(Received for publication, August 14, 1995; and in revised form, December 15, 1995)
From the
DSPA1 (Desmodus rotundus salivary plasminogen
activator), a plasminogen activator from the saliva of the vampire bat Desmodus rotundus, is an effective thrombolytic agent. An
unusual type of posttranslational modification, in which L-fucose is O-glycosidically linked to threonine 61
in the epidermal growth factor domain was found for natural DSPA
1
and its recombinant form isolated from Chinese hamster ovary cells.
In the present study a combination of carbohydrate and amino acid
composition analysis, amino acid sequencing, and mass spectrometry
revealed that the L-fucose is bound to residues 56-68 of
DSPA1. The amino acid sequence of this glycosylation site agreed
with the suggested consensus sequence Cys-Xaa-Xaa-Gly-Gly-Ser/Thr-Cys
described for other proteins. A new strategy for the identification of
the modified amino acid was established. Direct evidence for the
occurrence of fucosyl-threonine was obtained by mass spectrometry after
digestion of the glycopeptide with a mixture of peptidases. On the
basis of these results, DSPA
1 is a suitable model for studying the
influence of O-fucosylation on clearance rates, particularly
in comparative studies with the identically fucosylated and
structurally related tissue plasminogen activator.
Plasminogen activators (PAs) ()are involved in many
physiological processes and play a key role in fibrinolysis. They are
serine proteases that catalyze the conversion of plasminogen to plasmin
by a specific proteolytic
cleavage(5, 6, 7, 8) . Plasmin,
which is also a serine protease, efficiently degrades the fibrin
network. Due to their thrombolytic properties PAs are used as
therapeutic agents in acute myocardial infarction.
DSPA1 (Desmodus rotundus salivary plasminogen activator) is a
plasminogen activator from the saliva of the vampire bat Desmodus
rotundus(9, 10) . In contrast to other
plasminogen activators DSPA
1 displays a remarkable selectivity
toward fibrin-bound plasminogen (1) and is therefore
potentially a safer and more efficacious thrombolytic agent than other
PAs. For further characterization the glycoprotein was cloned and
expressed in baby hamster kidney cells (11, 12) as
well as Chinese hamster ovary (CHO) cells(13) . Like human
tissue plasminogen activator (t-PA), DSPA
1 is a multidomain
protein consisting of a finger domain, a serine protease, a kringle
domain, and an epidermal growth factor region. This type of multidomain
structure is found in several proteins that are active in coagulation
or fibrinolysis(14) . Comparison of the amino acid sequence of
t-PA and DSPA
1 reveals a homology of 72% for the corresponding
domains. In contrast to t-PA, which contains two kringle segments, only
one kringle region is present in DSPA
1. In t-PA the cleavage
sequence of Arg
-Ile, which is responsible for the
conversion of the single chain form to the higher active two-chain
form, is located in the kringle 2 domain. rDSPA
1 lacks this domain
and is the first example of a naturally occurring PA acting as a single
chain enzyme. DSPA
1 contains the consensus sequence of two N-glycosylation sites, which corresponds to t-PA minus the
kringle 2 region.
Epidermal growth factor domains are found in many
proteins involved in coagulation or fibrinolysis(14) . In
recent years three different types of posttranslational modifications
have been identified within this region(15) .
-Hydroxylation of Asp and Asn residues in the amino acid sequence
-Cys-Xaa-Asp/Asn-Xaa-Xaa-Xaa-Xaa-Tyr/Phe-Xaa-Cys-Xaa-Cys- is
established for many proteins, e.g. protein C, factor IX,
factor X, protein S, protein Z, and
others(16, 17, 18, 19, 20, 21) .
In addition to
-hydroxylation, two other types of
posttranslational modification have been described, both involving
glycosylation. Thus the disaccharide Xyl-Glc and the trisaccharide
Xyl
-Glc are present in bovine factor VII, factor IX,
protein Z, and thrombospondin (22, 23, 24) and in human factor VII and
factor IX(25) ; secondly O-linked L-fucose
was first discovered in the EGF domain by Kentzer (26) and
confirmed by Buko (27) for human pro-urokinase. Human factor
VII(28) , human factor XII(29) , and t-PA derived from
different cell lines (30) all contain O-linked L-fucose. Factor IX contains a tetrasaccharide bound via
fucose to serine(31) . In all published accounts, L-fucose is attached to a suggested consensus sequence
-Cys-Xaa-Xaa-Gly-Gly-Thr/Ser-Cys- and is found only in proteins of
human origin, with the exception of bovine factor VII(32) .
Since this consensus sequence is present in DSPA1, the possible O-fucosylation of natural and CHO cell-derived rDSPA
1 was
investigated, and its occurrence is described in this report.
Peptides were separated by a chromatography workstation (Bio-Rad)
equipped with a Eurospher C-100 column (4.6 250-mm, Knauer) at
room temperature with a flow rate of 1 ml/min. The applied gradient
consisted of solvent A (H
O, 0.1% trifluoroacetic acid) and
solvent B (CH
CN/H
O (70/30), 0.1%
trifluoroacetic acid). The two-stage gradient started 3 min after
injection and went from 0 to 12% solvent B in 10 min and then up to 50%
B in 110 min. Absorbance of the effluent was monitored at 215 nm.
Chymotryptic digestion (approximately 0.25 µg/nmol of peptide)
of the HPLC-purified tryptic peptide was performed in 1%
NHHCO
overnight. Peptides were separated as
described above.
Pronase E (1 mg/ml) was preincubated at 60 °C
in 1% NHHCO
, pH 7.0, for 30 min. Digestion was
performed for 48 h at 37 °C in 1% NH
HCO
, pH
7.0, containing 10 mM CaCl
, the Pronase E
(approximately 100 µg/nmol of peptide) being added in two portions,
one at 0 h and the other at 24 h. Proteins were removed by
centrifugation using a Centricon 3 microconcentrator at 2000
g.
Peptide hydrolysis was performed with 6 M HCl at 110 °C for 24 and 48 h. Amino acids were derivatized with o-phthaldialdehyde and 3-mercaptopropionic acid. Effluent was monitored by fluorescence at an emission wavelength of 330 nm with excitation at 450 nm.
Digestion with
-L-fucosidase from C. lampas was carried out
with 0.05 units/
3 nmol of peptide in a buffer containing 200
mM sodium citrate-phosphate, pH 4.1, for 18 h at 37 °C
according to the manufacturer's protocol.
To elucidate the covalent attachment of fucose to the EGF
domain, rDSPA1 was chemically reduced, carboxymethylated, and
enzymatically digested with trypsin. The resulting peptides were
separated by reversed-phase HPLC (RP-HPLC) (Fig. 1), and their
carbohydrate content was analyzed by monosaccharide composition
analysis. Besides two theoretical N-glycosylation sites, we
found a peptide (P1) that carried only one type of monosaccharide.
Glycan composition analysis by HPAEC-PAD yielded a signal corresponding
to the retention time of L-fucose (Fig. 2A). To
verify this result, monosaccharide analysis by GC-MS was used.
Attachment of L-fucose was confirmed by calculating the
retention time relative to an internal standard and by analysis of the
mass spectrometric fragmentation pattern (Fig. 2B). The
molecular mass of the fucosylated peptide (P1) was determined by
MALDI-TOF-MS. In the positive ion mode, a signal at m/z = 1781.3 was detected (Fig. 3A). By increasing
the laser intensity a second signal consistent with the loss of fucose
was observed. Measurements in the negative ion mode yielded a signal at
m/z = 1778.9, indicating an intact glycopeptide (Fig. 3B). The expected mass for the carboxymethylated
tryptic peptide comprising residues 56-82 (numbering from the N
terminus) was 3186.5 (M + H)
or 3349.6 (M +
H)
with one fucose residue attached. The observed mass
of 1781.3 was consistent with a sodium adduct of a peptide containing
residues 56-68 plus one fucose residue (Table 1). Amino
acid composition analysis and sequence determination of the first amino
acids confirmed the suggested peptide, located in the EGF domain.
Figure 1:
Detail of the RP-HPLC
separation of tryptic peptides from 300 µg of rDSPA1. Peak
P1, marked by an arrow, was shown to contain a
-L-fucose residue and was selected for further
investigation. Chromatographic conditions are described under
``Experimental Procedures.''
Figure 2:
Monosaccharide composition analysis
performed by HPAEC-PAD and GC-MS. A, glycopeptide P1 was
isolated, hydrolyzed using acidic conditions, and subjected to
HPAEC-PAD. Calculation of the retention time of the released
monosaccharide (peak X) relative to an internal standard (peak Y) suggested the presence of fucose. B, for
GC-MS analysis the released monosaccharide was subsequently reduced and
peracetylated. The mass spectrum of the peracetylated alditol displayed
a typical fragmentation pattern (M - 59) for a
deoxyhexose under electrical ionization conditions and confirmed the
attachment of fucose.
Figure 3: Mass spectrometric analysis of peptide P1. Mass determination using the positive ion mode (peak A) or the negative ion mode (peak B) was consistent with a sodium adduct of a peptide comprising residues 56-68 with one fucose residue attached. The calculated mass for peptide P1 is given in Table 1.
To
determine the anomeric configuration of the glycosidic linkage, the
tryptic glycopeptide was digested with -L-fucosidase from
chicken liver and from C. lampas. Three peptides were isolated
by RP-HPLC after treatment with
-L-fucosidase from
chicken liver; these contained presumably no phenylalanine (amino acid
residue 68) and had lost some fucose, as indicated by mass
spectrometry. Incubation of peptide P1 with
-L-fucosidase
from C. lampas also generated some new peptides, which were
not further analyzed. Peptidase activity is presumably due to
impurities in the enzyme preparation. To circumvent the problem of
degradation, the peptide was treated with chymotrypsin. Chymotryptic
peptides were separated by RP-HPLC (Fig. 4A) and subjected
to monosaccharide composition analysis. Fucose was found to be attached
to two peptides, which showed small differences in retention times.
Mass determination of the major peak P2 resulted in signals at
m/z = 1172.1 and m/z =
1148.1, using the positive or negative ion mode, respectively. This
agreed with the calculated mass for a sodium adduct of the peptide
comprising residues 56-63 plus one fucose residue and its
deprotonated form, respectively (Table 1).
Figure 4:
Detail of the RP-HPLC separation of
chymotryptic peptides. A, treatment of tryptic peptide P1 with
chymotrypsin generated some new peptides, which were separated by
RP-HPLC. Peptides P2 and P3 contained a fucose residue as indicated by
monosaccharide analysis. B, RP-HPLC separation of chymotryptic
peptides after digestion of peptide P2 with -L-fucosidase
from chicken liver. Removal of L-fucose was about 60%; thus
peptide P2* is the unchanged P2, whereas peptide P4 represents the
defucosylated form.
It is known that
the Asn-Gly sequence can be converted to Asp-Gly by deamidation in
alkaline solution(30, 33) . The minor peak P3 was
identified by MALDI-TOF-MS as the deamidation product of peptide P2.
Incubation of peptide P2 with -L-fucosidase from chicken
liver produced two peptides, which were separated by RP-HPLC (Fig. 4B). Mass spectrometric analysis revealed that
peptide P2* corresponded to the fucosylated peptide P2, whereas peptide
P4 represented the defucosylated form of P2. Comparison of the peak
areas for P2* and P4 indicated nearly 60% removal of L-fucose.
To identify the amino acid covalently linked to L-fucose, the chymotryptic peptide was exhaustively digested with a mixture of peptidases (Pronase E). Proteins and large peptides were removed by ultrafiltration before the mixture was subjected to mass determination. We detected a signal at m/z = 287.2 in the positive ion mode (Fig. 5A). This is consistent with the calculated mass for fucosyl-threonine as an adduct of sodium (m/z = 288.2). Measurements in the negative ion mode (Fig. 5B) resulted in a signal at m/z = 264.2, corresponding to the mass of deprotonated fucosyl-threonine (m/z = 264.2).
Figure 5:
Mass spectrometric analysis of
fucosyl-threonine. A, a signal at m/z = 287.2 consistent with fucosyl-threonine as a sodium
adduct was observed using the positive ion mode. B,
measurements in the negative ion mode resulted in a signal at m/z = 264.2 corresponding to
fucosyl-threonine. Ions at m/z = 153.7 and m/z = 307.3 represented matrix ions
DHB and the dimer
(DHB
)
. Both spectra were recorded in the
linear mode.
To
determine whether rDSPA1 was partially or completely modified by O-linked L-fucose, tryptic peptides were analyzed by
MALDI-TOF-MS. Starting from the elution time of the fucosylated peptide
P1, the subsequent 10 ml of the RP-HPLC run were fractionated and
subjected to mass determination. No signal for the defucosylated
peptide was observed. Furthermore, we compared the elution profile of
the defucosylated peptide, obtained by treatment of the tryptic
peptides with
-L-fucosidase from C. lampas, with
the profile of untreated peptides. A very weak signal from the
untreated peptides corresponded to the retention time of the
defucosylated peptide, but mass spectrometry showed that this signal
did not represent the defucosylated peptide.
The small scale tryptic maps for natural and
rDSPA1 showed a very similar pattern when developed under
identical conditions (Fig. 6). Mass spectrometry of peptides R1
and R2 of rDSPA
1 yielded ions at m/z =
1781.0 and m/z = 1781.7, indicating the
presence of the fucosylated peptide and the deamidated fucosylated form
containing amino acid residues 56-68. All peptides of natural
DSPA
1 that eluted between 85 and 110 min were subjected to mass
spectrometric analysis. The observed masses of 1780.9 Da for peptide N1
and 1782.0 Da for peptide N2 correspond to the sodium adduct of the
fucosylated peptide and its deamidated form characterized from
rDSPA
1. The covalent attachment of fucose to the peptides R1, R2,
N1, and N2 was confirmed by monosaccharide analysis. Mass spectrometric
data, monosaccharide analysis, and the nearly identical retention times
on RP-HPLC for the peptides R1/N1 and R2/N2 demonstrated that natural
and recombinant DSPA
1 were O-fucosylated in the same
peptide domain.
Figure 6:
Detail of the RP-HPLC tryptic maps
obtained by small scale tryptic digestion of recombinant (A)
and natural (B) DSPA1. A, peaks labeled R1 and R2 represent the O-fucosylated
peptide and its deamidated fucosylated form, respectively. B,
mass spectrometry and monosaccharide analysis of peaks marked N1 and N2 revealed the presence of the corresponding
fucosylated peptides for natural
DSPA
1.
In the present study we established that natural DSPA1
and CHO cell-derived rDSPA
1 are modified by the presence of a
single L-fucose residue, which is O-glycosidically
linked to threonine 61 in the epidermal growth factor domain. The
fucosylated tryptic peptide was isolated by RP-HPLC and identified by a
combination of amino acid and carbohydrate analysis as well as mass
spectrometry. Monosaccharide composition analysis by HPAEC-PAD and
GC-MS revealed the presence of the deoxyhexose L-fucose. The C
terminus of the tryptic peptide was phenylalanine, a typical cleavage
point for chymotrypsin but less favorable for trypsin. Cleaving at the
C-terminal side of phenylalanine, tryptophan, tyrosine, leucine, or
methionine is frequently observed, even when chymotryptic activity has
been chemically inhibited(34, 35) . Cleavage of bonds
adjacent to aromatic residues may therefore be due to small amounts of
-trypsin rather than to chymotrypsin(36) .
The
-anomeric configuration of the glycosidic linkage and the presence
of fucose in the L-enantiomeric form were demonstrated by
susceptibility to
-L-fucosidase from chicken liver and
from C. lampas. Comparison of RP-HPLC profiles of peptides
treated and untreated with L-fucosidase, followed by mass
spectrometric analysis, indicated that threonine 61 is always
fucosylated.
Different techniques of mass spectrometry have been previously reported for the identification of modified peptides(27, 31, 32) . In this study mass determination was performed by MALDI-TOF-MS. The quality of spectra recorded in the positive and the negative ion modes were comparable, with no basic differences in sensitivity (the same number of ions was formed) or undesired fragmentation. Loss of fucose during mass spectrometry (29) can be minimized by attenuating the laser intensity and working at the ionization threshold.
Characterization
of the fucose binding size is complicated by its lability under
alkaline or acidic conditions. -Elimination applied to the O-glycosylated amino acid residues, followed by comparative
amino acid analysis, has been reported(30, 32) . In
the present study an alternative approach was developed for the
identification of the modified amino acid. The fucosylated peptide was
exhaustively digested with Pronase E to cleave all peptide bonds,
followed by mass spectrometric analysis. Attachment of L-fucose to threonine was demonstrated directly by the
detection of fucosyl-threonine with MALDI-TOF-MS. This new strategy may
represent the most favorable method for the identification of this type
of glycosylation site.
Attachment of O-linked L-fucose has been described previously for human proteins.
This modification was also observed in bovine factor VII but not
described in detail(32) . The proteins examined were either
obtained from human plasma or expressed in different cell lines. In the
present study we demonstrated that the vampire bat protein DSPA1
and its recombinant form rDSPA
1, expressed in CHO cells, are
posttranslationally O-fucosylated. In all reports so far, the
attachment of L-fucose is restricted to the suggested
consensus sequence -Cys-Xaa-Xaa-Gly-Gly-Thr/Ser-Cys- located in EGF
domains (Table 2). This motif is also valid for DSPA
1, since
fucosylated threonine 61 is bound to a corresponding sequence in the
EGF domain. The result shows that O-linked L-fucose
is not limited to proteins of human origin but may be more widespread
than earlier suspected. Stults and Comming (37) suggested that
many glycoproteins carry O-linked fucose, based on
investigations using a CHO cell line deficient in N-acetylglucosaminyltransferase I (Lec1). In this study Lec1
cells were metabolically radiolabeled with
[6-
H]fucose.
-Elimination of the
radiolabeled proteins resulted in the release of
[6-
H]fucitol. However, the fucose attachment site
was not assigned to a specific sequence or domain. Apart from this
report, O-fucosylation has been observed only in EGF domains,
and all of the modified proteins are involved in blood coagulation or
fibrinolysis.
Presumably O-linked L-fucose has
some physiological relevance, but no specific function has so far been
discovered. It is noteworthy that L-fucose is an important
constituent of many carbohydrate antigenic epitopes such as the ABO
system and Lewis X structures, recognized by
selectins(38, 39) . It remains to be determined
whether O-linked L-fucose also represents a lectin
recognition signal. Preliminary data indicate that this modification
may influence the clearance rate of t-PA. Hajjar and Reynolds (40) reported binding of t-PA to suspended HepG2 cells,
followed by a rapid internalization and degradation of t-PA. Inhibition
studies and treatment with -L-fucosidase suggested the
involvement of O-linked L-fucose. It is not yet known
whether binding of t-PA to the low density lipoprotein receptor-related
protein/
2-macroglobulin receptor (LPR receptor) (41) depends on O-fucosylation. Thus O-linked L-fucose-mediated clearance may be a new mechanism or an
essential part of a described clearance pathway. Comparative studies by
Witt et al.(42) in dogs showed that the clearance of
rDSPA
1 is 4 times less than that of t-PA. In both proteins, O-linked L-fucose may contribute to clearance, but it
cannot account for the significantly different clearance rates. The
half-lives of these proteins therefore seem to be regulated by
different mechanisms. The role of O-linked L-fucose
as a recognition signal and its contribution to clearance rates remain
to be established.
Dedicated to Professor Heinz Egge on the occasion of his 65th birthday.