Neutral N-glycans of the gastropod Arion lusitanicusMartin Gutternigg, Karin Ahrer, Heidi Grabher-Meier, Sabine Bu¨rgmayr and Erika Staudacher Department fu¨r Chemie, Universita¨t fu¨r B
Trang 1Neutral N-glycans of the gastropod Arion lusitanicus
Martin Gutternigg, Karin Ahrer, Heidi Grabher-Meier, Sabine Bu¨rgmayr and Erika Staudacher
Department fu¨r Chemie, Universita¨t fu¨r Bodenkultur Wien, Vienna, Austria
The neutral N-glycan structures of Arion lusitanicus
(gas-tropod) skin, viscera and egg glycoproteins were examined
after proteolytic digestion, release of the glycans from the
peptides, fluorescent labelling with 2-aminopyridine and
fractionation by charge, size and hydrophobicity to obtain
pure glycan structures The positions and linkages of the
sugars in the glycan were analysed by two dimensional
HPLC (size and hydrophobicity) and MALDI-TOF mass
spectrometry before and after digestion with specific
exoglycosidases The most striking feature in the adult
tis-sues was the high amount of oligomannosidic and small
paucimannosidic glycans terminated with 3-O-methylated
mannoses The truncated structures often contained
modi-fications of the inner core by b1,2-linked xylose to the
b-mannose residue and/or an a-fucosylation (mainly a1,6-)
of the innermost GlcNAc residue Skin and viscera showed
predominantly the same glycans, however, in different
amounts Traces of large structures carrying 3-O-methylated galactoses were also detected The egg glycans contained mainly ( 75%) oligomannosidic structures and some pau-cimannosidic structures modified by xylose or a1,6-fucose, but in this case no methylation of any monosaccharide was detected Thus, gastropods seem to be capable of producing many types of structures ranging from those typical in human to structures similar to those found in nematodes, and therefore will be a valuable model to understand the regulation of glycosylation Furthermore, this opens the way for using this organism as a host for the production of recombinant proteins The detailed know-ledge on glycosylation also may help to identify targets for pest control
Keywords: Arion lusitanicus; gastropod; glycosylation; N-glycans; snail
Gastropods are intermediate hosts for schistosomes,
which are pathogenic to humans and domestic animals In
addition to schistosomiasis, diseases such as fascioliasis,
clonorchiasis and paragonimiasis represent only a few of
the snail transmitted diseases with worldwide medical and
economic impact Other potential candidates for pest
control are those gastropods, mainly slugs, which cause
damage to vegetables The worst case is a complete crop
failure but even their eating or moving tracks reduce the
commercial value of lettuce Structural features, which do
not occur in higher animals, are valuable candidates as a
target for pest control The most effective way would be
inhibition of enzymes that are not typical of mammals and
that are responsible for structures important for slug/snail
survival or reproduction This would be a convenient way
to reduce the population of these animals without high
amounts of conventional chemical pesticides
Analysing the complete set of N-glycan structures of a
species gives an overview on its biosynthetic capacity for
glycosylation It is the first step for the identification of glycosylation related target enzymes for inhibition
So far, N-glycan structures derived from the hemocyanins
of the snails Helixpomaia, Lymnaea stagnalis, Rapana venosa and the keyhole limpet Megathura crenulata have been published The Helixpomatia glycans show complex structures containing a common core with an a1,6-linked fucose to the reducing GlcNAc and a b1,2-linked xylose to the b-mannose residue One or both a-mannose residues may be substituted by GalNAcb1,4GlcNAcb1,2elements which contain two to four b1,3- or b1,6-linked galactoses with or without 3- or 4-O-methyl groups [1] Lymnaea stagnalishemocyanin contains low and high molecular mass biantennary oligosaccharides They lack the a1,6-linked fucose to the inner GlcNAc residue, but some antennae terminate with an a1,2-linked fucose Similarly to Helix pomatia, the basic element of the antennae is Galb1,3Gal-NAcb1,4GlcNAc [2,3] The two N-glycans of the functional unit RvH1-a of Rapana venosa hemocyanin are biantennary nonfucosylated oligosaccharides with 3-O-methylated ter-minal b1,3-linked galactose residues One of these residues also carries a sulfate group on the a1,6-linked core mannose and a 3-O-methylated GlcNAc residue b1,2-linked to the b-mannose of the core [4] Megathura crenulata hemocyanin
is substituted by a novel type of N-glycan with galactoses directly linked in b1,6-linkage to mannose residues [5] Recently a core structure terminated with two 3-O-methy-lated mannose residues linked to the major soluble protein
of the organic shell matrix of Biomphalaria glabrata was identified [6]
Furthermore some characteristics of a few enzymes which are involved in gastropod glycan biosynthesis have
Correspondence to E Staudacher, Department fu¨r Chemie,
Universita¨t fu¨r Bodenkultur Wien, Muthgasse 18,
A-1190 Vienna, Austria.
Fax: + 43 136006 6059, Tel.: + 43 136006 6063,
E-mail: erika.staudacher@boku.ac.at
Abbreviation: endoglycosidase H, endo-b-N-acetylglucosaminidae H.
Enzymes: endo-b-N-acetylglucosaminidae H (EC 3.2.1.96).
Note: The abbreviations for the glycan structures are detailed in
Figs 2and 4.
(Received 22December 2003, revised 2February 2004,
accepted 18 February 2004)
Trang 2been determined However, the information gained is
restricted in most cases to the enzyme specificity in vitro
and some biochemical parameters Lymnaea stagnalis has
been shown to contain the key enzyme for the formation
of complex N-glycans, GlcNAc-transferase I, which has
been proven to be a prerequisite for the action of
GlcNAc-transferase II, fucosylGlcNAc-transferases and xylosylGlcNAc-transferase
[7] This organism has also been shown to contain
GlcNAc-transferase II and xylosyltransferase [7], a
b1,4-GalNAc-transferase which shows similar characteristics to
mammalian b1,4-galactosyltransferase [8], a
b1,3-galacto-syltransferase and an a1,2-fucosyltransferase [9,10]
Hybridization experiments using a bovine
b1,4-galactosyl-transferase cDNA probe resulted in the isolation of a clone
encoding a b1,4-GlcNAc-transferase which is similar to the
mammalian galactosyltransferase in acceptor specificity but
requires a different nucleotide sugar It is definitely not
involved in the biosynthesis of the chitobiose core of
N-glycans [11–13] The function of this enzyme in vivo is
not clear The prostate glands of these snails also contain a
b1,4-glucosyltransferase forming Glcb1,4GlcNAc units
[14] Furthermore an a1,3-fucosyltransferase catalysing
the transfer of fucose from GDP-fucose to a
Galb1,4Glc-NAc acceptor forming the LewisX-unit has been found in
the connective tissue of Lymnaea stagnalis [10] and an
a1,3-fucosyltransferase catalysing the transfer of fucose
from GDP-fucose to the asparagine-linked GlcNAc has
been found in the albumin and prostate glands of the same
snail [15] However, no LewisX-containing structures, core
a1,3-fucosylated structures, or glucosylated units have been
detected in the glycans of this snail so far An
a1,2-L-galactosyltransferase which seems to be involved in the
elongation of the storage polysaccharide of the snail was
found in Helixpomatia [16] Although in vitro this
galactosyltransferase catalyses the transfer of a fucose into
a1,2-linkage from GDP-fucose to a Galb1,3Gal-O-Me
substrate, nothing is known about this ability in vivo
A number of exoglycosidases have been described from
gastropodian sources Some of them are commercially
available and widely used as tools in glycomic research The
majority of these enzymes seem to be part of the
degrada-tion and recycling processes of the cells and not be involved
in the N-glycosylation pathway
In the present study we present for the first time the
neutral N-glycan structures of a whole gastropod, the slug
Arion lusitanicus, in two developmental stages, to show its
capability for N-glycan biosynthesis and processing
Materials and methods
Materials
Slugs were collected by M Pintar (Department for
Integrative Biology, Institute for Zoology, Universita¨t fu¨r
Bodenkultur Wien, Vienna, Austria) and his students in
local gardens and were frozen immediately at)80 C
Eggs were collected by the authors, lyophilized and kept
at)20 C until use
Sephadex G25 fine and Sephadex G15 were purchased
from Amersham Biosciences, and Dowex 50W·2was from
Fluka (Fluka Chemie, Vienna, Austria) Standard
pyridy-laminated glycans were prepared in the course of previous
studies [17,18] All other materials purchased were of the highest quality available from Merck or Sigma
Preparation of N-glycans Thawed slugs (10 individuals for each preparation) were washed to remove the extraneous mucous components and dissected into three fractions; the skin and inner organs (viscera) were lyophilized separately, while the intestinal tract was discarded The dry material (skin, viscera or eggs) was suspended in 200 mL of 50 mM Tris/HCl buffer
pH 7.5, homogenized with an IKA Ultra Turrax T25 (IKA-Labortechnik, Janke and Kunkel GmbH, Staufen, Germany) at 15 000 r.p.m for 2· 20 s and centrifuged at
5000 g for 10 min The supernatant was adjusted to 80% (w/v) of ammonium sulfate and centrifuged at 27 500 g for
40 min The precipitate was dialyzed against water, con-centrated on rotary evaporation and made up to 150 mMof Tris/HCl, 1 mM CaCl2, pH 7.8 Thermolysin (ICN Biomedicals, Vienna, Austria) was added at a 40 : 1 (w/w) ratio of protein/enzyme and incubated for 20 h at
50C The digest was dialyzed against 2% (v/v) acetic acid and applied to a column of 100 mL of Dowex 50W·2 equilibrated in 2% (v/v) acetic acid The column was washed with 150 mL of the same solution and the (glyco)peptides were eluted with 0.4Mammonium acetate,
pH 6.0, concentrated and applied onto an Sephadex G25 column (1· 120 cm) equilibrated in 1% (v/v) of acetic acid Carbohydrate containing fractions detected by the orcinol-sulfuric acid method according to Winzler [19] were pooled, lyophilized and dissolved in approximately 1 mL citrate-phosphate buffer, pH 5.0 The N-glycans were released by incubation with 0.7 U of peptide:N-glycosidase A (Roche)
at 37C for 24 h, purified on Sephadex G15, Dowex 50W·2and Lichroprep RP (Merck) according to [18] and labelled with 2-aminopyridine as described previously [20,21]
Analysis of monosaccharides Monosaccharide analysis was carried out by hydrolysis of the glycans with 4Mtrifluoroacetic acid at 100C followed
by derivatization with 3-methyl-1-phenyl-2-pyrazolin-5-one and separation on reverse-phase HPLC according to Fu and O’Neill [22] or by conversion of the monosaccharides into their corresponding alditol acetates, which were then analysed by gas chromatography/mass spectrometry
as described [21]
Separation and analysis of N-glycans Fluorescently labelled oligosaccharides were separated into neutral and negatively charged fractions on an Econo-Pac High Q Cartridge (5 mL, Bio-Rad Laboratories) at a flow rate of 1 mLÆmin)1 Solvent A was 50 mM Tris/HCl,
pH 8.5; solvent B was 1M NaCl in solvent A The run was started with 5 min at 100% solvent A followed by a linear gradient of 5% per min to 50% solvent B, continued with 10% per min to 100% solvent B and terminated by
1 min at 100% solvent B Fluorometric detection was carried out at excitation and emission wavelengths of 320 and 400 nm, respectively
Trang 3The neutral fraction was further fractionated by a two
dimensional mapping technique starting with separation
according to hydrophobicity on an Hypersil ODS column
(0.4· 2 5 cm, 5 l, Forschungszentrum Seibersdorf, ARC
Seibersdorf research GmbH, Seibersdorf, Austria) [21]
Fluorometric detection was performed at excitation and
emission wavelengths of 320 and 400 nm, respectively, and
peaks were collected and dried prior to subfractionation by
size, in the second dimension The method was modified
from the procedure of Khoo et al [23] using a Palpak
type N column (4.6· 250 mm, Takara, Japan) at a flow
rate of 1 mLÆmin)1 Solvent A was 75 : 25 (v/v) acetonitrile/
stock solution [3% (w/v) acetic acid-triethylamine buffer at
pH 7.3 with 10% (v/v) acetonitrile] Solvent B was 50 : 50
(v/v) acetonitrile/stock solution The run was started with
5 min at 10% solvent B followed by a linear gradient of
2.8% per min to 80% solvent B, and terminated by 8 min at
80% solvent B Fluorimetric detection was performed
at excitation and emission wavelengths of 310 and
380 nm, respectively
Columns were calibrated in terms of glucose units with a
pyridylaminated partial dextran hydrolysate (3–11 glucose
units) Peaks from either size fractionation or reverse-phase
chromatography were analysed by MALDI-TOF and
subjected to exo- or endoglycosidase digestions
MALDI-TOF MS analysis
MALDI-TOF MS was carried out as described previously
[24] The sample (1 lL, 0.2–0.8 pmol) was spotted onto a
target and dried, followed by the addition of 0.8 lL of
matrix [2% (v/v) 2,5-dihydroxybenzoic acid in water
containing 30% (v/v) acetonitrile] The plate was transferred
immediately to a desiccator and vacuum was applied until
all solvent had evaporated Spectra were recorded on
a DYNAMO linear MALDI-TOF mass spectrometer
(Thermo BioAnalysis, Hemel Hempstead, UK) operated
with a dynamic extraction setting of 0.1 External mass
calibration was performed with pyridylaminated N-glycan
standards derived from bovine fibrin About 20 individual
laser shots were summed
In some cases, on-target digestions with exoglycosidases
were carried out using 6-aza-2-thiothymine [0.5% (w/v) in
water] as the matrix [25]
Exo- and endoglycosidase digests
Endoglycosidase H (recombinant from Escherichia coli,
Roche) was used at a concentration of 2mU in 0.15M
citrate-phosphate buffer, pH 5.0 containing 0.1M NaCl;
a-mannosidase (jack bean, Sigma) at 2mU in 50 mM
sodium acetate, pH 4.5 containing 0.2mMZnCl2;
a-fuco-sidase (bovine kidney, Sigma) at 2mU in 50 mM sodium
citrate, pH 4.5; a1,2-fucosidase (recombinant, Sigma) at
0.2mU in 50 mM sodium phosphate pH 5.0;
b-galactosi-dase (bovine testis, Roche) at 1.6 mU in 50 mM sodium
citrate, pH 5.0 and b-hexosaminidase (bovine kidney,
Sigma) at 25 mU in 20 lL of 0.1 M sodium citrate,
pH 5.0) Incubations were carried out in 20 lL of
appro-priate buffer at 37C overnight
For chemical release of fucose a1,3-linked to the
inner GlcNAc-residue, the dry sample was incubated for
48 h at 0C with 2 0 lL of 48% (v/v) hydrofluoric acid The acid was then removed under a stream of nitrogen [26]
Results
Adult tissues Oligomannosidic structures The N-glycan pattern of the labelled glycans on reverse-phase chromatography can be divided into four regions, I–IV (Fig 1) The first region (4–6.8 glucose units) contains mainly oligomannosidic structures (M5–M9; abbreviations of glycan structures are given in Fig 2 ), which were confirmed by their elution behaviour on HPLC in comparison with standard glycans, their mass on MALDI-TOF and their sensitivity to a-mannosidase and endoglycosidase H (Table 1 and data not shown) Using MALDI-TOF, moderate digestion with a-mannosidase gave a ladder of structures with masses with
a distance of 162.1 mass units, this effect could also be observed on Palpak-HPLC Endoglycosidase H digest on MALDI-TOF caused a shift by 281 mass units, indicating the loss of a GlcNAc-residue containing the fluorescent group Using HPLC, just the pyridylaminated GlcNAc-residue is still visible by the detector Structural isomers of
M7 and M8 were identified by their elution behaviour on reverse-phase
Methylated oligomannosidic structures Region II of the reverse-phase pattern (Fig 1) contained, in the preparations
of the adult snails, methylated mannosidic structures with mainly five to seven mannose residues and two or more, often three, methyl groups (abbreviations of glycan structures are given in Fig 2) Methylated M4, M8and M9structures were also found, however, in very low amounts (Table 1) All these structures were sensitive to endoglycosidase H (Fig 3) To confirm the presence of 3-O-methylmannose residues,
we performed carbohydrate composition analysis by gas chromatography/mass spectrometry Incomplete
methy-Fig 1 HPLC analysis of pyridylaminated neutral N-glycans of Arion lusitanicus on a reverse-phase column (A) Isomaltose standard, 4–14 glucose units, (B) skin, (C) viscera and (D) eggs Regions I–IV are indicated with arrows I, oligomannosidic structures; II, methylated oligomannisidic structures; III, a1,6-fucosylated structures; IV, large galactose containing structures.
Trang 4lated structures were subjected to an a-mannosidase digest
which made it possible to identify the position of the
unmethylated mannose in most cases For example, if the
terminal mannose of the a1,3-arm of a M9 structure was
not methylated, three mannoses could be released If one of
the terminal mannoses of the a1,6-arm lacked the methyl
group, two mannoses could be released, but we saw in our
experiments that the middle arm appeared to be less
accessible to the enzyme and so only one mannose was
cleaved in this case Due to their insensitivity to
a-mannosidase, the majority of the methylated
oligo-mannosidic (M5, M6and M8) glycans were determined to
be methylated on each terminal mannose Structures lacking
one methyl group were present only in a few percent of the
oligomannosidic methylated glycans (<10%), whereas
structures lacking two methyl groups were detectable only
in trace amounts If one methyl group was missing, it was in
most cases the middle antennae which was unmethylated,
whereas, if just one methyl group was present no preferences could be determined
a1,6-fucosylated structures The third region of the reverse-phase pattern was characterized by structures with an a1,6-fucose linked to the inner GlcNAc (Fig 1, Table 1) This fucose could be easily removed by a-fucosidase from bovine kidney A shift of)146.1 mass units on MALDI-TOF and the characteristic shift to earlier elution times on reversed phase chromatography confirmed the loss of a fucose linked a1,6 to the inner core The main compound was dimethylated Me2MMF6in skin and viscera (abbreviations of glycan structures are given in Fig 4) However, in viscera the monomethylated variant MeMMF6, and in skin a xylosylated variant Me2MMXF6 were also detected No a1,6-fucosylated glycans lacking the methyl groups could be determined in adult tissues (Table 1)
Fig 2 Structures of paucimannosidic (four
mannose residues or less) and oligomannosidic
glycans The abbreviation system applied
herein (according to [18]) names the terminal
residues, starting with the residue on the
6-linked antenna and proceeding counter
clockwise.
Trang 5Paucimannosidic structures Examination of regions I and
II of the reverse-phase pattern suggested the presence of
small paucimannosidic structures Therefore a further
preparation removing the oligomannosidic structures by digestion with endoglycosidase H prior to fractionation on reverse-phase was performed Using this strategy in both tissues, small truncated structures were found (Table 1) They contained up to four mannose residues and additional xylose and/or fucose residues linked to the inner core Similar to the previously described oligo-mannosidic structures, MMX occurred in a nonmeth-ylated, a mono- and a di-3-O-methylated form at the terminal mannose residues
In most of the cases the expected GlcNAc-residue linked
to the a1,3-mannose was missing This GlcNAc I (incor-porated by N-acetylglucosaminyltransferase I in b1,2-link-age to the a1,3-linked mannose) has been proven in other sources to be a prerequisite for the further transfer of core-modifying enzymes (fucosyltransferases and xylosyltrans-ferase) It can be speculated that the snail enzymes do not need this GlcNAc I residue for their action or, more probably, that a Golgi-hexosaminidase removes the Glc-NAc I in an early processing stage of the developing N-glycan as it has been shown previously for insects and nematodes [27,28]
The paucimannosidic glycans eluting in the first two regions on reversed phase HPLC and carrying a fucose residue were subjected to more intensive investigation The small size of the glycans and successful b-hexosaminidase and/or a-mannosidase digests led to the conclusion that these fucose residues were linked to the core While a1,6-linked fucose at the inner GlcNAc residue increases elution time drastically on a reverse-phase column [29], glycans with an a1,3-fucose linked to the same GlcNAc elute earlier at the positions found for the paucimannosidic
Table 1 Neutral N-glycan profiles of Arion lusitanicus Wherever the
detected traces are less then 0.2% an exact quantitation is not possible.
Therefore the amount is considered to be 0.1%.
Mannosidic structures
Methylated mannosidic structures
a1,6-fucosylated structures
Methylated a1,6-fucosylated structures
Other paucimannosidic structures
Other methylated paucimannosidic structures
Complex type structures with methylated galactoses
Fig 3 MALDI-TOF MS spectra of pyridylaminated oligosaccharides from region II Before (A) and after (B) digest with endoglycosidase H Structures labelled with an asterisk were not cleaved by endoglycosi-dase H.
Trang 6glycans under study The investigated fucose residues could only be cleaved by HF-treatment of the glycans and not by the usual amounts of commercially available fucosidases (Fig 5), which confirmed the occurrence of a low amount of a1,3-fucosylation of the core in both adult tissues
Complexstructures Less than 4% of the structures of adult tissues contained various larger N-glycans with a number of galactose residues terminated with methyl groups, eluting in region IV of the reversed phase pattern (Fig 1) The linkage of the methyl groups was identified
by gas chromatography/mass spectrometry to be 3-O-methylation In contrast to [4], we could not observe a removal of the methylated galactose by bovine testis galactosidase, therefore subsequent exoglycosidase diges-tions were not possible Due to the low amount and the heterogeneity of the fractions a detailed analysis of those glycans was omitted From our data we suspect that the structures may be similar to those described by Lommerse
Fig 4 Structures of paucimannosidic glycans
with or without core fucosylation or
xylosyla-tion The abbreviation system applied herein
(according to [18]) names the terminal
resi-dues, starting with the residue on the 6-linked
antenna and proceeding counter clockwise In
the case of the core fucose, which occurs in
more than one type of linkage, the linkage is
depicted as a superscript.
Fig 5 HPLC analysis of pyridylaminated MMXF 3 on a reverse-phase
column (A) Isomaltose standard, 3–11 glucose units, (B) MMXF3,
(C) MMXF 3 after incubation with a-fucosidase from bovine kidney
and (D) MMXF3after incubation with hydrofluoric acid.
Trang 7et al [1] for Helixpomatia aD-hemocyanin, where one or
both antennae of biantennary xylosylated glycans
termin-ate with a varying number of methyltermin-ated galactose
residues
Eggs
The egg glycans differed from those derived from adult
tissues (Table 1) While in preparations of adult slugs the
unmethylated, oligomannosidic structures were restricted to
8.8% and 47% in skin and viscera, respectively, in the eggs
75% of the total N-glycans were oligomannosidic
struc-tures, dominated by M5–M8glycans The remaining 25% of
structures were equally divided into MMX and a series of
a1,6-fucosylated small glycans, some of them carrying
the GlcNAc I The most striking result, however, was the
complete absence of methylated structures in eggs No
oligomannosidic or paucimannosidic structures were
sub-stituted by methyl groups
Discussion
In order to obtain information about the N-glycan
biosyn-thesis capacity/capability of the gastropod Arion lusitanicus
we performed a protein preparation of whole animals
(except the digestion system) separated into viscera and skin
fractions
The tissues were homogenized and the proteins were
precipitated and digested with thermolysin After
purifica-tion of the (glyco)peptides by ion exchange chromatography
and gelfiltration, the N-glycans were released by PNGase A
in order to ensure that a1,3-fucosylated structures were
also released [30] The oligosaccharides were labelled with
2-aminopyridine and separated by anion exchange
chro-matography into neutral and negatively charged fractions
To obtain individual structures the neutral fraction was
further fractionated on reverse-phase HPLC and the
collected peaks were subfractionated on a Palpak column
Aliquots of all fractions were analysed by MALDI-TOF
mass spectrometry Further information was gained by
gas-chromatography/mass spectrometry of the alditol acetates
Elution behaviour on both HPLC systems compared with
standard oligosaccharides, in combination with the mass
information from MALDI-TOF, led to the conclusions
about the structure which were confirmed by digestion with
specific exoglycosidases For relative quantitation of the
structures see Table 1
In the course of our work we found that the percentages
of structures vary slightly with the area where the slugs had
been collected (due to nutritional conditions), the age (size)
of the individuals and their physiological status (carrying
eggs or not) However, skin and viscera preparations
contained the same spectrum of N-glycans Therefore it
can be ruled out that unusual structures are due to food or
environmental contaminants
The most obvious structural feature of these slug adult
tissues is the high degree of structures with terminal
3-O-methylated mannose residues (>80% in skin and 50%
in viscera) and traces of structures with 3-O-methylated
galactoses (Table 1) Methylated sugars were first described
in the early 1970s in the polysaccharides of procaryotes,
lower eucaryotes, algae and fungi with soil habitat In
gastropod hemocyanin 3-O-methylated mannose and 3-O-methylated galactose were found in 1977 [31] Since that time a number of methylated sugars have been found in polysaccharides from plants and procaryotes In molluscs 3-O-methylated mannose and/or 3-O-methylated galactose were found in some hemocyanins [32], 6-O-methylation of mannose was found in the giant clam Hippopus hippoppus [33] and 3-O-methyl galactose and 3-O-methyl GlcNAc in Rapana venosa[4] In nematodes 2-O-methylated fucose was found in Toxocara [34] and Caenorhabditis elegans [35] The high degree of methylation and its occurrence on so many different structures in Arion lusitanicus leads to the assumption that methylation is an important regulating event in this organism The enzyme(s) responsible appear to
be very active and widely distributed along the modifying oligosaccharide steps during the biochemical pathway of the N-glycans As this modification, as far as we know now, is restricted to lower animals it may be an interesting target for pest control
However, the slugs also contain another set of N-glycans, the occurrence of which seems to be highly regulated The traces of Me2GlcM9may be a relic of the early events of glycan processing or play an important role in folding or assembly of a special protein, as has been speculated recently for GlcM9of Antheraea pernyi and Bombyxmori arylphorin [36]
Besides the usual set of oligomannosidic structures, Arion lusitanicus tends to accumulate short antennary chains similar to plants, insects and C elegans, lacking the GlcNAc I residue which has been shown to be neces-sary for the action of a number of modifying enzymes (core-fucosyltransferases, xylosyltransferase and GlcNAc-transferases II–V) [7,37] A highly active Golgi-located b-N-acetylhexosaminidase has been suggested, which removes the GlcNAc residue from the Mana1,3-antenna after fucosylation and xylosylation; such an enzyme has already been described in insects and C elegans [27,28] Due to the small size of the glycans, heterogeneity is mainly caused by modification of the core A remarkable amount of xylose linked b1,2to the b-mannose and/or fucosylation of the reducing GlcNAc, was detected Mainly a1,6-linked fucose was observed a1,3-linked fucose, like that typical for plants, occurred only in trace amounts A corresponding a1,3-fucosyltransferase has been detected in Lymnaea stagnalis[15], but here it is the first time that one of its products has been found in a snail It can be speculated that this structural feature is limited to some very specialized cells and does not occur randomly in the organism There was no evidence for the presence of difucosylation
of the inner GlcNAc-residue found in lepidopteran insects [29] and squid rhodopsin [38], or of difucosylation in combination with a core xylose as is present in Schistosoma japonicumeggs [39] Terminal fucosylation such as the a1,2-fucosylation seen in another gastropodian source (Lymnaea stagnalis) [3] or LeXdeterminants were also not found Arion lusitanicus contains an enormous potential for generating a large set of structural elements commonly found in eukaryotic N-glycosylation: they sialylate [40], they carry a1,6-linked as well as a1,3-linked fucose as shown for some insects, nematodes and trematodes, and b1,2-linked xylose, as found in plants and trematodes, and they are able
to methylate terminal sugars (mannose and galactose) as
Trang 8found in nematodes Thus they combine structural features
from mammals, plants, insects, nematodes and trematodes
This is the first known complete system where it is possible
to investigate the regulation of N-glycan modification in its
fullest variety An understanding of this complex system,
i.e why a distinct structure occurs on a certain protein, will
improve our knowledge on the rules of glycan modification
and help to optimize the production of recombinant
glycoproteins
In addition, the snail system itself may be useful for the
production of a large variety of glycoproteins For example
it may present the first opportunity to produce some
structures similar to those in pathogenic nematodes or
trematodes Proteins modified in the snail system could for
instance be used for the elucidation of the immune response
to those nonmammalian structures Furthermore the
snail-produced glycans may be a safe way to stimulate and
improve the human immune response to recognize and fight
against those pathogenic nematodes and trematodes
Acknowledgements
This project was financed by the Austrian Fonds zur wissenschaftlichen
Forschung Project number P13928-BIO We want to thank Dr
Manfred Pintar (Department for Integrative Biology, Institute for
Zoology, Universita¨t fu¨r Bodenkultur, Wien) for identification and
classification of the slugs, Daniel Kolarich and Dr Friedrich Altmann
for support on the MALDI-TOF and Dr Iain Wilson for reading the
manuscript The technical help of Thomas Dalik, Susanna Eglseer and
Denise Kerner is highly appreciated.
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