporation into the fila- the host by mediating adhesion with the host. Compara-
ment. tively few proteins are located in the inner membrane of
E. coli. These proteins, unlike adhesions, are nonpatho-
genic. However, the literature on E. coli membrane glyco-
4.2 Nonenzymatic glycosylation proteins is scanty.

Nonenzymatic glycosylation is a chain of chemical reac- An envelope-specific, phenol-soluble glycoprotein iso-

tions affecting the free amino groups in proteins of eukar- lated from E. coli B was reported previously [100]. Later a
yotes. It is a condensation reaction between reducing report claiming the glycosylation of the plasmid-encoded
sugars and primary (a or e) amino groups in proteins. It F-like conjugative plasmid pili of E. coli K-12 strains was
proceeds in two main (early and late) stages, the former published [99]. Recently, a glycoprotein produced by the
involving the formation of aldimines (Schiff bases), while enterotoxigenic strain (ETEC) of E. coli has been identified
the latter transforms the aldimines into more stable Ama- [101]. This is found to act as an adhesin that may partici-
dori products. The terminal adducts irreversibly bound pate in the disease process. The critical step in the ETEC
to proteins are designated as advanced glycation end infection process is the initial colonization of the intestinal
products (AGEs). Besides reducing sugars, many other mucosa by bacterial cells, which is followed by the pro-
sugar and nonsugar compounds have been found to duction of at least one of the two major enterotoxins:
cause AGE modifications in proteins [95]. The substrates heat stable toxin and heat-labile toxin. The initial coloniza-
for this glycation may include a variety of compounds, tion was mediated through fimbrial colonization factor
containing the 2NH2 groups, including DNA and amino- antigens (CFAs). The enterotoxin thus produced was
lipids and amino acids such as lysine. The majority of thought to act on the intestine, leading to water-loss
studies have been carried out with higher eukaryotes, associated with ETEC infection [102]. In addition to this,
owing to the clinical importance of this process and its the ETEC strain H10407 is also capable of invading
relation to ageing [96]. Nonenzymatic glycosylation in epithelial cells derived from human ileocecum and colon.
yeast has also been reported [97]. This invasion was mediated by the membrane glyco-
protein of E. coli. Two separate chromosomally encoded
Earlier studies with E. coli were confined to the nonenzy-
invasion loci (tia and tib) responsible for directing the non-
matic glycosylation of DNA [98]. Recently, it has been
adherent and noninvasive E. coli to adhere to and invade
shown by Mironova et al. [95] that nonenzymatic glyco-
cultured human intestinal cells, have been cloned [103].
sylation is initiated during the normal growth of E. coli.
The tib locus has been found to direct the synthesis of
Two main types of post-translational alterations, a cova-
TibA, a 104 kDa outer membrane protein, by the tibA
lent dimerization and truncation, in the cysteine-less
gene. This TibA, is originally synthesized as a pre-TibA
recombinant human interferon-gamma (rhlFN-g), expres-
precursor protein, which undergoes post-translational
sed in the E. coli strain LE392 have been observed. It has
glycosylation to form the mature TibA glycoprotein, with
been shown that dimmer and truncated species were
covalently attached carbohydrate moieties. These sugar
formed under in vivo conditions, independently of the
components are believed to mediate closer contact be-
presence of glucose in the growth medium. Fluorescent
tween the bacterium and the host cell [38].
analysis provided further evidence for AGE formation
in untransformed E. coli cells suggesting nonenzymatic Recently, we have isolated and purified a novel mem-
glycosylation [95]. With the mechanism described, an brane glycoprotein (Gp45) from E. coli [107]. This mem-
unglycosylated protein core can be translocated through brane-anchored glycoprotein of apparent molecular mass
the cell membrane and the corresponding glycoconju- of 45 kDa contained approximately 60% carbohydrate.
gates may pass through this membrane in their lipid- For the isolation of Gp45, cell extract or membrane frac-
linked stage. Thus, despite the lack of compartmentaliza- tion was treated with sodium deoxycholate and precipi-
tion in prokaryotic systems, the bacterial glycoprotein tated with TCA. The supernatant fraction of TCA contain-
biosynthesis resembles the mechanism of glycosylation ing the Gp45 was further purified on DEAE-Sephadex
in eukaryotes. A-25. SDS-PAGE showed a single band at the 45 kDa
376 R. K. Upreti et al. Proteomics 2003, 3, 363–379

position that stained with periodic acid Schiff reagent S-layer coated liposomes can be used as a versatile system
(PAS). Monosaccharide analysis revealed the presence for entrapping and binding of various target molecules [111].
of mannose, glucose, galactose, galactosamine and glu- The S-layer subunits (including glycoproteins) not only pro-
cosamine as the major sugar constituents, while the vide the mechanical strength to the liposomes while cry-
amino acid analysis showed the presence of acidic amino stallizing on its surface, but also provide excellent sites
acids as the major constituents. Significant incorporation for cross-linking and covalent attachment of molecules. In
of radiolabelling in the Gp45 isolated and purified from such cases glycoproteins are a better choice compared to
E. coli grown in media containing [14C]-glucosamine sug- other protein molecules. These liposomes may be used as
gested its biosynthesis in the organism. When cells were a carrier and/or drug delivery, drug-targeting system [112].
grown in basal minimal media containing [4C]-glucosa- Furthermore, the S-layer lattice may be used for the forma-
mine in the presence of Bacitracin, the radiolabelling tion of isoporous ultrafiltration membranes with well-de-
in the dialyzed TCA supernatant fraction was negligible fined molecular sieving and antifouling characteristics.
compared to the controls without Bacitracin. PAS staining
Surface and other glycoproteins are important virulence
also did not reveal the presence of Gp45 in Bacitracin
factors in pathogenic microorganisms, for example, TibA
treated preparations. This primarily suggested that Baci-
of E. coli, 60 kDa immunodominant glycoprotein of
tracin inhibited the biosynthesis of glycoprotein. However,
Streptococcus sp., antigen A of C. fetus, surface pili of
the mechanism of glycosylation needs further explora-
N. gonorrhea. These glycoproteins offer a good possibil-
tion. The amount of Gp45 (per gram of cell mass) found
ity of being used as a target for vaccine development. In
to be the same in different media and culture age, further
addition, the S-layer glycoprotein self-assembly prod-
indicated that it is an indispensable structural constituent
ucts are shown to possess an intrinsic adjuvant property
of the bacterial cell envelope. The function of Gp45 in
and have also been used as vaccine carriers [113]. Var-
E. coli remains to be explored. Being localized in the cell
ious bacterial glycoproteins having enzymatic activity
envelope, it is possible that it may have an enzymatic
may have industrial importance due to their inherent
activity or it is an adhesin. This glycoprotein characteristi-
thermostability and resistance towards proteolytic deg-
cally resembles the anorexigenic membrane glycoprotein
radation. The fascinating possibilities for the expression
isolated from eukaryotic cell membrane [104–107]. The
of prokaryotic glycoproteins has further opened the
cross-reactivity amongst the antibodies and antigen from
opportunities in the direction of the production of recom-
E. coli Gp45 and from eukaryotic glycoproteins as well as
binant glycosylated and tailor-made glycoproteins in lar-
their precipitin pattern also indicated the reaction of com-
ger quantities, particularly in the case of industrially and
plete identity. The parenteral administration of Gp45 iso-
medically important glycans. Other important biological
lated from E. coli was also found to cause significant loss
activities associated with bacterial glycoproteins need
of appetite in murine models with no toxic effect.
further exploration for their industrial applications. For
example, antitumour activity of streptococcal acid glyco-
protein (SAGP) produced by Streptococcus pyogenes
6 Industrial potential of bacterial [114] and our recent finding of anorexigenic activity of a
glycoproteins glycoprotein isolated from cell envelope of E. coli [107].
General application potential of bacterial surface proteins
and glycoproteins have been discussed to some extent
7 Concluding remarks
[5, 108, 109]. S-layers are composed of identical subunits
of proteins, mainly glycoproteins, which assemble into Over the last two decades, a significant change of per-
oblique, square or hexagonal lattices. The periodic struc- ception has taken place regarding bacterial glycopro-
ture of S-layer lattice functional groups of amino acid side teins. For many years, protein glycosylation was assumed
chains are arranged in defined and regularly repeated to be limited to eukaryotes; but now plenty of information
positions and orientations. Therefore, they can be ex- on the structure, function and biosynthesis of prokaryotic
ploited as a matrix for the immobilization of enzymes, glycoproteins has accumulated, with S-layer glycans
antibodies or ligands and covalently bound macromole- being one of the best studied examples. Among Gram-
cules [110]. With glycosylated S- layers, not only the pro- positive bacteria, S-layer glycoproteins have been identi-
tein moiety but also the carbohydrate residues are avail- fied only in bacilli. In Gram-negative organisms their pres-
able for immobilization of various macromolecules. In ence is not fully investigated. Extensive biochemical
addition, they may further be used for potential applica- studies of the S-layer glycoprotein from halobacteria
tions like bioanalytical mono and multi-enzyme sensors, have, at least in part, unravelled the glycosylation path-
enzyme and affinity membranes, immunoassay dipstics way in archaea. However, molecular biological analyses
and affinity microparticles [108, 123]. of these pathways need further exploration.
Proteomics 2003, 3, 363–379 Bacterial glycoproteins 377

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