(Received for publication, October 17, 1995; and in revised form, February 16, 1996)
From the
A novel fucosyltransferase (cFTase) activity has been enriched
over 10-fold from the cytosolic compartment of Dictyostelium based on transfer of
[
H]fucose from
GDP-[
H]fucose to
Gal
1,3GlcNAc
-paranitrophenyl
(paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity
behaved as a single component during purification over DEAE-, phenyl-,
Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel
filtration resins. The purified activity possessed an apparent M
of 95
10
, was
Mg
-dependent with a neutral pH optimum, and exhibited
a K
for GDP-fucose of 0.34
µM, a K
for pNP-LNB of 0.6
mM, and a V
for pNP-LNB of 620
nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of
the Superdex elution profile identified a polypeptide with an apparent M
of 85
10
, which coeluted
with the cFTase activity and could be specifically photolabeled with
the donor substrate inhibitor
GDP-hexanolaminyl-azido-
I-salicylate. Based on substate
analogue studies, exoglycosidase digestions, and co-chromatography with
fucosylated standards, the product of the reaction with pNP-LNB was
Fuc
1, 2Gal
1,3GlcNAc
-pNP. The cFTase preferred substrates
with a Gal
1,3 linkage, and thus its acceptor substrate specificity
resembles the human Secretor-type
1,2-FTase. Afucosyl isoforms of
the FP21 glycoprotein, GP21-I and GP21-II, were purified from the
cytosol of a Dictyostelium mutant and found to be substrates
for the cFTase, which exhibited an apparent K
of 0.21 µM and an apparent V
of 460 nmol/min/mg protein toward GP21-II. The highly purified
cFTase was inhibited by the reaction products
Fuc
1,2Gal
1,3GlcNAc
-pNP and FP21-II. FP21-I and
recombinant FP21 were not inhibitory, suggesting that acceptor
substrate specificity is based primarily on carbohydrate recognition. A
cytosolic location for this step of FP21 glycosylation is implied by
the isolation of the cFTase from the cytosolic fraction, its high
affinity for its substrates, and its failure to be detected in crude
membrane preparations.
The glycosylation of proteins traversing the secretory pathway of eukaryotic cells has been well studied(1, 2, 3) . Evidence has been steadily accumulating that some amino acid side chains of proteins of the cytosol and nucleoplasm are also modified, with either a single sugar, O-GlcNAc(3, 4) , or oligosaccharides(5, 6, 7, 8, 9) . The frequency of O-GlcNAc modifications of cytosolic and nucleoplasmic proteins may approach that of phosphorylation(10) , and there is evidence that cytosolic glycosylation may be regulatory(5, 8, 10, 11, 12) , based on the existence of glycoforms which vary with different physiological states and protein localizations, the presence of specific cytosolic glycosidases, half-lives of sugar modifications which vary relative to the host protein, and potential competition for attachment sites between O-GlcNAc and phosphate groups.
FP21 is a protein found both in the cytosol and the nucleus, and is
modified by an oligosaccharide in the cellular slime mold Dictyostelium discoideum(7, 8) . FP21 is a
highly conserved protein whose amino acid sequence shows 68% identity,
78% similarity, and one amino acid residue difference in length between Dictyostelium and humans. FP21 has been detected in the cyclin
A/cdcK2 complex associated with the G1 checkpoint of the human HeLa
cell cycle(13) , the kinetechore complex (14) of
budding yeasts, ()and in association with certain membranes
of Dictyostelium. (
)FP21 has a particularly high
abundance in the inner ear organ of Corti (15, 16, 17) . An FP21 gene is present in a
green alga virus genome(18) , and two copies are frequently
found in eukaryotic genomes, including Dictyostelium. (
)Dictyostelium FP21 possesses a single tetra- or
pentasaccharide, which appears to contains Fuc, (
)Xyl, and
Gal (8) and is probably O-linked based on
susceptibility to release by mild base (7) or
hydrazinolysis.
Two FP21 isoforms have been purified which
vary in their proportions of Xyl and Gal(8) .
Since a
GDP-Fuc synthesis mutant in Dictyostelium exhibits slow growth
which can be partially rescued by exogenous Fuc, we have
investigated the fucosylation of FP21 for its possible involvement in
this phenotype. Using afucosyl-FP21 from the fucosylation mutant as an
acceptor substrate, an
-cFTase (cytosolic fucosyltransferase)
activity dependent upon GDP-
-Fuc was detected in the cytosolic
S100 fraction but not in the particulate or membrane
fraction(7) . FP21 accounted for >80% of the acceptor
activity in crude S100 extracts of the fucosylation mutant, as if it
were the primary acceptor substrate for this enzyme. This activity was
also able to attach Fuc to lacto-N-biose
(Gal
1,3GlcNAc
-) attached to a hydrophobic aglycone moiety, as
determined by cross-competition studies. This enzyme activity appeared
to mediate attachment of a peripheral Fuc, and was novel in terms of
its apparent cytosolic, rather than Golgi, compartmentalization, and
its submicromolar K
for GDP-Fuc. As shown
here, a 1.2 million-fold purified preparation of the enzyme activity
retains its high affinity for GDP-Fuc, displays a high affinity for
afucosyl FP21, links Fuc in a
1,2 linkage to a
1,3-linked Gal
on type 1 and 3 acceptors, and copurifies with a polypeptide which can
be photoaffinity-labeled with a donor substrate analog.
Reactions involving protein acceptor substrates were conducted similarly, except that protein acceptors were introduced from concentrated stock solutions, Tween 20 was present at 0.05% (v/v), BSA was reduced to 0.5 mg/ml, and the reaction was initiated by the addition of the cFTase. Incorporation into protein substrates was determined after SDS-PAGE of the samples, within 1 day of fixation with Coomassie Blue and destaining, by excising the appropriate region of the gel and scintillation counting, as described(7) .
The final Superdex
200 activity pool, purified 1.2 10
-fold, is
referred to as preparation A. Activity purified 317-2200-fold through
DEAE, phenyl, and Superdex resins is referred to as preparation B,
activity purified 106-fold through DEAE, phenyl, and Reactive Blue-4
resins is referred to as preparation C, and activity purified 27-fold
through the DEAE-resin is referred to as preparation D.
Figure 7:
SDS-PAGE analysis of protein substrates
and inhibitors. GP21-I (GI) and GP21-II (GII),
isolated from the GDP-Fuc synthesis mutant strain HL250, FP21-I (FI), and FP21-II (FII), isolated form the normal
strain Ax3, and rP21 (R), expressed in E. coli with
an N-terminal oligo-His tag, were purified under nondenaturing
conditions as described under ``Experimental Procedures,''
and 2-10 µg were subjected to SDS-PAGE followed by staining
with Coomassie Blue. M standards with values
10
are labeled on the right. The lower arrow on the left marks the position of the
GP21s and FP21s, and the upper arrow marks the position of
rP21.
To test the effect of detergents, enzyme purified through the Reactive Blue-4 step (preparation C) was diluted 100-fold in buffer D and incubated in varying concentrations of 16 detergents as described under ``Experimental Procedures.'' Only Tween 20 and Tween 80 sustained activity at all concentrations tested. Activity losses ranged from 30 to 99% for the other detergents; in general, greater losses were observed as detergent concentrations diminished below the critical micelle concentration. At later stages of purification, 0.05% Tween 20 was superior to BSA in preserving enzyme activity at 4 °C.
Figure 1:
Purification of
the cFTase. The data shown in this figure correspond to the
purification described in Table 1. Protein, which was monitored
by absorbance at 280 nm using a 0.5-cm path length flow cell, and
cFTase activity, which was assayed as dpm in the presence of 1
µM GDP-Fuc and 0.36 mM pNP-LNB, are plotted as a
function of elution volume. Panel A, typical example of an
S100 fraction which was pumped onto the DEAE-Sepharose Fast Flow resin.
After washing, the column was eluted with a 0-0.25 M gradient of NaCl, whose concentration at the entry point of the
450-ml column is plotted. Fractions from the main DEAE column activity
peak which contained >500 dpm activity/aliquot were pooled and
frozen. Panel B, activity pools from 10 DEAE columns were
pooled and pumped onto the phenyl-Sepharose 6 Fast Flow (high sub)
resin. After washing, the column was eluted with a 0-60% (v/v)
gradient of ethylene glycol, whose concentration at the entry point of
the 175-ml column is plotted. Panel C, fractions from the main
phenyl column activity peak which contained >4000 dpm/aliquot were
pooled, concentrated, diluted to reduce the ethylene glycol
concentration, and pumped onto the Reactive Blue-4 agarose dye resin.
After washing, the column was eluted with a 0.1-1.5 M NaCl gradient, whose concentration at the entry point of the 25-ml
column is plotted. Fractions from the main peak which contained
>2000 dpm/aliquot were pooled, concentrated, and diluted to 0.1 M NaCl prior to application to the GDP-adipate and
GDP-hexanolamine columns (not shown). Panel D, the GDP-eluate
from the GDP-hexanolamine column was concentrated and applied to the
Superdex 200 HPLC gel filtration column and eluted isocratically. 95.0%
of the activity emerged as peak I centered at 30.4 ml, and 3.7% emerged
as a minor peak centered at 37.7 ml, as peak II. Calibration of the
Superdex column with M standards (see
``Experimental Procedures'') indicated that peaks I and II
had apparent M
values of 95
10
and 40
10
,
respectively.
The DEAE pool
adsorbed quantitatively to phenyl-Sepharose 6 Fast Flow (high sub) and
>99% of the eluted activity emerged as a single peak with slight
trailing close to the end of a 0-60% gradient of ethylene glycol (Fig. 1B). cFTase activity was assayed in eluted
fractions after dilution in buffer D, and recovery ranged from 50 to
90%, or 53% for the trial reported (Table 1). Quantitative
adsoprtion to phenyl-Sepharose 6 Fast Flow (low sub), or to phenyl
Sepharose CL-4B, required addition of 10-20%
(NH)
SO
to the DEAE pool, and thus
these resins were not used.
The pool of phenyl-purified enzyme was
concentrated by ultrafiltration. Recovery of activity after
concentration varied from 10 to 90%, or 27% in the reported trial (Table 1), and activity was not detected in the ultrafiltrate. At
this stage of purification, the activity typically displayed an
apparent M value of 95
10
after HPLC gel filtration (see below), and thus poor recovery
appeared to be associated with insolubility or denaturation. In early
trials with the other phenyl resins involving long column residence
times, substantial activity with an M
value of 40
10
was correlated with poor recovery of
activity,
as discussed further below.
The phenyl-pool activity adsorbed efficiently to the the Reactive Blue-4 dye resin, and 97% of the eluted activity emerged as a monodisperse peak early in a gradient of 0.1-1.5 M NaCl (Fig. 1C). The remaining activity emerged later as a small peak, and overall activity recovery was 92%. cFTase activity could also be adsorbed to RB-1, RB-72, RG-5, RR-120, RG-19, and CB dye resins, but the Reactive Blue-4 resin was selected on the basis of more selective binding relative to BSA. RY-3 and RY-86 dye resins did not adsorb activity. Concentration of activity by ultrafiltration resulted in a 33% loss of activity.
The Reactive Blue-4-pool activity was unretarded by
GDP-adipate Sepharose CL-4B. 290 µg of protein was recovered from
the column after elution with 2 mM GDP, but was not examined
further. Activity in the flow-through fraction was, however,
quantitatively adsorbed to GDP-hexanolamine Sepharose. The selective
binding to GDP-hexanolamine was predicted based on the inhibitor
characteristics of a series of GDP-Fuc analogues (see below), and is a
behavior typical of mammalian
1,2-FTases(30, 31, 32, 33) .
The GDP-hexanolamine column was eluted with a 0-2 mM GDP
gradient, and the activity eluted in the 0-1 mM range of
the gradient. <5% additional activity was eluted by various
combinations of high GDP and NaCl concentrations, Tween 20 and ethylene
glycol. After ultrafiltration, the enzyme activity was stable at 4
°C for at least 1 days.
The GDP affinity pool was applied to a
Superdex 200 gel filtration column by HPLC. 95% of the recovered
activity eluted as a monodisperse peak (peak I) centered at a M position of 95
10
(Fig. 1D). An additional 3.7% eluted at M
position 40
10
(peak II),
and the remaining activity eluted near the void volume. In pilot
studies, >90% of activity applied to the GDP-hexanolamine column was
recovered after gel filtration. The 35% recovery in the reported trial (Table 1) did not seem to be due to retention on the column (see
above), and may have been due to 3 days storage of the GDP-hexanolamine
fractions at 4 °C prior to further processing. After gel
filtration, activity decayed with a 12-h half-life at 4 °C, but was
stable for 3 days in 0.05% (v/v) Tween 20 at 4 °C. Activity was
only partially stabilized by 2.0 mg/ml BSA, and strongly destabilized
by purified cytochrome c (data not shown). Finally, activity
was stabilized by freezing at -80 °C.
HPLC gel filtration
fractions were analyzed by SDS-PAGE and silver staining (Fig. 2). Three major silver-stained bands were visible in the
fractions of highest activity (peak I), centered at 30.4-ml elution
volume. Only the amount of the most heavily stained band, at M position of 85
10
,
correlated precisely with the activity of the fractions. To obtain
further evidence that this polypeptide, FT85, was equivalent to the
enzyme, selected HPLC gel filtration fractions were UV-irradiated in
the presence of the photoactive donor substrate analogue
GDP-hex-
I-ASA, at 30 µM, in the presence or
absence of 690 µM GDP-Fuc. Photolabeling under similar
conditions has previously been useful in identifying fucosyl- and
mannosyltransferases(23, 35) . SDS-PAGE followed by
autoradiographic analysis of a fraction from peak I (30.4-ml elution
volume) revealed that the most intensely photolabeled polypeptide,
PL85, migrated at the position of FT85 (Fig. 3, lane
e), and labeling of this band was inhibited by GDP-Fuc (lane
f). PL85 was not detected in the fraction eluting at 28.6 ml (lane c), which possessed <0.2% of the peak activity, but
was faintly detectable in the fraction eluting at 33.1 ml, which
possessed 3.1% of the peak activity (not shown). Two additional, much
less intensely photolabeled bands were also detected in the major
activity peak at M
positions 40 and 29
10
, and are discussed below. The relative intensities of
these bands is clearer in Fig. 3, lane i, which is a
shorter autoradiographic exposure. Multiple minor bands were also
photoreactive in lane e, but in each case labeling was only
slightly inhibited by GDP-Fuc (lane f), showing that their
reactivity was nonspecific and that photoprotection by GDP-Fuc was not
due to general UV absorbance by the nucleotide moiety.
Figure 2:
SDS-PAGE analysis of Superdex 200 gel
filtration fractions. 8% (by volume) of even-numbered fractions from Fig. 1D, were reduced and alkylated with 40 mM iodoacetamide, run on a 7-20% gradient polyacrylamide gel,
and stained with silver. Lanes are labeled as the elution volumes given
in Fig. 1D. The samples had retained full cFTase
activity at the time of analysis. The polypeptide corresponding to FT85
is identified on the left. M values
10
are indicated on the right.
Figure 3:
Photoaffinity labeling of purified cFTase.
Peak I fractions of Fig. 1D (elution volume
28.6-32.2 ml) were pooled, concentrated, and an aliquot was
subjected to SDS-PAGE followed by staining with Coomassie Blue (lane a). The positions of FT85, FT40, and FT29 are indicated
in the left margin. M values
10
are in shown in lane b. Aliquots of
fractions from 28.6 ml (lanes c and d), 30.4 ml (peak
I) (lanes e, f, i, and j), and 37.7 ml (peak
II) (lanes g and h) elution volumes (Fig. 1D) were photoaffinity labeled with
GDP-hex-[
I]ASA in the absence(-) or
presence (+) of a 23-fold concentration excess of GDP-Fuc, as
indicated at the top. Lanes i and j are a shorter
autoradiographic exposure of lanes e and f. The
positions of PL85, PL40, and PL29, which are inferred to be identical
with FT85, FT40, and FT29, are indicated. The samples in lanes a-j had lost about 50% of their cFTase activity prior to photolabeling
and SDS-PAGE, which explains the reduced abundance of FT85 and
increased abundance of FT40 and FT29 in lane a relative to Fig. 2. Lanes k-o, peak I fractions from a separate gel
filtration of the same cFTase preparation A were photolabeled after
>90% loss of cFTase activity. The amount of original activity which
was photolabeled corresponded to 2% of the original activity which was
photolabeled in lanes c-j. The relative intensity of labeling
of PL40 in the sequential fractions is approximately proportionate to
the original activity in the fractions.
Together with the
results shown in Fig. 2and other data, this figure shows that
FT40, and possibly FT29, are breakdown products of the intact cFTase
polypeptide, FT85. These and other results (see text) suggest that
reduction of cFTase activity occurs as FT85 is proteolytically degraded
to FT40 and possibly FT29.
The minor
cFTase activity peak eluting at 37.7 ml (Fig. 1D, peak
II; M position of 40
10
)
contained a single, specifically-labeled photoreactive species,
referred to as PL40, at an M
position of 40
10
(Fig. 3, lane g). Negligible
PL85 was detectable in this fraction. The amount of PL40 varied with
the level of cFTase activity in neighboring fractions (data not shown).
PL40 was not detectable by silver staining (Fig. 2, 37.7 ml), as
expected based on its low apparent abundance relative to PL85 (Fig. 3, compare lanes e and g). PL40,
together with PL29, were also observed in peak I (Fig. 3, lane e). PL40 appears to be a proteolytic fragment of PL85
based on the following observations. 1) The ratio of PL40 to PL85 in
the major peak I was time dependent. After 7 days of storage at 4
°C, during which activity diminished by >90%, only PL40 could be
detected (Fig. 3, lanes k-o). 2) The original
photolabeling of peak I described above in Fig. 3had been
performed after 4 days of storage at 4 °C, during which activity
had decreased by 50%. SDS-PAGE analysis of the pooled peak I fractions
at this time revealed that the level of FT85 had decreased concomitant
with the appearance of the new bands labeled at FT40 and FT29 (compare Fig. 3, lane a, with Fig. 2, 30.4 ml). Thus the
appearance of PL40 in the main activity peak is correlated with the
appearance of FT40. 3) Pilot studies on phenyl-purified enzyme showed
that cFTase activity became smaller as determined by HPLC gel
filtration as activity decreased,
and that the M
95
10
activity peak I was
less stable than the M
40
10
activity peak II. (
)We conclude that PL85 is
equivalent to FT85, and that FT85 is susceptible to proteolytic
degradation to FT40, which is equivalent to PL40. FT40 possesses
similar K
values for its substrates and similar
substrate specificity, but has a reduced specific activity compared to
FT85 (see below). This model explains both the time-dependent
appearance of FT40 in the M
95
10
activity peak after gel filtration, as well as the time-dependent
appearance of the M
40
10
activity peak II before gel filtration.
In summary, the cFTase
activity was purified 1.2 million-fold at 4.3% yield. Some of the
losses could be attributed to specific proteolysis, which may explain
the small proportion of activity (<5%) which could be resolved from
the main peak during DEAE, phenyl, Reactive Blue-4, and gel filtration
chromatographies. The FT85 polypeptide represents about 30% of the
total protein. The cFTase consisted of a single 95 10
M
activity after the first chromatography
step and, aside from inconsistent generation of the apparently
proteolytically-derived 40
10
M
activity, no other molecular species expressing this activity
were detected during the purification.
Figure 4:
Kinetic properties of the cFTase with
respect to pNP-LNB concentration. Approximately 125 pg of preparation A
was incubated for 1 h with varying concentrations of pNP-LNB, in the
presence of 1.0 µM GDP-[H]Fuc. Inset, double-reciprocal plot of the data, which are fitted to
a straight line. The apparent K
with
respect to pNP-LNB was 0.6 mM, and the apparent V
was 620 nmol/min/mg
protein.
Figure 5:
Kinetic properties of the cFTase with
respect to GDP-Fuc concentration. Preparation A was incubated with
varying concentrations of GDP-[H]Fuc in the
presence of 2.0 mM pNP-LNB for 1 h. Inset,
double-reciprocal plot of the kinetic data, which are fitted to a
straight line. The estimated apparent K
with respect to GDP-Fuc was 0.34 µM, and the
apparent V
was 560 nmol/min/mg
protein.
The cFTase
preferred a disaccharide acceptor as negligible activity was observed
with monosaccharide-pNP substrates. 2-O-methylation or
1,2-fucosylation of the nonreducing terminal Gal of pNP-LNB
abolished activity (Table 4). The
1,3-linkage of the Gal was
important for enzyme recognition, as Gal
1,4GlcNAc and
Gal
1,6GlcNAc were at best very weak inhibitors (Table 3),
and Gal
1,4GlcNAc
-pNP exhibited only several percent of the
activity of pNP-LNB, even above the K
of pNP-LNB (Table 4). Finally, the enzyme was not specific for the identity
or linkage of the penultimate sugar of the disaccharide, as
Gal
1,3GalNAc
-benzyl was a good substrate (Table 4).
The [H]fucosyl-pNP-LNB
reaction product was chromatographed with pNP-LNB and authentic
Fuc
1, 2Gal
1,3GlcNAc
-pNP on Dionex PA-1 and PA-100 HPLC
columns (Fig. 6). >95% of the radioactivity coeluted with
Fuc
1,2Gal
1,3GlcNAc
-pNP, detected by integrated
amperometry, which itself eluted earlier than pNP-LNB, as
expected(38) . Together, these observations identify the
pNP-LNB reaction product of the cFTase as
Fuc
1,2Gal
1,3GlcNAc
-pNP. The cFTase is thus an
1,2-FTase which, as described above, shows a marked preference for
type 1 and type 3 disaccharide acceptors.
Figure 6:
Co-chromatography of the pNP-LNB cFTase
reaction product with Fuc1,2Gal
1,3GlcNAc
-pNP. pNP-LNB
was fucosylated in the presence of GDP-[
H]Fuc by
preparation A of the cFTase and recovered on a C
Sep-Pak
and by gel filtration. The
H-reaction product was combined
with 10 µg each of pNP-LNB and synthetic
Fuc
1,2Gal
1,3GlcNAc
-pNP, and the mixture was applied to a
PA-1 column and eluted isocratically with 150 mM NaOH. Peak 1
is unknown material present in the reaction product, peak 2 is at the
position of Fuc
1,2Gal
1,3GlcNAc
-pNP, and peak 3 is at the
elution position of pNP-LNB. No sugars or radioactivity were detected
from 25-60 min. >95% of the radioactivity coeluted with
Fuc
1,2Gal
1,3GlcNAc
-pNP on this column, and also on a
PA-100 column eluted with a gradient of NaAc in 0.1 N NaOH
(not shown).
Figure 8:
Kinetic properties of the cFTase with
respect to GP21 isoform concentrations. Approximately 125 pg of
preparation A was incubated for 10 min with 2.0 µM GDP-[H]Fuc and varying concentrations of
GP21-I or -II. Inset, double-reciprocal plot of the kinetic
data. The data for GP21-II are fitted to a straight line, whereas the
data points for GP21-I, which are nonlinear, are interpolated. The
nested inset expands the plot at lower values of 1/V and 1/S.
The cFTase exhibits an apparent K
of 0.21
µM with respect to GP21-II and an apparent V
of 460 nmol/min/mg
protein.
Fucosylation of the FP21 polypeptide depended on its glycosylation status. Recombinant Dictyostelium FP21 (rP21), purified under nondenaturing conditions from E. coli with a short oligo-His tag at its N terminus (Fig. 7), exhibited no acceptor activity with preparation A (data not shown), consistent with previous evidence that the single Fuc on FP21 is located at the nonreducing terminus of an oligosaccharide(7, 8) , which is expected to be absent from rP21. FP21-I and FP21-II, isolated from normal Dictyostelium cells, were also not substrates, consistent with previous studies on crude extracts that soluble FP21 is fully fucosylated(7) .
pNP-LNB and GP21 would reciprocally inhibit each other's
fucosylation if they were substrates of the same enzyme. Both GP21-I
and -II inhibited pNP-LNB fucosylation in a concentration-dependent
manner (Table 6), as expected based on analysis of crude S100
extracts(7) . At a pNP-LNB concentration slightly below its K, a concentration of GP21-I slightly above
GP21-II's K
value inhibited pNP-LNB
fucosylation approximately 40%, whereas a higher concentration of
GP21-II was required for 40% inhibition. Conversely, much higher
concentrations of pNP-LNB were required to inhibit GP21-I than GP21-II
fucosylation. Thus both GP21 isoforms and pNP-LNB appeared to be
fucosylated by the same enzyme, but GP21-I appeared to have a higher
affinity than GP21-II for the cFTase.
Recognition of GP21 by the cFTase appeared to depend primarily on carbohydrate rather than peptide determinants, based on the inhibitory potential of the different protein analogues. rP21 failed to inhibit the fucosylation of GP21-I, GP21-II, and pNP-LNB (Table 6), and in fact mildly stimulated the fucosylation of GP21-I and GP21-II. Since this stimulatory effect was not observed for pNP-LNB fucosylation, it may reflect substrate-substrate interactions, as 1) rP21 stimulated the fucosylation of GP21-I more than that of GP21-II, 2) GP21-I fucosylation was cooperative with respect to its own concentration (see above), and 3) GP21 and FP21 are known to aggregate in the concentration range examined(8) . FP21-I was only a weak inhibitor (Table 6) compared to FP21-II (see below), showing an effect only at the highest inhibitor:substrate ratio tested against 0.028 µM GP21-I (Table 6). The absence of inhibitory effects by both rP21 and FP21-I suggested that the enzyme does not recognize the polypeptide backbone of FP21.
In contrast, FP21-II was
a good inhibitor of the fucosylation of all three substrates, pNP-LNB,
GP21-I, and GP21-II, with >50% inhibition observed at
10-100-fold concentration excess over the GP21-II and GP21-I
substrate concentrations tested. The effect of FP21-II may represent
product inhibition, as the reaction product,
Fuc1,2Gal
1,3GlcNAc
-pNP, was found to be an inhibitor of
pNP-LNB fucosylation, when tested over a similar range of
inhibitor:substrate ratios (Table 6). The failure of FP21-I to
exert inhibition, except at the highest concentrations, may be due to
its extra mole of Gal (8) which, if applied following
fucosylation, may mask recognition of the FP21-I oligosaccharide. If
rP21 and the GP21 and FP21 isoforms differed only in their
glycosylation, the simplest interpretation is that the specificity for
fucosylation and inhibition of fucosylation resides in the sugar
portion of the substrate.
Multiple FTases have been described in mammalian cell
extracts and secretions which catalyze the formation of Fuc1,2Gal
linkages (30, 31, 32, 33, 40) .
Separate
1,2-FTases appear to be encoded by the H and Secretor
loci in humans(33, 39) , and DNAs encoding the H and
Secretor enzymes have been
cloned(40, 41, 42, 43) . Like all
other known Golgi glycosyltransferases(45, 46) , the
Secretor and H enzymes are synthesized as type 2 membrane proteins
consisting of a large C-terminal catalytic ectodomain, attached via a
linker region to a transmembrane domain and a small N-terminal
endodomain. The soluble, secretory forms of Golgi glycosyltransferases
are apparently derived by proteolytic cleavage within the linker
region(45) . It is not clear whether the secretory form is
physiologically meaningful. The K
values of Golgi
and Golgi-derived FTases for GDP-Fuc are in the range of 10-100
µM, and apparent K
values for
oligosaccharide acceptor substrates range from 0.1 to 20
mM(30, 31, 32, 33, 40) .
The order of magnitude of these K
values is
consistent with the expected concentrations of these substrates in the
Golgi apparatus.
The Dictyostelium cFTase, which is also
capable of catalyzing the formation of a Fuc1,2Gal linkage, shows
both similarities to and differences from the mammalian
1,2-FTases. The cFTase occurs in soluble form after gentle cell
lysis that maintained the latency of Golgi enzymes, and its activity
could not be detected in particulate extracts of the cell(7) .
Its polypeptide could be purified to apparent homogeneity using
conventional and affinity chromatography and electrophoresis methods
which have been successfully applied to secreted forms of mammalian
enzymes. Although the cFTase appears to be fairly hydrophobic when
chromatographed on phenyl-Sepharose columns and certain detergents
stabilized its activity after purification, detergents were not used
during purification. The apparent M
of the cFTase
polypeptide (FT85), at 85
10
, is approximately
twice that of known Golgi enzyme cleavage products. The implication
that the cFTase diverges from the Golgi type 2 membrane protein
structural paradigm and resides in the cytosol must await confirmation
from sequencing and cloning studies.
The K of
the purified cFTase for GDP-Fuc, at 0.34 µM, is unusually
low compared to Golgi FTases, but similar to the value of a cytosolic O-GlcNAc transferase for its donor substrate
UDP-GlcNAc(34) . The high affinity of the cFTase for GDP-Fuc is
consistent with its proposed cytosolic localization, because in the
cytosol the cFTase would be in competition with the Golgi GDP-Fuc
transporter (2) for this substrate. Although the cFTase differs
from mammalian Golgi
FTases in its high affinity for GDP-Fuc, its
nucleotide binding is more similar to
1,2-FTases than
1,3-FTases in its preference for GDP-hexanolamine over GDP-adipate (31) . The proportionately high sensitivity of the cFTase to
inhibition by other guanine nucleotides (Table 2) raises
interesting regulatory questions about the effects of their
intracellular pools.
The K of the purified
cFTase for the type 1 disaccharide conjugate pNP-LNB
(Gal
1,3GlcNAc
-pNP) is similar to that of mammalian
1,2-FTases. The monosaccharide conjugate pNP-Gal, and the type 2
disaccharide conjugate Gal
1,4GlcNAc
-pNP are only very poorly
if at all fucosylated, and Gal
1,6GlcNAc is at best a poor
inhibitor. In contrast, the enzyme will fucosylate the type 3
disaccharide Gal
1,3GalNAc
-pNP, thus tolerating alternate
sugars of opposite anomeric linkage penultimate to the terminal,
fucosylated Gal. The acceptor specificity preferences of the cFTase are
similar to that of highly purified porcine submaxillary
1,2-FTase (30, 44) and the Secretor-type
1,2-FTase purified
from human serum or plasma(31, 32, 33) , and
distinct from the H-type
1,2-FTase, which has a broader
specificity range(31, 32, 33, 40) .
Speculation that the Secretor-type gene preceded a gene duplication
event leading to divergent evolution of the H-type gene (39) is
reinforced by the present identification of a Secretor-type enzyme in
the cellular slime molds. The specificity of the cFTase is compatible
with what is known about the FP21 oligosaccharide, which contains Gal.
However, the underlying sugar is not GlcNAc or GalNAc, as amino sugars
are not detected in FP21(8) .
The cFTase exhibits varying V and K
values for
different aglycone moieties attached to the disaccharide. Of the three
which have been tested, pNP has the highest activity, benzyl is
intermediate, and the 8-methoxycarbonyloctyl moiety exhibits an order
of magnitude lower activity compared to pNP when present at
concentrations near or below their K
values. This
suggests that acceptor substrate recognition involves more than the
terminal disaccharide, which is consistent with the lower than expected
inhibitory potential of free lacto-N-biose and the 3000-fold
lower apparent K
of the native substrate GP21.
Effects of alternate aglycone moieties have also been observed for
mammalian FTases (31, 33) and other Golgi
glycosyltransferases(47) , and polypeptide domains have been
shown to be important determinants of recognition by certain
glycosyltransferases(48) . However, rP21 and FP21-I are not
inhibitors of the cFTase, suggesting that the FP21 polypeptide is not
an important determinant for cFTase recognition. The higher affinity of
the enzyme for GP21-I compared to GP21-II, as determined by
cross-inhibition studies (Table 6), in contrast to the higher
inhibitory potential of FP21-II compared to FP21-I, may be most easily
explained by glycosylation differences which have been described for
the two FP21 isoforms(8) . Why the different isoforms are
differentially glycosylated remains to be determined. Since GP21 is the
major acceptor of the reaction in crude extracts from the GDP-Fuc
synthesis mutant(7) , high affinity recognition of the FP21
carbohydrate implies that the carbohydrate structure is of limited
distribution, and may be required because FP21 is not concentrated in a
compartment. This is reminiscent of the relationship between
UDP-Glc:glycoprotein Glc-1-phosphotransferase, another cytosolic
glycoprotein glycosyltransferase involved in peripheral modifications,
and phosphoglucomutase, which appears to be its predominant acceptor
substrate(5) .
The V of the purified
cFTase with respect to pNP-LNB was 620 nmol/min/mg protein, at 1.0
µM GDP-Fuc. The V
would be expected
to extrapolate to 830 nmol/min/mg at saturating GDP-Fuc, or about 2.5
µmol/min/mg with respect to the content of FT85 in the highly
purified preparation A. This value is greater than the estimated
specific activity of a highly purified Se-type FTase from
serum(33) , but less than that of a purified porcine Golgi
1,2-FTase(30) . Projecting back to the cell, the specific
activity suggests that there are on the order of 200 copies/cell of the
cFTase in the cytosol. If the approximately 2
10
copies/cell of the acceptor substrate FP21
were
dissolved in the full volume of the cell, FP21 would be at a
concentration of about an order of magnitude above its K
for fucosylation. If the cFTase were operating at its V
, there would be enough enzyme to be able to
fucosylate all FP21 in about 1 h.