Glycobiology vol. 8 no. 8 pp. 799–811, 1998
Fucoseβ-1-P-Ser is a new type of glycosylation: using antibodies to identify a novel
structure in Dictyostelium discoideum and study multiple types of fucosylation
during growth and development
Geetha Srikrishna, Liying Wang and Hudson H.Freeze1
The Burnham Institute, La Jolla Cancer Research Center, La Jolla, California
92037, USA
Received on November 21, 1997; revised on February 13, 1998; accepted on
February 17, 1998
1To
whom correspondence should be addressed at: The Burnham Institute,
10901 North Torrey Pines Road, La Jolla, CA 92037.
Three antibodies that recognize distinct fucose epitopes were
used to study fucosylation during growth and development of
Dictyostelium discoideum. mAb83.5 is known to recognize an
undefined “fucose epitope on several proteins with serinerich domains, while mAb CAB4, and a component of antihorse-radish peroxidase, specifically recognize Fucα1,6GlcNAc and Fucα1,3GlcNAc residues respectively in the
core of N-linked oligosaccharides. We show that mAb 83.5 defines a new type of O-glycosylation. Serine-containing peptides incubated with GDPβ[3H]Fuc and microsomes formed
two fucosylated products. A neutral product accounting for
30% of the label did not react with the antibody, while the rest
of the label was incorporated into a charged product which
contained all the mAb83.5 reactive material. β-Elimination of
the labeled peptide or endogenous products produced
[3H]Fuc-1-P, indicating phosphodiester linkage to serine.
Fucβ-1-P and GDP-βFuc at 100 µM blocked mAb83.5 binding to endogenous and peptide products, but their α-linked
anomers did not. Electrospray ionization mass spectra of the
neutral and anionic labeled products showed major peaks of
mass units corresponding to O-Fuc-Ser peptide and O-Fucphospho-Ser peptide, respectively. The activity of Fuc-phosphotransferase exactly paralleled the accumulation of reactive glycans during growth and development. The expressions
of N-glycan core Fucα1,6GlcNAc and Fucα1,3GlcNAc and
their respective fucosyl transferase activities were also synchronous, but their developmental regulation differed from
one another. Fucα1,6GlcNAc was expressed maximally during growth but declined during development. In contrast core
Fucα1,3GlcNAc epitopes were expressed almost exclusively
during development. These findings provide direct evidence
for a novel type of O-phosphofucosylation, demonstrate the
existence of an O-fucosyl transferase, and identify two different types of core fucosylation in the N-glycans of Dictyostelium.
Key words: antibodies/Dictyostelium discoideum/
differentiation/fucosylation/fucosyl transferases
 1998 Oxford University Press
Introduction
Dictyostelium discoideum is a free-living, haploid amoeba which
is often used as a model system to study a myriad of basic
biological questions. These include evolution, genome organization, cytoskeleton dynamics, water and ion transport, and signal
transduction. In the laboratory, the unicellular amoebae can be
grown in nutrient media (axenic growth) or in association with
bacteria (bacterial growth). Growing ameobae phagocytose
bacteria, but starvation induces chemotatic aggregation into
groups of 105 cells that become mutually adhesive and undergo
a dramatic synchronous morphogenesis and cell-type differentiation into a multicellular organism. Briefly, the aggregating cells
become encased in an extracellular matrix that isolates them from
the environment. The newly created multicellular organism is
highly mobile and phototatic, and under the proper environmental
conditions cells differentiate into two major cell types, spore cells
and stalk cells. The individual spores are covered by a multilayered coat of mucin-like glycoproteins and cellulose that act as
a permeability barrier to prevent dessication. These spores set
upon a tapering stalk composed of evacuolated stalk cells, each
surrounded by cellulose, but lacking the mucin-like glycoproteins
that accompany spore cells (Loomis, 1975). Stalk and spore cells
synthesize different types of glycans (Freeze, 1997). Most
developmental studies have focused on inducers of protein
expression and cell differentiation that affect pattern formation,
as well as descriptions of cell movement within the aggregate and
its control during morphogenesis. A detailed and comprehensive
review on the biology of vegetative and developmental stages of
Dictyostelium has recently appeared (Maeda et al., 1997).
Our understanding of glycobiology of multicellular organisms
is based on recent studies in a limited number of model systems
such as Drosophila and the mouse. The most popular approach to
assess the physiological function of the glycans is to ablate one
or more of the relevant glycosyl transferases (Varki and Marth,
1995). Dictyostelium has also been used as a model to study
glycobiology during growth and development. However, a
fundamental problem is that relatively few structures have been
solved. As an alternative approach, several laboratories have
generated monoclonal antibodies against undefined carbohydrate
antigens expressed at different stages in development since many
Dictyostelium glycans are highly immunogenic in mammals (for
a review, see Freeze, 1997). Though the structures they recognize
are unknown, these antibodies have been very useful to study
developmental regulation of the respective epitopes and physiological consequences of their loss in mutant strains (Freeze,
1997). The primary defects in some mutants have been localized
to specific enzymes in the N- and O-linked glycosylation
pathways (Freeze et al., 1983a,b, 1989; Gooley et al., 1992).
Structures that have so far been resolved include Man-6-SO4 in
N-linked chains (Freeze and Wolgast, 1986), GlcNAc-O-threonine (Jung et al., 1997), GlcNAc-1-P in phosphodiester linkage
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G.Srikrishna, L.Wang and H.H.Freeze
to serine (Gustafson and Milner, 1980; Mehta et al., 1996), and
Fucα1,6GlcNAc in the core of N-linked oligosaccharides
(Srikrishna et al., 1997).
Previous studies suggest the presence of at least five types of
fucosylation in Dictyostelium. (1) “Core fucosylation in
N-linked chains, defined as “resistance of glycopeptides to
Endoglycosidase H digestion, is high in vegetative growth and
decreases during development, while “peripheral fucosylation
of N-linked chains displays the opposite pattern (Ivatt et al.,
1984). Neither type of fucosylation has been structurally
characterized. Fucose residues in α1,6 linkage to core GlcNAc
are widespread in many animal glycoproteins, but those in α1,3
linkage have so far only been documented in plant and some
insect proteins. (2) In elegant studies, West and coworkers have
demonstrated the association of fucose with a cytosolic protein
FP21, modified by an unusual cytosolic fucosyl transferase
(Kozarov et al., 1995). (3) O-Linked fucose analogous to that
seen in EGF domains of human blood proteins and other
mammalian proteins (Harris and Spellman, 1993) has been found
on spore coat protein SP96 (Riley et al., 1993). (4) Another
“unknown O-linked fucose epitope recognized by mAb83.5 is
also present on spore coat proteins (West and Erdos, 1988). (5)
Prespore cell surface glycoprotein PSA (SP29) contains glucosamine, fucose, and phosphate (Haynes et al., 1993). However, PSA
is not recognized either by 83.5, or by MUD62, a similar but
independently isolated antibody (Grant and Williams, 1983).
Western blots show that fucose epitopes on PSA and SP96 are not
localized on the same proteins. Also distinct mutants lacking the
two epitopes have been generated (Champion et al., 1991).
To further characterize developmentally regulated fucosylation in Dictyostelium, we used three fucose-specific antibodies.
The first of these is mAb83.5, which was generated against slug
stage proteins (West et al., 1986). The undefined “fucosecontaining epitope is found on a lysosomal enzyme cysteine
proteinase 7 (CP7, cprG) expressed only in bacterially grown
cells (Mehta et al., 1996), as well as in a set of three proteins SP96
(cotA), SP75 (DP87, cotD), and SP80 that are expressed late in
development (West and Erdos, 1988). These three proteins are
stored in prespore vesicles (PSVs) prior to their secretion and
subsequent incorporation into the spore coat. Mutants lacking the
mAb83.5 reactive fucose epitope have more porous spore coats
(Gonzalez-Yanes et al., 1989), suggesting that it may play an
important role in protecting the integrity of the spore coat. CP7,
SP96, SP75, and SP80 are phosphorylated and all, except SP80,
have been cloned (Fosnaugh and Loomis, 1989; Osaki et al.,
1993; Ord et al., 1997). Predicted sequence shows that each has
a serine-rich domain that is likely to be a site for phosphorylation
or glycosylation since these regions also resemble mucins (West
et al., 1996). The second antibody is CAB4. We recently showed
that it specifically recognizes Fucα1,6GlcNAc in the core of
mammalian N-linked chains, but not Fucα1,3GlcNAc
(Srikrishna et al., 1997). It also does not recognize fucose in other
linkages. The third is an affinity purified component of anti-HRP
which recognizes Fucα1,3GlcNAc found in the core of insect and
plant N-linked glycans, but not Fucα1,6GlcNAc (G.Srikrishna,
unpublished observations). Our studies with these antibodies
reveal a novel type of O-fucosylation in Dictyostelium, and
provide more direct evidence for the presence of the two specific
types of core fucosylation. They also demonstrate differential
expression of the respective fucosyl transferases during growth
and development.
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Results
Fucosyl transferase assay for generation of mAb 83.5
epitope
mAb 83.5 was raised against slug stage proteins (West et al., 1986).
Its recognition of a “fucose epitope on CP7 and spore coat
proteins is blocked by high concentrations of L-fucose (West et al.,
1986; Mehta et al., 1996). However, we found that the antibody
does not react with any of 15 different proteins or neoglycoproteins
that contain α fucose in linkages commonly seen in N- or O- linked
sugar chains, and glycolipids (data not shown; for a list of
oligosaccharides tested for reactivity, see Srikrishna et al., 1997).
Therefore, it was likely that mAb 83.5 recognized fucose in a novel
type of linkage. A specific fucosyl transferase assay was designed
to generate the antigen, and the high specificity of the antibody was
used to detect the product. Since the amount of endogenous
acceptors varied, we looked for an artificial acceptor that could also
be used to identify the epitope structure. All three mAb 83.5
reactive proteins, CP7, SP75, and SP96 have prominent serine rich
domains (West et al., 1996; Ord et al., 1997) . Based on serine
motifs in these domains, three octanoylated serine-containing
synthetic peptides, GSSSSSSG, GSGSGSGS, and SGSQSGSQ,
were tested as acceptors using microsomes from 20 h developed
cells and unlabeled or 3H-labeled GDPβFuc. The soluble peptide
products were purified using C-18 spin columns and either counted
directly or applied to ELISA plates for detection with mAb 83.5.
Blank incubations lacked peptide, and recovery was quantified by
estimating preparation losses using previously purified material.
The reactions were linear with time and protein. Km for GDPβFuc
was 17 µM calculated from mAb 83.5 reactivity using the -SGpeptide, and only GDPβFuc, but not its α anomer, served as a
donor in the reaction. None of the native peptide acceptors reacted
with the antibody (not shown). Each peptide product showed
comparable 3H incorporation and reactivity with mAb83.5. Kinetic
analysis of each peptide acceptor gave similar Km values
(75–100 µM) calculated from either radiolabeled product or
ELISA reactivity. These results suggested that the major radiolabeled product was probably identical to the antigenic product.
Since none of the three serine-motifs was highly preferred over the
others, the remaining experiments use C8-GSGSGSGS peptide.
mAb83.5 defines a new type of protein modification
Since generation of the fucose epitope required only the peptide
and no prior glycosylations or additional sugar nucleotides, it is
likely that the antibody recognizes fucose directly bound to the
peptide. We used acid and base hydrolysis, QAE-Sephadex,
anion-exchange HPLC, ESI-MS, and MALDI-TOF-MS to
characterize the products further. β-Elimination of the labeled
fucosylated peptide product by treatment with 0.1N NaOH at
room temperature for 4 h, (conditions under which GlcNAc-1-P
was completely liberated from the peptide), released 60–70% of
the label from the peptide showing that fucose is probably in an
O-linkage to serine. Label released by β-elimination bound to
QAE-Sephadex and was eluted by 125 mM NaCl suggesting that
it has 2–3 negative charges; the charges were neutralized by
alkaline phosphatase treatment, suggesting the presence of a
phosphomonoester (not shown). The purified peptide product and
β-eliminated products before or after phosphatase digestion were
analyzed by TLC. The peptide product ran close to the solvent
front. Following β-elimination, the product comigrated with
standard Fucβ-1-P (Figure 1), and with L-fucose after phosphatase
Fucosylation in Dictyostelium
Fig. 1. β Elimination of peptide products liberates Fuc 1-P. Label was
released from peptide product(s) by incubation with 0.1N NaOH at room
temperature for 4 h. Released label was isolated on C18 spin columns, and
repeatedly lyophilized from water to remove ammonium formate. It was then
analyzed on a cellulose TLC run in ethyl acetate/acetic acid/water, 6:4:3
(v/v) 0.5 cm strips were cut, wetted with water, and counted. The position of
authentic Fucβ-1-P was visualized by performing Dische-Shettles reaction
(Beeley, 1985) on material eluted from 0.5 cm strips. Arrows mark the
positions of [3H]GDPβFucose, and of the peptide product before β
elimination. Alkaline phosphatase digestion converts released label to
L-fucose (not shown).
digestion (not shown). These results showed that at least 60–70%
of the label was consistent with it being [3H] Fuc-1-P in a
phosphodiester O-linkage to the peptide.
HPLC analysis (Figure 2) showed two products. Peak 1 was a
neutral molecule that accounted for 28% of the label, and did not
react with the antibody. The same product was seen in the
run-through of a QAE Sephadex column (not shown). Peak 2
represented <5% of the total radiolabeled products, did not react
with the antibody, and was not analyzed further. Peak 3 contained
67% of the label and all of the mAb83.5 reactive material. This
peak eluted at the same position as GDP[3H]βFuc which has two
negative charges. This major product was however distinct from
GDPβFuc, since thin-layer chromatograms showed vastly
different Rf values for the two compounds (Figure 1). Since the
same native peptide modified by a single GlcNAc-1-P linked to
serine (Mehta et al., 1997) elutes between the neutral molecule and
the major fucosylated product, it suggested that the latter had at
least two negative charges. This distribution of charge matches
with 70% of label being liberated from the peptide products by
mild alkali hydrolysis. Prolonged incubations with alkali at higher
temperatures releases O-linked fucose (Stults and Cummings,
1993; Moloney et al., 1997), but they partially hydrolyze Fuc-1-P,
and were not routinely used. Also, subquantitative release of the
label could be expected if the C-terminal serine was O-fucosylated
(Montreuil et al., 1986). However, multiple charges on the serine
peptide as revealed by HPLC and QAE analysis, show that more
than one serine residue was modified, since the label released by
alkali is associated only with Fuc-1-P.
HPLC peaks 1 and peak 3 were subjected to acid hydrolysis.
Mild acid hydrolysis sufficient to cleave labile phosphodiester
bonds released >85% of the label from peak 3 (Figure 3B), while
peak 1 was unaffected (Figure 3A). Strong acid hydrolysis
released a great majority of the label from peak 1 (not shown).
These results are consistent with the presence of Fuc-1-P in a
phosphodiester linkage to the peptidyl serine in peak 3 product.
The calculated mass for the unmodified GS- peptide is 720.8.
Figure 4 shows ESI-MS spectra of HPLC purified peak 1 and
Fig. 2. Anion exchange HPLC of serine peptide products. Fucosylated
product(s) generated on peptide acceptors were isolated from C18 spin
columns, and subjected to anion-exchange HPLC on Microsorb-MV
amine-bonded silica column. The product(s) were loaded in 0.01M
ammonium formate pH 6.0 with 40% acetonitrile, washed for 5 min, and
eluted with a linear gradient of 0.01–0.25 M ammonium formate pH 6.0 for
1 h with a flow rate of 0.5 ml /min. Individual fractions (0.5 ml) were
collected, and aliquots were counted for radioactivity measurement. A, B,
and C represent the positions of known standards: (A) [3H] fucose, (B)
C8-GSGSGSGS peptide modified by phophodiester-linked [3H]
GlcNAc-1-P, (C) GDP[3H]βfucose. The three radioactive peaks marked 1, 2,
and 3 were individually pooled, desalted on C18 spin columns and eluted
with 50% MeOH. Equimolar amounts of products from individually pooled
peaks were tested for reactivity with mAb 83.5 by ELISA. Vertical bars
represent antibody reactivity.
peak 3. Peak 1 gave a negative ion mass of 863 consistent with
one fucose unit in O-linkage to the peptide. Peak 3 gave a negative
ion mass of 1166. These results combined with acid lability of the
label in peak 3 are consistent with two Fuc-phosphoryl groups in
O-linkage to the peptide. Mild acid hydrolysis of peak 3 followed
by digestion with alkaline phosphatase resulted in reappearance
of the original peptide with a mass of 719. Spectra run in the
positive ion modes gave identical results, with one or more
sodium adducts. MALDI-TOF analysis showed negative ion
masses of 864 for peak 1 and 1171.7 for peak 3, similar to the
ESI-MS data (not shown).
To verify that similar products were made on endogenous
acceptors, cell extracts generated at 22 h of development were
labeled with GDP[3H]βFuc and analyzed. Before β-elimination,
a majority of the label remained at the origin (Figure 5). After
β-elimination approximately 45% of the label comigrated with
Fuc-1-P and 30% with free fucose. About 20% remained closer
to the origin, and probably represents label associated with
N-glycans. Phosphatase digestion converted all of the label in the
position of Fuc-1-P to L-fucose (not shown). Thus, Fuc-1-phosphotransferase and O-Fuc transferase activity appear to be major
consumers of GDP[3H]Fuc for endogenous acceptors incubated
under these conditions, and generate products similar to those
made on peptides.
High concentrations of L-fucose are known to inhibit the
binding of the mAb83.5 to spore coat proteins (West et al., 1986).
If Fuc-1-P was indeed the epitope recognized by the antibody, we
reasoned that binding of the antibody to both peptide products and
endogenous proteins should be competed much better by lower
concentrations of Fuc-1-P. Figure 6 shows inhibition of the
binding of mAb83.5 to endogenous proteins by different sugars.
The β anomers of Fuc-1-P, and GDP β Fuc were much more
effective than their corresponding α anomers in blocking the
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G.Srikrishna, L.Wang and H.H.Freeze
Fig. 3. Acid hydrolysis of HPLC purified serine peptide products. Peptide
products separated by anion exchange HPLC (peaks 1 and 3, Figure 2) were
desalted and subjected individually to mild acid hydrolysis (A, peak 1; B,
peak 3) as described in Materials and methods. The hydrolyzed material
were loaded on C18 spin columns, washed with 0.1 M ammonium formate
pH 6.0, and eluted with 50% MeOH. Radioactivity in individual fractions
was measured by scintillation counting.
binding. L-Fucose was also 10- to 20-fold less effective, and
Glc-1-P, a sugar phosphate control, did not inhibit binding at any
concentration studied. Identical inhibition results were seen with
the binding of mAb83.5 to peptide products (not shown). These
results suggest that GDP-β L-Fuc directly donates Fucβ-1-P to
serine residues on the peptides, and to serine or threonine residues
on endogenous acceptors to create the essential minimal epitope
for mAb 83.5 recognition.
Fuc-1-P epitope and the corresponding
Fuc-phosphotransferase activity are developmentally regulated
mAb83.5 was next used to study the developmental expression of
Fuc-1-P and Fuc-1-phosphotransferase. In agreement with past
results, the predominant reactive bands are spore coat proteins
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Fig. 4. Electrospray ionization-mass spectrometry of HPLC purified serine
peptide products. Peptide products separated by anion exchange HPLC
(peaks 1 and 3, Figure 2) were desalted on C18 spin columns. Aliquots were
subjected to ESI-MS, recorded in both negative and positive ion modes.
Spectra recorded in the negative ion modes are given here. C8-GSGSGSGS
has a calculated mass of 720.8. (A) Peak 1 product gives a mass of 863.0
consistent with one fucose in O-linkage to the peptide. (B) Peak 3 product
gives a mass of 1166 consistent with two Fuc-phosphoryl groups in
O-linkage to the peptide. (C) Mild acid hydrolysis of peak 3 product
followed by digestion with alkaline phosphatase resulted in reappearance of
the native peptide with a mass of 719. MALDI-TOF analyses gave identical
results (not shown).
Fucosylation in Dictyostelium
weight cysteine proteinase CP5 is seen, which is known to react
with both mAb 83.5 and mAb AD7.5 (Souza et al., 1995).
Fuc-1-phosphotransferase activity exactly parallels the
accumulation of the Fuc-1-P-containing glycans (Figure 7B). It
transiently increases during bacterial growth around the time of
bacterial clearing, corresponding to the expression of the CP7.
Between 8 and 16 h there is a 100-fold increase in specific activity
correlating with the expression of the spore coat proteins.
Two linkage-specific antibodies recognize fucoses in the
core of N-linked glycans
Fig. 5. β Elimination of endogenous products liberates both fucose and
Fuc-1-P. Endogenous acceptors were labeled as described in Materials and
methods. Label was released by β elimination and the assay mixture was
analyzed on TLC as indicated above. Released label comigrated with
authentic Fucβ-1-P and l-fucose. Alkaline phosphatase digestion converts
released label to l-fucose ( not shown).
Fig. 6. Binding of mAb 83.5 to endogenous products is more effectively
inhibited by Fucβ-1-P and GDP β-L Fuc than by their α anomers or by
l-fucose. Endogenous products were coated onto an ELISA microwell plate
and the wells were incubated with 1 µg/ml of 83.5 in the presence of varying
concentrations of β or α-l-fucose-1-phosphate, GDPβ-l-fucose, l-fucose and
Glucose-1-phosphate. The plates were developed with alkaline phosphatase
conjugated anti-mouse IgG, and p-nitrophenyl substrate. Binding in the
absence of inhibitor was considered 100%. Each point is the mean of two
determinations, varying by <10%. Identical results were obtained when
fucosylated serine peptides were used as antigen, and GDPα-l-Fuc follows
the inhibition pattern of Fucα-1-P (not shown).
SP75 and SP96 (Figure 7A), with SP80 between. They begin to
appear around 16 h of development and peak at 20 h. mAb 83.5
also recognizes distinct proteins appearing near the end of growth
on bacteria or in axenic medium. In bacterially grown cells, the
predominant band is the 38 kDa cysteine proteinase 7 (CP7), as
shown by reaction to anti-GlcNAc-1-P antibody, AD7.5, in
agreement with previous results (Mehta et al., 1996). The
∼55 kDa band may represent a precursor form of CP7 (Ord et al.,
1997). Both of these bands progressively decrease in intensity
during development, as cysteine proteinase activity decreases.
CP7 is not expressed in axenic cells, but another lower molecular
mAb CAB4 was raised against Dictyostelium cell surface
glycoproteins expressed at mid-development (Crandall and
Newell, 1989). We recently showed that they specifically
recognize core Fucα1,6 GlcNAc in N-linked chains produced by
mammalian cells, regardless of other substitutions on the chain
(Srikrishna et al., 1997). These antibodies do not cross-react with
proteins or neoglycoproteins that contain α-fucose in other
linkages commonly seen in N- or O- linked chains. This provides
a proven probe for core Fucα1,6 GlcNAc-containing glycans.
Later in development, Dictyostelium accumulates cellulose
and other highly processed N-linked chains typical of plant
glycoproteins (Loomis, 1975; Amatayakul-Chantler et al., 1991;
Sharkey and Kornfeld, 1991). Developed cells might therefore
synthesize N-glycans containing core Xylβ1,2Manβ-, and Fucα1,3GlcNAcβ-epitopes characteristic of plant glycoproteins.
Antisera against plant glycoproteins such as HRP or pineapple
stem bromelain contain antibody populations that recognize each
of those modifications (Faye et al., 1993). The two populations
could be separated from each other by affinity purification using
bee venom phospholipase (PLA2), which has only core Fucα1,3GlcNAcβ-, not Xylβ1,2Manβ. The affinity-purified antifucose antibody specifically recognized Fucα1,3GlcNAcβ in the
core of plant N-glycans, and on PLA2, but not Fucα1,6GlcNAcβ
in the core of mammalian N-glycans, or Fucα1,3 GlcNAc on
outer lactosamine groups, e.g., as in human α1acid glycoprotein.
This anti-fucose component of anti-HRP therefore provides a
specific probe for N-linked glycans containing Fucα1,3GlcNAcβ. Details of the purification, fractionation, and characterization
of the anti-HRP components will be described elsewhere.
Both anti-core Fucα1,6GlcNAcβ and Fucα1,3GlcNAcβ antibodies were individually used to probe expression of each
fucosylated glycan during growth and development by immunoblots
and immunoassays. Antibodies that recognize Xylβ1,2Manβ in
control plant proteins did not show any reactivity with Dictyostelium
proteins (not shown).
Reciprocal expression of core Fucα1,6GlcNAc and core
Fucα1,3GlcNAc glycans during growth and development
Many proteins expressed during growth on bacteria and in axenic
medium react strongly with the anti- core Fucα1,6GlcNAc
antibody, CAB4. Expression progressively decreases as development begins with a transient increase around 16 h of development
(Figure 8A). Binding is specifically inhibited by 50 µM porcine
fibrinogen, which contains core Fucα 1,6 GlcNAc (not shown).
In contrast, core Fucα1,3GlcNAc antigen is essentially absent
during growth on axenic medium, and appears only at the very
end of growth on bacteria (Figure 9A). Its expression increases
dramatically during development, especially on several proteins
(>80 kDa) that accumulate at 16–24 h. Binding is completely
inhibited by 10 µM HRP, which carries core Fucα1,3 GlcNAc,
showing that recognition is specific (not shown).
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G.Srikrishna, L.Wang and H.H.Freeze
Fig. 7. (A) Immunoblots of Dictyostelium discoideum lysates made from cells at various stages of axenic or vegetative growth (left) or development were probed with
mAb 83.5. Cell densities indicated during axenic growth are ×106/ml, and during bacterial growth are ×108/plate. Thirty micrograms of protein from each lysate was
separated by electrophoresis on 12% SDS–polyacrylamide gels, blotted onto nitrocellulose membranes, and blocked with 10% milk in TBS. The membranes were
incubated with 83.5 (1 µg IgG /ml), and further developed with alkaline phosphatase conjugated goat anti-mouse IgG and BCIP/NBT substrate. (B) A comparison of
Fuc-phosphotransferase activities in membranes isolated at various time points in growth and development. Each point is the mean of two determinations.
Design of ELISA-based core fucosyl transferase assays
We developed two ELISA-based assays to measure the activities
of the core fucosyl transferases. Initial experiments showed that
covalently bound HRP glycopeptides were readily detected by
glycan-directed anti-HRP. Covalently bound asialo-agalactotransferrin glycopeptides (GlcNAc-terminated biantennary
804
chains) is a substrate for either core Fuc transferase. Glycopeptides (native, or conjugated to BSA) immobilized on
microtiter plates were incubated with GDPβFuc and microsomes
and the antigenic products generated were individually detected
by the specific antibodies. Glycopeptides coated directly on the
plates or linked to BSA give similar results (not shown).
Fucosylation in Dictyostelium
Fig. 8. (A) Immunoblots of Dictyostelium discoideum lysates made from cells at various stages of axenic or bacterial growth or development, probed with CAB4
(anti core Fucα1,6GlcNAc antibody), as described in Figure 7A. (B) A comparison of core Fucα1,6 transferase activities in membranes isolated at various time
points in growth and development. Each point is the mean of two determinations.
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G.Srikrishna, L.Wang and H.H.Freeze
Fig. 9. (A) Immunoblots of Dictyostelium discoideum lysates made from cells at various stages of axenic or vegetative growth or development, probed with anti
core Fucα1,3GlcNAc component of anti-HRP, as described in Figure 7A. (B) A comparison of core Fucα1,3 transferase activities in membranes isolated at
various time points in growth and development. Each point is the mean of two determinations.
Identification of the coreα1,3 Fuc T product by the anti-Fucα1,3GlcNAcβ fraction of anti-HRP was specifically inhibited by
HRP, but not by pFg. Similarly, identification of the coreα1,6
806
FucT product by CAB4 was specifically inhibited by pFg, but not
HRP (not shown). As positive controls for the individual core
fucosyl transferases, we used mung bean seedlings (core α1,3
Fucosylation in Dictyostelium
FucT) and human skin fibroblasts (core α 1,6 Fuc T). The specific
activity of the enzyme in the total mung bean extracts as measured
by the solid phase assay was 11.3 pmol/min/mg protein and
corresponds well with activity of 23 pmol/min/mg protein
reported earlier using a radiolabeled product measurement assay
(Staudacher et al., 1995).
Under the assay conditions, formation of the respective
fucosylated products was proportional to increasing amount of
enzyme and to time of incubation. Km values for GDPβFuc were
1.5 µM for core α1,3 Fuc T, and 3.1 µM for core α1,6 Fuc T.
GDPαFuc did not serve as a donor for either enzyme.
Developmental expression of core fucosyl transferases
The expression of both Fuc transferase activities was measured
throughout growth and development. Their expression correlated
generally well with the reciprocal expression of the respective
fucose epitopes during development (Figures 8B, 9B). Core α1,3
Fuc T activity was low during growth and increased dramatically
by 12 h into development; core α1,6 Fuc T activity on the other
hand was maximal during growth and decreased during development. High activities of core α1,6fucosyl transferase during early
axenic growth (Figure 8B) with a corresponding low level of
expression of core fucosylated proteins (Figure 8A) could be due
to nonavailability of acceptor proteins during early growth. Also
a transient fall in enzyme activity at cell densities of 1.7 × 108
during bacterial growth may be artifactual because there is no
corresponding decrease in expression of core fucosylated proteins. A comparison of the activities of the two core fucosyl
transferases in membrane preparations from various sources is
given in Table I.
Table I. Comparison of two core fucosyl transferase activities in various
membrane preparations
Source of membranes
Mung bean seedlings
Human skin fibroblasts
Specific enzyme activity (pmol /min /mg protein)a
Core α1,3Fuc T
Core α1,6 Fuc T
11.3
<0.001
0.004
0.27
Dictyostelium:
Axenic growth
0.48
4.33
24 h development
2.67
0.35
aEach
value is the mean of two determinations.
Discussion
The goal of this study was to (1) define fucosylated structures
recognized by three different antibodies, (2) use each antibody to
study the developmental regulation of the fucose epitopes they
recognize, and (3) develop sensitive antibody-based fucosyl
transferase assays in crude microsomes.
Our studies show that the minimal epitope recognized by mAb
83.5 is Fuc-1-P, since no prior or additional glycosylations were
required for antibody reactivity. However, it is likely that two or
more Fuc-1-P residues may enhance antibody recognition. The
success of using the peptide acceptors makes it likely that Fuc-1-P
is processively added to the Ser or Thr-rich domains in the
proteins; however, threonine acceptors have not been tested, and
the actual glycosylation sites in the native acceptors are unknown.
Since these domains resemble mucin-like glycoproteins, and
three different serine peptides were equally good acceptors, there
is probably no one consensus sequence. The protein’s three
dimensional structure may influence addition of Fuc-1-P, as it
does the addition of GlcNAc-1-P to Ser in Dictyostelium cysteine
proteinases (Mehta et al., 1997). We do not know which serine
residues were preferentially modified, whether Fuc-1-P can be
further elongated, and if additional modifications enhance or
decrease antibody binding. Since two residues of Fuc-1-P are
added to the peptide acceptors, it is likely that Fuc-1-P addition
is processive on a single acceptor. In the native proteins, more
than one serine or threonine is probably modified. SP96 and
SP75, the major mucin-like glycoproteins that react with mAb
83.5 are highly modified proteins (Fosnaugh and Loomis, 1989;
West et al., 1996). Phosphofucosylation of these proteins may be
analogous to phosphoglycan assembly in the serine/threonine
rich domains of acid phosphatase and “mucin-like proteophosphoglycans of Leishmania, which are modified by Manα1-P-Ser,
and elongated by other sugars (Mengeling et al., 1997).
This is the first report of Fucβ-1-P-Ser modification of
proteins. Distribution of this epitope in spore coat proteins
suggests that the negative charge may be crucial in preserving the
integrity of the spore coat. mAb83.5 antigen-defective mutants
have more porous spore coats and decreased spore viability
(Gonzalez-Yanes et al., 1989). Fuc-1-P may have limited
distribution, since immunoblots and ELISAs of various mammalian cell lines and SDS-solubilized tissues did not have any
detectable reactivity using mAb 83.5 (not shown). Although
O-fucose is found in mammalian proteins with EGF modules
such as tissue plasminogen activator (tPA) and other coagulation
proteins (Harris and Spellman, 1993), mAb 83.5 does not
recognize this modification either in human tPA or in our
synthetic peptide products. However, synthesis of O-fucose in
Dictyostelium suggests that this modification has been conserved
for selected proteins in higher organisms.
The N-linked oligosaccharides of Dictyostelium are derived
from a lipid linked precursor, consisting of GlcNAc2Man9Glc3,
common to most eukaryotic cells. After the glycans are transferred, two types of processing occur and correspond to two major
stages in the developmental program. There is very little mannose
processing during vegetative growth and through early development, and complex-type N-linked chains are probably not found
(Amatayakul-Chantler et al., 1991; Sharkey and Kornfeld, 1991).
High levels of core fucosylation, as deduced from Endo H
resistance, are seen in vegetative growth on both axenic cultures
and bacterial lawns, when it steadily rises during mid and late
exponential growth (Tschursin et al., 1989). Our studies show that
this core fucosylation is indeed Fucα1,6GlcNAc. In the synthesis
of mammalian N-linked oligosaccharides, addition of fucose is
believed to be a terminal event occurring exclusively on complex
or hybrid structures (Kornfeld and Kornfeld, 1985). However,
there is now growing evidence that core fucosylated high
mannose structures do occur in mammalian cells. Lin et al.
identified core Fucα1,6GlcNAc in the N-glycans of GlcNAc
transferase 1 deficient Lec-1 CHO cells that cannot synthesize
complex or hybrid N-glycans (Lin et al., 1994). More recently,
Endo et al. documented the presence of novel fucosylated high
mannose type sugar chains in the oligosaccharides of the rat
hepatoma alkaline phosphatase (Endo et al., 1996).
Later in development, Dictyostelium induces two N-glycantrimming α-mannosidases that generate a high proportion of
chains carrying 3–5 mannose residues similar to those found in
insects and plant glycoproteins (Sharkey and Kornfeld, 1991).
From our studies it is evident that when mannose-trimmed
807
G.Srikrishna, L.Wang and H.H.Freeze
substrates become available during late development, there is a
synchronous increase in plant-like core fucosylation, i.e., core
Fucα1,3GlcNAc. Though there is core α1,3 FucT activity during
axenic and bacterial growth (Figure 9B), there is no corresponding expression of core Fucα1,3GlcNAc containing proteins
(Figure 9A). This is probably because of the unavailability of
mannose-trimmed substrates during growth.
Fluorograms of 3H-fucose-labeled total glycoconjugates at
different stages in development (Lam and Siu, 1981) show that low
molecular weight proteins (<80 kDa) are predominantly fucosylated in early development. In our immunoblots, many of these
bands correspond to those modified by core Fucα1,6GlcNAcβ
(Figure 8A). These low molecular weight fucosylated proteins
progressively decrease by mid-development, and high molecular
weight ones (>80 kDa) appear during late development. Again our
immunoblots show that most of these bands in late development
correspond to those spore coat proteins modified by Fuc-1-P, and
by core Fucα1,3GlcNAcβ (Figures 7A, 9A). This is also consistent
with earlier studies showing intense 3H-fucose labeling of spores
but not stalks (Gregg and Karp, 1978).
Standard methods to measure core fucosyl transferase activities utilize labeled GDPβFuc and count the labeled products
after removing excess labeled sugar donor on a Dowex column
(Staudacher et al., 1995), or by binding the labeled products to a
lentil-lectin Sepharose column (Voynow et al., 1988). Although
useful, these methods require product isolation and more
importantly, often lack specific product identification. To cite an
example, Cummings and coworkers emphasize the importance of
specific product identification in the assay of GDPFuc:Galβ1,4GlcNAc-R (Fuc to GlcNAc) α1,3 fucosyl transferase which generates the blood group antigen Lewisx (Yan et al.,
1994). Galβ1,4GlcNAc-R is also the acceptor for human
H-fucosyl transferase which generates the H-antigen, Fucα1,2Galβ1,4GlcNAc-R. Similarly, both core Fuc transferases
can transfer fucose from GDPFuc to the common Gn2Man3Gn2
glycopeptide acceptor, and the Fucα1,3GlcNAc product cannot
be distinguished from Fucα1,6GlcNAc containing product by
merely measuring radioactivity, especially if both enzymes are
present in the same incubation. The availability of linkage-specific antibodies that recognize core fucose epitopes prompted us
to develop solid-phase assays that not only eliminate the use of
radioactivity, but also improve specific identification of the
product. Under conditions used for fucose transfer to endogenous
and the Ser-Gly peptide acceptors to generate 83.5 antigen,
fucosylation of N-glycans of endogenous acceptors also occurs
(Figure 5). While labeling was essential in tracking the product,
specific identification of the Fuc-1-P epitope was possible by
ELISA using 83.5. Also, as little as 10 fmol of fucose
incorporation was detectable by ELISA. This sensitivity can
easily be increased several orders of magnitude by using
4MU-phosphate or a chemiluminescent substrate.
Materials and methods
Anti-Dictyostelium mAb83.5 was a kind gift from Dr. Christopher West of the University of Florida College of Medicine.
CAB antibodies were generously provided by Dr. Peter Newell,
University of Oxford, United Kingdom. Polyclonal rabbit
anti-HRP, HRP-agarose, PLA2-agarose, lactoferrin, bromelain,
jack bean β galactosidase, α and β L-Fuc-1-phosphates and
GDPαFucose were obtained from Sigma. Human transferrin and
GDPβFucose were from Calbiochem, La Jolla, CA. Proteinase K
was obtained from Boehringer Mannheim. N-Octanoyl (C8)
808
serine peptides were from Tana Laboratories, Houston, TX.
Bonded C18 reverse phase silica gel was from Analtech, Newark,
DE. Microsorb-MV amine-bonded silica column was from
Rainin Instrument Co. Inc., Emeryville, CA. Cellulose TLC
plates were from Eastman-Kodak Company, Rochester, NY.
Xenobind covalent binding plates were obtained from Xenopore
Corporation, Hawthorne, NJ. Mung beans were purchased at a
local food store.
Growth and harvest of cells of D.discoideum and
preparation of cell lysates
Dictyostelium discoideum strain AX4 were grown axenically in
HL-5 medium or in association with Klebsiella aerogenes on agar
plates. Cells were generally subcultured to densities of 3 ×
105 cells/ml from high density cultures. At this stage cells enter
exponential growth and begin to plateau at 0.8–2.0 × 107 cells/ml.
Growth ceases completely in axenic cultures at about 2–3 ×
107 cells/ml. From axenic cultures, cells were harvested both at
exponential and stationary phases of growth. From bacterial
cultures, cells were harvested at different time points prior to and
at the time of clearing of the bacterial lawns (0 time point), and
washed free of residual bacteria. Development was initiated after
the cells had cleared the bacteria. They were washed, plated on
2% buffered agar plates, and harvested at various time points that
correlated with their well-established developmental stages. The
cell samples were disrupted by freeze-thaw, in 10 mM Tris–HCl
pH 7.5 with 1 mM DTT, and lysates were analyzed after
quantitation of protein.
Preparation of membrane extracts from mung bean
seedlings, human skin fibroblasts, and Dictyostelium
Mung bean seedlings: (positive control for GDP-fucose:N-acetyl
β-D-glucosaminide α1,3fucosyl transferase or core α1,3 Fuc T).
Membrane extracts were made from mung bean seedlings as
described by Staudacher et al. (1995). Briefly, 25 g of mung beans
were soaked in tap water overnight at 37C and then germinated
on moist cheese cloth in darkness at 37C for 2 days. The coats
were manually removed. The seedlings were homogenized in
50 mM TrisHCl buffer pH 7.3 containing 250 mM sucrose and
0.5 mM DTT. Triton X-100 was added to the resulting suspension
to a final concentration of 1%. The suspension was stirred in the
dark at 4C for 24 h, and a high speed supernatant was harvested.
Human skin fibroblasts: (positive control for GDP-fucose:Nacetyl β-D-glucosaminide α1,6-fucosyl transferase, or core
α1,6Fuc T). Normal human skin fibroblasts obtained from
American Type Culture Collection were grown to confluency in
α-minimal essential medium supplemented with 10% fetal
bovine serum and 2 mM glutamine. Cells were harvested and
membrane extracts were made essentially as described by
Voynow et al. (1991). Briefly, 2 × 107 cells in a frozen pellet were
sonicated first in 0.1% Triton X-100, and after centrifugation, the
pellet was resuspended and resonicated in 50 mM sodium
cacodylate buffer pH 6.0 with 10mM NaCl, 40 mM MgCl2, 10%
glycerol, and 2% Triton CF 54. A high speed supernatant was then
harvested.
Dictyostelium membranes. Membranes were prepared from AX4
strain of Dictyostelium discoideum cells grown axenically in
HL-5 medium, or in association with Klebsiella aerogenes on
agar plates. Cells were plated from axenic cultures on 2%
buffered agar to initiate development, and harvested at various
Fucosylation in Dictyostelium
time points during development. Cells were immediately washed
in either 50 mM Tris buffer pH 7.5 or in 10 mM sodium phosphate
pH 6.5 thrice, and sonicated in the same buffer. After a low speed
centrifugation to remove nuclear pellets, the supernatants were
centrifuged at 80,000 × g for 20 min to collect microsomes which
were resuspended in the initial buffer, aliquoted, and stored at
–80C after protein measurement.
Immunoassays
Proteins were immobilized on ELISA plates, and unbound sites
were blocked with 3% bovine albumin (BSA) in TBS overnight.
The wells were washed and the antigens were then allowed to
react with the primary antibodies in TBS containing 1% BSA and
0.1% Tween 20 for 1 h at room temperature. The wells were then
washed and incubated with alkaline phosphatase conjugated goat
anti-mouse IgG, followed by development with p-nitrophenyl
phosphate substrate, and read in an ELISA plate reader.
SDS–polyacrylamide gel electrophoresis and Western blot
analysis
Proteins were separated by SDS-PAGE in 12% gels and transferred
to nitrocellulose membranes. The membranes were blocked overnight with 10% fat-free milk in TBS, washed with TBS containing
0.05% Tween 20, and incubated with the primary antibodies for 1
h at room temperature. This was followed by reaction with alkaline
phosphatase conjugated goat anti-mouse IgG. Bound proteins were
visualized by incubating with 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium substrate.
Fucosyl transferase assay for the generation of mAb 83.5
antigen, and analysis of the products
N-Octanoyl(C8)GSGSGSGS peptide was the acceptor for most
assays. In some experiments similar peptides, (C8)GSSSSSSG or
(C8)SGSQSGSQ (Mehta et al., 1997), were used. The standard
incubation mixture contained, 0.1 M MES, pH 6.8, 100 µM
acceptor peptide, 50 µM GDP Fuc, 10 mM MnCl2, 0.1% Triton
X-100, 5 mM AMP, and 100 µg membrane protein in 50 µl. After
incubation at 22C for 60–90 min, the reaction was terminated by
heating at 100C for 3 min, diluted to 250 µl with water, and
centrifuged at 10,000 × g for 5 min to remove precipitated
proteins. The supernatant was loaded on a C18 spin column and
extensively washed with 0.1 M ammonium formate pH 6.0, and
the peptide was eluted with 50% methanol. The product was dried
by lyophilizing twice in water, or in a SpeedVac and was detected
by its reactivity with mAb83.5. One unit of FucT activity was
defined as amount of enzyme catalyzing the transfer of 1.0 pmol
of fucose per min under the assay conditions.
Lipid ELISA for the detection of 83.5 FucT product
Lyophilized peptide product was reconstituted in 45% methanol;
25 µl aliquots each containing 0.1–0.2 pmol were applied to
individual wells of a 96-well microtiter plate, and allowed to dry
at room temperature. The wells were blocked overnight with 5%
BSA in PBS and incubated with 1.0 µg/ml of 83.5 mAb for 1 h
at room temperature in PBS containing 1% BSA and 0.1%
Tween-20. They were then developed with goat anti-mouse
alkaline phosphatase conjugate and p-nitrophenyl substrate.
Labeling endogenous acceptors
Solubilized membranes made from Dictyostelium cells at 20 h of
development were used as source of the endogenous acceptors.
The Fuc transferase reaction was carried out as described above.
Supernatant remaining after centrifugation of the reaction
mixture was discarded, and the protein pellet was washed thrice
in cold PBS to remove any remaining GDP Fuc. Pellet was
quickly solubilized by sonication in 0.05M NaOH at 4C for
8 sec, and neutralized to a pH of 7.0. The solubilized proteins
were processed through a Microcon-10 ultrafiltration cartridge to
remove salt and residual GDP Fuc, and analyzed further.
Analysis of products generated on serine peptides
β-Elimination of the fucosylated peptide and endogenous products
was carried out by treatment with 0.1N NaOH at room temperature
for 4 h. Thin-layer chromatography on cellulose plates, anionexchange HPLC, and QAE-analyses were performed as described
previously (Mehta et al., 1997). To identify the products on TLC,
individual lanes were cut into 0.5 cm strips, and counted after
wetting with water. Standard fucose-1-phosphate was detected
either using phopho-molybdate spray (Mehta et al., 1997) or by
cutting out 0.5 cm strips and analyzing by the Dische-Shettles
Reaction (Beeley, 1985). Mild acid hydrolysis of the peptide
products was done using 0.01N HCl for 15 min at 100C, and
strong acid hydrolysis using 2 M TFA at 100C for 2 h.
Electrospray-ionization Mass Spectrometry (ESI-MS) was carried
out at the Mass Spectrometry Facility at Scripps Research Institute,
La Jolla, CA. MALDI-TOF-MS was performed on a Kratos
MALDI I system using the following matrices (1) DAN, a
saturated solution of 1,5 diaminonaphthalene in 50% acetonitrile
or (2) THAP, a mixture of trihydroxy acetophenone in 50%
acetonitrile and 60 mM ammonium formate in the ratio of 1:1.
Generation of acceptor peptides (asialo, agalacto
N-glycopeptides) from human transferrin for core fucosyl
transferase assays
A modification of the method of Schachter et al. (1983) was used
to generate glycopeptide substrates from human transferrin, which
contains predominantly biantennary N-linked oligosaccharides.
These glycopeptides were used directly, or conjugated to BSA.
Glycopeptides equivalent to 1 mg transferrin were conjugated to 2
mg of BSA by overnight incubation at room temperature in the
presence of 30 mg of 1-ethyl-3(3-dimethylaminopropyl) carbodimide/HCl in 1 ml of PBS. The reaction mixture was extensively
dialyzed against water and lyophilized.
Core fucosyl transferase assays
Transferrin glycopeptides were immobilized to the wells of a
covalent binding plate as per the manufacturer’s protocol. Briefly,
200 µl of a 5 µM solution of the glycopeptides in 0.1 M
bicarbonate buffer pH was added to each well (1 nmol per well),
and allowed to bind at 37C for 2 h. The wells were washed with
PBS containing 0.1% Tween 20 and blocked with 3% BSA in
PBS at 37C for 2 h. For the fucosyl transferase assays,
membrane extracts were diluted to a final protein concentration
of 100 µg/ml in 0.1 M MES buffer pH 6.8 for (core α1,3 FucT)
or 0.1 M sodium cacodylate pH 5.6 (for core α1,6 Fuc T), each
containing 0.2% Triton X-100. Fifty microliters of the diluted
extract was incubated directly in the wells with 50 µl of buffer
containing a final concentration in the assay of 80 µM GDP Fuc,
10 mM MnCl2, and 5 mM AMP (for core α1,3 FucT) and 2.5 mM
809
G.Srikrishna, L.Wang and H.H.Freeze
GTP, 80 µM GDP Fuc, and 4 mM MgCl2 (for core α1,6 FucT).
To account for background reactivity with the antibody of any
endogenous fucosylated proteins in the extract which may
directly bind to the plate, control wells were incubated with
extracts alone in the same volume of sample dilution buffer.
Incubations were performed at room temperature for 2 h. After
aspirating off the reaction mixture, the wells were washed with
PBS containing 0.1% Tween 20. Products generated by the
respective fucosyl transferases were detected by incubating the
wells with 1 µg/ml CAB4 (for core α1,6FucT product) or with
1 µg/ml of anti-Fucα1,3GlcNAc component of anti-HRP (fore
core α1,3FucT product) for 2 h at room temperature. This was
followed by incubation with alkaline phosphatase conjugated
anti-mouse IgG (for core α1,6FucT product) or anti-rabbit IgG
(for core α1,3FucT product) and p-nitrophenyl phosphate
substrate, and the plates were read at 405 nm. Absorbance
measured in the control wells due to reactivity of endogenous
proteins was subtracted for calculation of enzymatic activity.
Amount of product generated in each well by the respective FucT
reactions was calculated from the reactivity of half a picomole of
standard human lactoferrin (containing 1 pmol of an N-glycan
modified by coreFucα1,6GlcNAc) or from the reactivity of 1
pmol of pineapple stem bromelain (containing 1 pmol of an
N-glycan modified by core Fucα1,3GlcNAc) measured under the
same conditions. One unit of FucT activity was defined as amount
of enzyme catalyzing the transfer of 1.0 pmol of fucose per min
under the assay conditions.
Acknowledgments
We thank Dr. Christopher West and Dr. Peter Newell for their
generous gifts of mAb 83.5 and mAb CAB4, respectively; The
Scripps Research Institute, La Jolla, for ESI-MS analysis; Dr. James
Etchison and Dr. Gordon Alton for performing the MALDI-TOF
analysis; Drs. Darshini Mehta and Marion Lammertz for helpful
discussions; and Susan Greaney for secretarial help. This work was
supported by R01-GM 32485 and R01-CA 38701.
Abbreviations
AMP, adenosine monophosphate; BSA, bovine albumin; ELISA,
enzyme-linked immunosorbent assay; ESI-MS, electrosprayionization-mass spectrometry; HPLC, high performance liquid
chromatography; HRP, horseradish peroxidase; mAb, monoclonal
antibody; MALDI-TOF-MS, matrix-assisted laser desorption timeof-flight mass spectrometry; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PLA2, phospholipase A2;
TBS. Tris buffered saline; TLC, thin layer liquid chromatography.
References
Amatyakul-Chantler,S., Ferguson,M.A.J., Dwek,R.A. and Rademacher,T.W.,
Parekh,R.J., Crandall,I.E. and Newell,P.C. (1991) Cell surface oligosaccharides on Dictyostelium during development. J. Cell. Sci., 99, 485–495.
Beeley,J.G. (1985) Analysis of constituents. In Burdon,R.H. and van Knippenberg,P.H. (eds.), Laboratory Techniques in Biochemistry and Molecular
Biology, Vol. 16. Elsevier, Oxford, pp. 100–152
Champion,A., Gooley,A.A., Callaghan,M., Carrin,M.I., Bernstein,R.L., Smith,E.
and Williams,K.L. (1991) Immunodominant carbohydrate determinants in the
multicellular stages of Dictyostelium discoideum. J. Gen. Microbiol., 137,
2431–2438.
Crandall,I.E. and Newell,P.C. (1989) Changes in cell surface glycoproteins during
Dictyostelium development analysed using monoclonal antibodies. Development, 107, 87–94
810
Endo,T., Fujiwara,T., Ikehara,Y. and Kobata,A. (1996) Comparative study of the
sugar chains of alkaline phosphatases purified from rat liver and rat AH-130
hepatoma cells. Occurrence of fucosylated high-mannose type and hybrid
type sugar chains. Eur. J. Biochem., 236, 579–590.
Faye,L., Gomord,V., Fitchette-Laine,A.-C. and Chrispeels,M.J. (1993) Affinity
purification of antibodies specific for Asn-linked glycans containing α1,3fucose or β1,2 xylose. Anal. Biochem., 209, 104–108.
Fosnaugh,K.L. and Loomis,W.F. (1989) Sequence of the Dictyostelium discoideum spore coat gene SP96. Nucleic Acids Res., 17, 9489.
Freeze,H.H. (1997) Dictyostelium discoideum glycoproteins: using a model
system for organismic glycobiology. In Montreuil,J., Vliengenthart,J.F.G. and
Schachter, H. (eds.), New Comprehensive Biochemistry, Vol 29B, Glycoproteins II. Elsevier, Cambridge, pp. 89–121.
Freeze,H.H. and Wolgast,D. (1986) Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification
of mannose-6-sulfate. J. Biol. Chem., 261, 127–134.
Freeze,H.H., Yeh,R., Miller,A.L. and Kornfeld,S. (1983a) Structural analysis of
the asparagine-linked oligosaccharides from three lysosomal enzymes of
Dictyostelium dscoideum. Evidence for an unusual acid-stable phosphodiester. J. Biol. Chem., 258, 14874–14879.
Freeze,H.H., Yeh,R., Miller,A.L. and Kornfeld,S. (1983b) The mod A mutant of
Dictyostelium discoideum is missing the alpha 1,3-glucosidase involved in
asparagine-linked oligosaccharide processing. J. Biol. Chem., 258,
14880–14884.
Freeze,H.H., Willies,L., Hamilton,S. and Koza-Taylor,P. (1989) Two mutants of
Dictyostelium discoideum that lack a sulfated carbohydrate antigenic
determinant synthesize a truncated lipid-linked precursor of N-linked
oligosaccharides. J. Biol. Chem., 264, 5653–5659.
Gonzalez-Yanes,B., Mandell,R.B., Girard,M., Henry,S., Aparicio,O., Gritzali,M.,
Brown,R.D., Erdos,G.W. and West,C.M. (1989) The spore coat of fucosylation mutant in Dictyostelium discoideum. Dev. Biol., 133, 576–587.
Gooley,A.A., Marshchalek,R. and Williams,K.L. (1992) Size polymorphisms due
to changes in the number of O-glycosylated tandem repeats in the
Dictyostelium discoideum glycoprotein PsA. Genetics, 130, 749–756.
Grant,W.N. and Willimas,K.L. (1983) Monoclonal characterization of slime
sheath: the extracellular matrix of Dictyostelium discoideum. EMBO J., 2,
935–940.
Gregg,J.H. and Karp,G.C. (1978) Patterns of cell differentiation revealed by
tritiated fucose incorpration in Dictyostelium discoideum. Exp. Cell. Res., 112,
31–46.
Gustafson,G.L. and Milner,L.A. (1980) Occurrence of N-acetylglucosamine-1-phosphate on proteinase 1 from Dictyostelium discoideum. J. Biol.
Chem., 255, 7208–7210.
Harris,R.J. and Spellman,M.W.(1993) O-linked fucose and other post translational modifications unique to EGF modules. Glycobiology, 3, 219–224.
Haynes,P.A., Gooley,A.A., Ferguson,M.A.J., Redmond,J.W. and Williams,K.L.
(1993) Posttranslational modifications of the Dictyostelium discoideum
glycoprotein PsA. Glycophosphatidylinositol membrane anchor and composition of O-linked oligosaccharides. Eur. J. Biochem., 216, 729–737.
Ivatt,R.L., Das,O.P., Henderson,E.J. and Robbins,P.W. (1984) Glycoprotein
biosynthesis in Dictyostelium discoideum: developmental regulation of the
protein-linked glycans. Cell, 38, 561–567.
Jung,E., Gooley,A.A., Packer,N.H., Slade,M.B., Williams,K.L. and Dittrich,W.
(1997) An in-vivo approach for the identification of acceptor sites for
O-Glycosyltransferases: motifs for the addition of O-GlcNAc in Dictyostelium discoideum. Biochemistry, 36, 4034–4040.
Kornfeld,R. and Kornfeld,S. (1985) Assembly of asparagine linked oligosaccharides. Annu. Rev. Biochem., 54, 631–664.
Kozarov,E., van der Wel,H., Field, M., Gritzali,M., Brown,R.D., and West,C.M.
(1995) Characterization of FP21, a cytosolic glycoprotein from Dictyostelium.
J. Biol. Chem., 270, 3022–3030.
Lam,T.Y., and Siu,C.-H. (1981) Synthesis of stage-specific glycoproteins in
Dictyostelium discoideum in during development. Dev. Biol., 83, 127–137.
Lin,A.I., Philipsberg,A. and Haltiwanger,R.S. (1994) Core fucosylation of
high-mannose type oligosaccharides in GlcNAc transferase I-deficient (Lec1)
CHO cells. Glycobiology, 4, 895–901.
Loomis,W.F. (1975) Dictyostelium discoideum: a developmental system. Academic Press, San Diego, pp. 72–85.
Maeda,Y., Inouye,K. and Takeuchi,I. (1997) Dictyostelium. A model system for
cell and developmental biology. Universal Academy Press, Tokyo, Japan.
Mehta,D.P., Ichikawa,M., Salimath,P.V., Etchison,J.R., Haak,R., Manzi,A. and
Freeze,H.H. (1996) A lysosomal cysteine proteinase from Dicyostelium
discoideum contains N-acetylglucosamine-1-phosphate bound to serine but
not mannose-6-phosphate on N-linked oligosaccharides. J. Biol. Chem., 271,
10897–10903.
Fucosylation in Dictyostelium
Mehta,D.P., Etchison,J.R., Wu,R. and Freeze,H.H. (1997) UDP-GlcNAc:ser-protein N-acetylglucosamine-1-phosphotransferase from Dictyostelium discoideum recognizes serine- containing peptides and eukaryotic cysteine proteinases. J. Biol. Chem., 272, 28638–28645.
Mengeling,B.J., Beverley,S.M. and Turco,S.J. (1997) Designing glycoconjugate
biosynthesis for an insidious intent: phosphoglycan assembly in Leishmania
parasites. Glycobiology, 7, 873–880.
Moloney,D.J., Lin,A.I. and Haltiwanger,R.S. (1997) The O-linked fucose
glycosylation pathway. Evidence for protein-specific elongation of O-linked
fucose in Chinese hamster ovary cells. J. Biol. Chem., 272, 19046–19050.
Montreuil,J., Bouquelet,S., Debray,H., Fournet,B., Spik,G. and Strecker,G.
(1986) In Chaplin,M.F. and Kennedy,J.F. (eds.), Carbohydrate Analysis, A
Practical Approach. IRL Press, Washington, DC, p. 149.
Ord,T., Adessi,C., Wang,L. and Freeze,H.H. (1997) The cysteine proteinase gene
cprG in Dictyostelium discoideum has a serine-rich domain that contains
GlcNAc-1P. Arch. Biochem. Biophys., 339, 64–72.
Osaki,T., Nakao,H., Orii,H., Morio,T., Takeuchi,I. and Tasaka,M. (1993) Developmental regulation of transcription of a novel pre-spore spcific gene (DP87)
in Dictyostelium discoideum. Development, 117, 1299–1308.
Riley,G.R., West,C.M. and Henderson E.J.(1993) Cell differentiation in controls
assembly of protein-linked glycans. Glycobiology, 3, 165–177.
Schachter,H., Narasimhan,S., Gleeson,P. and Vella,G. (1983) Glycosyltransferases involved in elongation of N-glycosidically linked oligosaccharides of the
complex or N-acetyllactosamine type. Methods Enzymol., 98, 98–134.
Sharkey,D.J. and Kornfeld,R. (1991) Developmental regulation of asparaginelinked oligosaccharide synthesis in Dictyostelium discoideum. J. Biol. Chem.,
266, 18485–18497.
Souza,G.M., Hirai,J., Mehta,D.P. and Freeze,H.H. (1995) Identification of two
novel Dictyostelium discoideum cysteine proteinases that carry N-acetylglucosamine-1-P modification. J. Biol. Chem., 270, 28928–28945.
Srikrishna,G.,Varki,N.M., Newell,P., Varki,A. and Freeze,H.H. (1997) An IgG
monoclonal antibody against Dictyostelium discoideum glycoproteins specifically recognizes Fucα1,6GlcNAcβ in the core of N-linked glycans. J. Biol.
Chem., 272, 25743–25752.
Staudacher,E., Dalik,T., Wawra,P., Altmann,F. and Marz,L. (1995) Functional
purification and characterization of GDP-fucose: β-N-acetylglucosamine
(Fuc to Asn linked GlcNAc) α1,3-fucosyl transferase from mung beans.
Glycoconj. J., 12, 780–786.
Stults,N.L. and Cummings,R.D. (1993) O-Linked fucose in glycoproteins from
Chinese hamster ovary cells. Glycobiology, 3, 589–596.
Tschursin,E., Riley,G.R. and Henderson,E.J. (1989) Differential regulation of
glycoprotein sulfation and fucosylation during growth of Dictyostelium
discoideum. Differentiation, 40, 1–9.
Varki,A. and Marth,J.D. (1995) Oligosaccharides in vertebrate development. Sem.
Dev. Biol., 6, 127–138.
Voynow,J.A., Scanlin,T.F. and Glick,M.C. (1988) A quantitative method for
GDP-L-Fuc:N-acetyl-β-D-glucosaminide α1,6 fucosyl transferase activity
with lectin affinity chromatography. Anal. Biochem., 168, 367–373.
Voynow,J.A., Kaiser,R.S., Scanlin,T.F. and Glick,M.C. (1991) Purification and
characterization of GDP-L-fucose-N-acetyl β-D-glucosaminide α1–6fucosyl
transferase from cultured human skin fibroblasts. Requirement of a specific
biantennary oligosaccharide as substrate. J. Biol. Chem., 266, 21572–21577.
West,C.M. and Erdos,G.W. (1988) The expression of glycoproteins in the
extracellular matrix of the cellular slime mold Dictyostelium discoideum. Cell.
Differ., 23, 1–16
West,C.M., Erdos,G.W. and Davis,R. (1986) Glycoantigen expression is regulated
both temporally and spatially during dvelopment in the cellular slime molds
Dictyostelium discoideum and D.mucoroides Mol. Cell. Biochem., 72,
121–140.
West,C.M., Mao,J., van der Wel,H., Erdos,G.W. and Zhang,Y. (1996) SP75 is
encoded by the DP87 gene and belongs to a family of modular Dictyostelium
discoideum outer layer spore coat proteins. Microbiology, 142, 2227–2243.
Yan,L., Smith,D.F., and Cummings,R.D. (1994) Determination of
GDPFuc:Galβ1–4GlcNAc-R(Fuc to GlcNAc)α1,3fucosyl transferase activity by a solid-phase method. Anal. Biochem., 223, 111–118.
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