From the Friedrich Miescher-Institut, P. O. Box 2543, CH-4002 Basel, Switzerland, the § Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, the ¶ Institute for Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, D-48149 Münster, Germany, and the ** Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston Massachusetts 02215
Received for publication, September 4, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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Thrombospondin-1 (TSP-1) is a multidomain
protein that has been implicated in cell adhesion, motility, and
growth. Some of these functions have been localized to the three
thrombospondin type 1 repeats (TSRs), modules of ~60 amino acids in
length with conserved Cys and Trp residues. The Trp residues occur in
WXXW patterns, which are the recognition motifs for
protein C-mannosylation. This modification involves the
attachment of an Thrombospondin (TSP)1-1
is a large homotrimeric glycoprotein present in the The three TSR modules, which are found in TSP-1 and -2 but not in the
other TSPs, are of particular importance to the present study. TSRs are
~60 amino acids in length and are characterized by conserved Cys,
Trp, Ser, and Arg residues (9). Molecules that have been proposed to
bind these modules in TSP-1 include fibrinogen/fibrin, CD36, heparan
sulfate, other glycosaminoglycans and sulfatide, fibronectin, TGF- Post-translational modifications in TSP-1 could complicate the use of
synthetic peptides or recombinant proteins. The sequence WXXW has recently been shown to be the recognition motif for
a glycosyltransferase that attaches an Here we demonstrate that human platelet TSP-1 and recombinant TSRs
expressed in various insect cells are C-mannosylated. These results increase the panel of proteins that are subject to
C-mannosylation and the range of organisms able to catalyze
this reaction. In the course of the mannosylation analyses, we
discovered that all three TSRs contained an O-linked
disaccharide (Glc-Fuc-O) on the homologous Thr or Ser
residue in the sequence CXX(S/T)CG of the
putative cell-binding site. This finding is the first report of this
disaccharide in a protein and demonstrates that protein O-fucosylation is not restricted to EGF-like modules. Taken
together the analyses show that the subtype of TSR found in TSP-1 is
subject to two types of unusual glycosylations in a 10-residue stretch, to which several functions have been localized previously.
Materials--
Monosaccharide standards for HPAE-PAD analysis
were obtained from Sigma. Human platelet TSP-1 was purified from the
supernatant of thrombin-stimulated platelets as described previously
(26). Recombinant TSR3 (see Fig. 1 for nomenclature of recombinant
proteins) and TSR123 were expressed in Sf9 or High-Five cells
using the vector pAcGP67P3.coco and pAcGP67P123.coco. These vectors are a modification of pAcGP67 (Invitrogen, Carlsbad, CA), in which modules
of interest are placed between the signal sequence of baculovirus GP67
and a His tag. Details about construction of the vectors and protein
purification have been published elsewhere (27). Recombinant TSR123 was
also expressed in Drosophila S2 cells. A DNA fragment
encoding all three TSRs of TSP-1 (amino acids 361-530) was prepared
using human TSP-1 cDNA as a template and the forward primer
475htsp1f (GAT GAT CCC GGG GAC GAC TCT GCG GAC GAT GGC) and the reverse
primer 476htsp1r (GAT ACC GGT AAT TGG ACA GTC CTG CTT G). The
polymerase chain reaction product was cloned between the
XmaI and the AgeI sites of the vector
pMT/BiP/V5-HisA (Invitrogen, Carlsbad, CA). The recombinant protein
included the vector-derived amino acid sequence RSPWG at the N terminus
and TGHHHHHH at the C terminus. The fidelity of the polymerase chain reaction product was verified by nucleotide sequencing. The expression vector was cotransfected into Drosophila S2 cells with the
selection vector pCoHYGRO according to the manufacturer's protocol
(Invitrogen). Transfected cells were selected with hygromycin B, and
the expression of recombinant peptide was monitored by Western blotting
using the polyclonal antibody R3 that was raised against a fusion
protein that contained all three TSRs of TSP-1 (4). For large scale preparation of recombinant protein, S2 cells were grown in serum-free media for 5 days. The culture supernatant was centrifuged to remove the
cells and dialyzed against 20 mM sodium phosphate, pH 7.8, and 500 mM NaCl. The dialysate was applied to a column of
ProBond resin (Invitrogen). The column was washed with 20 mM sodium phosphate, pH 7.8, and 500 mM NaCl
followed by 20 mM sodium phosphate, pH 6.0, and 500 mM NaCl. The column was eluted stepwise with 20 mM sodium phosphate, pH 6.0, 500 mM NaCl
containing 50, 200, or 500 mM imidazole. The protein
eluting with 500 mM imidazole was dialyzed against 20 mM sodium phosphate, pH 7.0, and 500 mM NaCl,
and 1% sucrose was added prior to storage.
Peptide Nomenclature--
The amino acid numbering of mature
human TSP-1 has been used (2). Tryptic peptides have been numbered
according to their occurrence in the mature polypeptide and have been
labeled with the prefix "T." The following affixes were used to
indicate subdigestions: chymotrypsin (Ch), endoproteinase AspN (D),
aminopeptidase M (AMP), carboxypeptidase (CPase). Different
subfragments have been numbered with arabic numbers according to their
order in the parent peptide. In case of heterogeneity in a peptide,
subforms have been indicated with Latin letters (e.g.
T55a-Ch-1).
Isolation of Peptides--
Proteins were reduced and
carboxamidomethylated as described (22) and cleaved with proteases at
37 °C. TSP-1 (0.4 or 5 mg) was digested twice with trypsin (1% w/w)
in 50 mM NH4HCO3, pH 7.8, for 2 and
18 h, and relevant tryptic peptides were subfragmented with
chymotrypsin (1% w/w) for 7 h. Recombinant TSR3 from Sf9 cells (70 µg in 400 µl of 50 mM Tris-HCl, pH 8.0) was
digested twice with trypsin (1.5% w/w) for 2 and 4 h and
subsequently with endoproteinase AspN (1.5% w/w) for 4 h.
Peptides from recombinant TSR3 from High-Five cells were prepared in
the same way, except that the tryptic peptide TSR3-H5-T55 was purified
before cleavage with endoproteinase AspN. Recombinant protein TSR123
from Drosophila S2 cells (100 µg in 150 µl of 50 mM Tris-HCl, pH 8.0) and TSR123 from High-Five cells (130 µg or 2 mg in the same buffer) were cleaved twice with trypsin (1.5%
w/w) for 2 h. Digests were fractionated by HPLC or LC-ESIMS using
Vydac C8 and C18 columns essentially as described previously (22). If
necessary, final purification of peptides was achieved by HPLC using 25 mM NH4 acetate, pH 6.0, containing 2 or 80%
CH3CN as buffer A and B, respectively.
Analysis of C-Mannosylated Peptides--
Purified
C-mannosylated peptides were examined by low energy CID
tandem-MS experiments of the [M + 2H]2+ ions, using a
Sciex API 300 mass spectrometer. The resolution of the first quadrupole
was set to 1300 for the selected mass, whereas the third quadrupole was
set at isotopic resolution over the entire mass range. Solid phase
Edman degradation and identification of PTH-(C2-Man-)Trp
were performed as described previously (22).
Nanospray Quadrupole-Time-of-Flight Mass Spectrometry of
O--
Glycosylated Peptides Subfragmentation of O-Glycosylated Peptides--
Peptides
T42-Ch-2, T49-Ch-2, and T55a-Ch-2 from platelet TSP-1 were
subfractionated as follows. Digestions were performed at 37 °C with
enzymes obtained from Roche Molecular Biochemicals. Where indicated,
purification was achieved by LC-MS. The amounts of peptide were
estimated from peak heights in HPLC experiments. The following buffers
were used: 50 mM NH4HCO3, pH 7.3, for aminopeptidase M, the same buffer at pH 7.8 for CPase B, 50 mM NH4 acetate, pH 5.0, for CPase Y, 10 mM Tris-HCl, pH 8.0, for endoproteinase AspN, and 5 mM NH4HCO3 for subtilisin. Peptide
T42-Ch-2 (200 pmol in 10 µl) was cleaved with 0.03 units of
aminopeptidase M for 17 h, and the product was isolated. In a
second experiment the purified product of the aminopeptidase digestion
was further digested in 5 µl with CPase B (0.7 µg) for 2 h,
dried, and cleaved with CPase Y (1 µg) for 44 h. Peptide
T49-Ch-2 (100 pmol in 10 µl) was treated with aminopeptidase M in the
same way. A second sample (200 pmol in 5 µl) was cleaved with
endoproteinase AspN (220 ng) for 2 h. The purified product was
then digested in 5 µl with 1 µg of CPase Y for 26 h. Peptide
T55a-Ch-2 (200 pmol in 10 µl) was cleaved with subtilisin (200 ng)
for 18 h. Purified fragment product in 10 µl was further cleaved
with aminopeptidase M, as above. The cleavage of another 200 pmol in 5 µl with CPase B and Y was carried out as described for peptide
T42-Ch-2.
The final products of these cleavages were analyzed in a nano-ESIMS
experiment, monitoring neutral loss of 308 or 146 Da from either the
[M + H]+ and/or [M + 2H]2+ ion, using the
API 300 mass spectrometer. Their identity was verified in a low energy
CID tandem-MS experiment, using the same apparatus. Methods for the MS
analyses have previously been described (28). The corresponding
peptides from recombinant TSR3 from Sf9 and High-Five cells were
treated and analyzed in the same way.
Monosaccharide Building Block Analysis of O-Glycans with HPAE-PAD
Chromatography--
Monosaccharide species from glycopeptides were
obtained by acid hydrolysis in 2 M trifluoroacetic acid for
2 h at 100 °C. Building block analysis of released sugars was
carried out on a DX 500 chromatography system (Dionex Corp., Sunnyvale,
CA) using a CarboPacTM PA-1 column (4 × 250 mm).
Oligosaccharides were detected using an ED40 electrochemical detector,
with potentials of the gold electrode E1 = 0.05 V,
E2 = 0.75 V, E3 = Platelet TSP-1 Is C-Mannosylated on Trp-368, -420, -423, and
-480--
Counting from the N terminus of mature human platelet TSP-1,
the tryptic peptides T42, T49, and T55, originating from TSR-1, -2 and
-3 respectively, contain potential C-mannosylation sites (Fig. 1). Reduced and
carboxamidomethylated TSP-1 was cleaved with trypsin and fractionated
by reversed phase LC-ESIMS (Fig. 2A). The MS data were
extracted for the theoretical mass of each of these peptides, taking
into account the possible presence of 0-3 hexosyl residues. This
allowed the detection of modified peptides T42, T49, and T55a, as well
as a minor amount of unmodified T55b (Fig. 2A). Nanospray
ESIMSMS analysis of the impure peptides revealed that in all three
peptides the third Trp residue in the WXXWXXW motif was unmodified (data not shown). This allowed the use of chymotrypsin to generate the N-terminal fragments of these peptides (affixed "Ch-1"), which contain the C-mannosylation
sites, and their C-terminal fragments (affixed "Ch-2") (Fig. 2,
B-D). Peptides T42-Ch-1, T49-Ch-1, and T55a-Ch-1 were
purified to apparent homogeneity, as judged from the single amino acid
sequence observed by Edman degradation.
Peptide T42-Ch-1, originating from the first TSR module, had a mass of
2230.0 Da, which was consistent with residues 355-371 containing one
hexosyl residue. The substituent could be present at the Trp residues
at positions 2, 11, or 14 of the peptide or it could be
O-glycosidically linked to one of the hydroxy amino acids
(Fig. 1). The latter could be excluded, because no 162-Da losses in MS
or MSMS experiments, indicating O-linked hexose, were
observed. It should be noted that under the MS conditions used, such
phenomena were indeed observed in other peptides (see below). The
ESIMSMS spectrum of peptide T42-Ch-1 is shown in Fig. 3A. The m/z values
of ions b2-b8 all showed the Trp at position 2 of the peptide
(Trp-355) to be unmodified. Modification of the Trp at positions 11 or
14 of the peptide (Trp-365 and -368, respectively) would result in MSMS
spectra that differed in the ions b11-13 and y4-6. Five of the six
ions that were expected for Trp-368 containing the 162-Da substituent
were observed (Fig. 3A), but none of those indicating
modification of Trp-365 were detected. In low energy CID tandem-MS,
ions containing a (C2-Man-)Trp show a characteristic loss
of 120 Da (22, 29, 30). The spectrum of T42-Ch-1 (Fig. 3A)
exhibited this phenomenon for the parent, y5, y6, and b14 ions, thus
providing evidence for the C-glycosidic linkage.
Edman degradation yielded PTH-Trp in cycles 2 and 11, and in
cycle 14 a PTH-derivative was observed, which comigrated exactly with
PTH-(c2-Man-)Trp obtained from residues 5-10 of human
RNase2 (Table I).
Peptide T49-Ch-1 (1666.8 Da), from the second TSR module, was 324 Da
heavier than expected from the cDNA sequence, suggesting the
presence of two hexosyl residues. Fig. 3B shows its
nanospray ESIMSMS. No losses indicating O-linked hexosyl
residues were observed. The presence of the ions y5, y6, b6, and b8
strongly indicated that each of the Trp residues at positions 5 and 8 of the peptide (Trp-420 and-423, respectively) was carrying a hexosyl
residue. Ions that would indicate that one of these residues was
unmodified and the other modified with a 324-Da disaccharide were not
detected. The characteristic 120-Da loss from the parent and b9 ions
occurred twice, in agreement with two C-hexosylated residues
in this peptide. Edman degradation of peptide T49-Ch-1 yielded
PTH-(C2- Man-)Trp in cycles 5 and 8 (Table I).
The mass of peptide T55a-Ch-1 (1861.8 Da) suggested that it contained
one hexosyl residue, which could be located on Trp residues at
positions 9 or 12 of the peptide (Trp-477 and -480, respectively) or on
Ser-481. However, no indication of an O-linked sugar was obtained. The two possible modifications of the Trp residues would yield different b9-b11 and y4-y6 ions. Five of the six ions that located the hexosyl residue to Trp-480 were observed (Fig.
3C), whereas only the ion at m/z 1287.7 would
indicate that Trp-477 was modified. However, a more likely explanation
for the latter would be loss of H2O from the
(C-hexosylated) y10 ion (m/z 1305.7), since it
was part of a continuous series of losses of H2O (* in Fig.
3C). The presence of a C-C linkage was concluded from the 120-Da losses from the parent, b12-b13, y5, y7-y10, and y12 ions. Edman degradation yielded PTH-Trp in cycle 9 and
PTH-(C2-Man-)Trp in cycle 12 (Table I).
The minor amount of unmodified peptide T55b (3062 Da) was analyzed by
nanospray ESIMSMS without further purification. The results (not shown)
confirmed its identity and the absence of modification of its Trp residues.
The stoichiometry of C-mannosylation of the TSR modules of
platelet TSP-1 was high. T42 and T49 were found in the
C-mannosylated form only. The degree of modification of
Trp-480 in peptide T55 was ~90%. Since both the modified and
unmodified peptides comigrated with other peptides, this percentage
could only be estimated from the area under the peak of [M + nH]n+ ions for n = 2 and 3, in the total ion current trace associated with the LC-ESIMS
separation shown in Fig. 2A. A summary of these results is
given in Fig. 4.
Platelet TSP-1 Contains an O-Linked Disaccharide on Ser-377,
Thr-432, and Thr-489--
The C-terminal, chymotryptic fragments of
peptides T42, T49, and T55a were isolated in pure form (T42-Ch-2,
T-49-Ch-2 and T55a-Ch-2; Fig. 2, B-D). The spectra of these
peptides yielded masses that were 308 Da higher than predicted from the
sequence. Characteristically, the theoretically predicted mass was
observed as well (Fig. 2, B-D; insets). This was
reminiscent of the behavior of O-linked saccharides, for
which facile dissociation in MS and low energy CID tandem-MS
experiments have been well documented (31, 32). Furthermore, the mass
of 308 Da exactly fitted the sum of the masses of a deoxyhexosyl (146 Da) and a hexosyl residue (162 Da). These findings strongly suggested
the presence of O-linked sugars in these peptides.
Peptide T42-Ch-2 contained five hydroxyamino acids that potentially
could be O-glycosylated (Fig. 1). Four of these could be
removed by digestion with aminopeptidase M. The resulting peptide, T42-Ch-2-AMP, still contained the 308-Da substituent (Table I). The
Q-TOF tandem-MS of the doubly charged molecular ion of the peptide at
m/z = 664.29 is shown in Fig.
5A. The observation of the B
fragment ions of the HexdHex disaccharide at m/z = 309.12, deoxyhexose at m/z = 147.08, and hexose at
m/z = 163.07, as well as the ions corresponding to
their neutral loss of water provided evidence for the presence of the
disaccharide moiety. The signals at m/z = 822.87 [M
dHex + 2H]2+, 897.36 [b6 dHexHex]+, and
1153.44 [b8 dHexHex]+ strongly indicated that the
disaccharide HexdHex is attached to Ser-377 through the dHex
residue.
Independent evidence for the site of modification was obtained by
treating this peptide with CPase B followed by CPase Y. This yielded a
peptide with a mass of 629.9 Da, having the sequence S(Cam)CG + 308 Da
(Table I). Since the cysteine residue had been carboxymethylated, it
could not carry the substituent, which therefore had to be present on
the Ser residue (Table I).
In peptide T49-Ch-2 five potential glycosylation sites, i.e.
Ser-427, Ser-428, Ser-430, Thr-432, and Thr-439 (Fig. 1), are present.
The Q-TOF tandem-MS of the doubly charged molecular ion of the peptide
at m/z = 903.88 is shown in Fig. 5B. The
observation of the B fragment ions of the HexdHex disaccharide at
m/z = 309.12, deoxyhexose at m/z = 147.06, and hexose m/z = 163.05, as well as the ions
corresponding to their respective neutral losses of water, provided
evidence for the presence of the disaccharide moiety. Its attachment to
Thr-432 could be concluded from the observation of three singly charged
glycosylated fragments: at m/z = 1286.59 [y9
HexdHex]+, 1472.54 [y11 HexdHex]+, and
1632.69 [y12 HexdHex]+. The signal at m/z = 822.87 [M dHex + 2H]2+ presented direct evidence for
the HexdHex disaccharide attachment to Thr-432 through the dHex
residue. No ions were detected that would indicate that either dHex,
Hex, or the disaccharide Hex-dHex were attached to any of the remaining
four potential glycosylation sites.
Aminopeptidase M treatment of peptide T49-Ch-2 yielded the peptide
T(Cam-)CGDGVITR + 308 Da, whereas digestion with endoproteinase AspN
followed by CPase B and Y, in a separate experiment, resulted in the
peptide SS(Cam-)CSVT(Cam-)C + 308 Da (Table I). The two peptides only
had the sequence -T(Cam-)C- in common, confirming that the substituent
was present on the Thr residue.
In peptide T55-Ch-2 two possible O-glycosylation sites,
i.e. Ser-487 and Thr-489, are present (Fig. 1). The Q-TOF
tandem MS of the doubly charged ion of this peptide (m/z
845.01) is shown in Fig. 5C. The observation of the B
fragment ions of the HexdHex disaccharide (m/z = 309.31) and the hexose (m/z = 163.24), as well as the
ions corresponding to the neutral loss of water, provided evidence for
the disaccharide. Its attachment to Thr-489 could be concluded from the
observation of two out of four glycosylated peptide fragments,
m/z = 952.50 [y8 dHex]+ and 1114.57 [y8
HexdHex]+. Furthermore, the signal at m/z = 952.50 strongly suggested that the HexdHex disaccharide was attached
to Thr-489 through the dHex residue. It is important to note that no
ions were detected, which would indicate that either dHex, Hex, or the
disaccharide HexdHex was attached to Ser-487.
Peptide T55-Ch-2 yielded the peptide T(Cam-)CGGGQVK +308 Da (1113.2 Da)
upon digestion with subtilisin followed by aminopeptidase M, and in a
second experiment the peptide DI(Cam-)SVT(Cam-)C + 308 Da (1161.5 Da)
was obtained after consecutive CPase B and Y digestion (Table I). The
two fragments only had the sequence -T(Cam-)C- in common, confirming
that the modification was present on Thr-489.
Fucose and Glucose Are the Major Constituents of O-Linked Glycans
on TSP-1--
Results of HPAE-PAD analysis of monosaccharides released
from O-glycosylated peptides of TSP-1 are summarized in
Table II. Deoxyhexoses (Fuc and Rha) and
hexoses (Glc, Gal, and Man), commonly occurring in O-linked
oligosaccharides, were used as standards for instrument calibration. In
all three peptides, very intense fucose and glucose peaks were observed
(Fig. 6), which led to the conclusion
that fucose and glucose are the major constituents of
O-linked glycans on TSP-1. The glucose peak was sometimes
followed by a much smaller peak with a retention time roughly the same as that of the mannose standard. Although the difference in retention time (30 s) and the shape of the peak suggested that it could not be
assigned to mannose, additional analysis should be carried out. A
summary of the analyses of the O-linked glycans is given in
Fig. 4.
Recombinant TSR3 from Sf9 or High-Five Cells Are
C-Mannosylated on Trp-480 and O-Glycosylated on
Thr-489--
Sf9 cells infected with recombinant baculovirus
carrying the construct encoding TSR3 (TSR3-Sf9) yielded protein
that was heterogeneous with respect to its mass. A small amount of a
species with the mass expected from the cDNA sequence (7250 Da,
~5% of the total) was detected by LC-ESIMS. In addition, four other
species that had masses of 7396, 7412, 7558, and 7720 Da were found.
Because the mass increments closely corresponded to that of integral
numbers of dHex and Hex, the recombinant protein seemed to be
glycosylated at multiple sites. A tryptic/endoproteinase AspN digest
was fractionated by LC-ESIMS (Fig. 7),
yielding multiple forms of both the N-terminal peptide,
TSR3-Sf9-T55-D-1, as well as the C-terminal one,
TSR3-Sf9-T55-D-2. All other observed peptides (Fig. 7,
fractions 1, 2, 4, and 6) had masses as predicted
from the cDNA sequence. Peptide TSR3-Sf9-T55-D-1a had a mass
of 1758.8 Da, in agreement with the presence of one hexosyl residue.
ESIMSMS and Edman degradation showed Trp-480 to be
C-mannosylated, whereas the other Trp residues were
unmodified (Table III). Peptide
TSR3-Sf9-T55-D-1b had a mass, 1596.0 Da, in agreement with the
theoretical value (Table III) and thus was unmodified. By integrating
the areas under the relevant peaks, it could be estimated that Trp-480
was 55% C-mannosylated. Both peptides contain the sequence
Asn-Gly, which is sensitive to deamidation at basic pH values. The
deamidated forms of these peptides eluted ~0.4 min after the main
peak (fractions 7a and 8a; Fig. 7) and had a mass that was 1 Da
higher.
Two major forms of the C-terminal peptide were isolated as follows:
TSR3-Sf9-T55-D-2a, which was 308 Da heavier than expected from
the cDNA sequence (Table III), and peptide
TSR3-Sf9-T55-D-2b, which was 146 Da heavier. A small amount of a
peptide with the mass expected from the cDNA sequence was detected
in fraction 4, together with another peptide. Peptides
TSR3-Sf9-T55-D-2a and -2b were examined by nanospray Q-TOF MS as
described above for the peptides from platelet TSP-1. In addition,
exopeptidase fragments were analyzed. The data (Table III) demonstrated
that a disaccharide of the form Hex-dHex- was O-linked to
Thr-489 in peptide TSR3-Sf9-T55-D-2a. A single dHex residue was
present at the same position in peptide TSR3-Sf9-T55-D-2b.
Integration of the peaks of these peptides (Fig. 7) showed that Thr-489
in TSR3 from Sf9 cells was 95% O-glycosylated. The
ratio between mono- and disaccharide was ~1:1 (Fig. 4). In a separate
experiment, tryptic peptides from this protein were analyzed without
previous cleavage with endoproteinase AspN. This yielded six different
TSR3-Sf9-T55 peptides, representing all possible combinations
between C- and O-glycosylation at Trp-480 and
Thr-489, respectively (data not shown). Taken together, these results
explained the five forms of TSR3-Sf9, i.e. 7250 Da
(no modifications), 7396 Da [+ (dHex-)Thr], 7412 Da
[+(C2Man-)Trp], 7558 Da [+(C2-Man-)Trp
and (dHex-)Thr, and +(HexdHex-)Thr], and 7720 Da [+(C-Man-)Trp and
(HexdHex-)Thr]. Monosaccharide analysis (Table II) showed that only
fucose could be positively identified in TSR3-Sf9-T55-D2b, whereas fucose and glucose were detected in TSR3-Sf9-D2a.
The same experiments, except for the monosaccharide analyses, were
repeated with recombinant TSR3 from High-Five cells (TSR3-H5) (data not
shown). The results revealed the same modifications, although at lower
stoichiometries (Fig. 4).
Recombinant Modules TSR123 from S2 Cells Are Not C-Mannosylated but
Are O-Glycosylated on Ser-377, Thr-432, and Thr-489--
Fig.
8A shows the part of the
LC-ESIMS chromatogram containing the peptides with the
C-mannosylation motifs and the O-glycosylations. All other observed major peptides had masses as predicted from the
cDNA sequence. The MS data were extracted for the theoretical masses of TSR123-S2-T42, -T49, and -T55, including the C-
and O-glycosylations described above. Only two species were
detected for each of the three tryptic peptides, the unmodified form
and one, which was 146 Da heavier (Table
IV). No evidence for
C-glycosylation was observed at all. To verify this, each of
the tryptic peptides was cleaved with chymotrypsin. The results of an
analytical LC-ESIMS experiments for each of the peptides is shown in
Fig. 8, B-D. The peptides produced from the part of
TSR123-S2-T42, -T49, and T55 containing the C-mannosylation
motifs all had masses in agreement with those calculated from the DNA
sequence (data not shown), and thus were unmodified. The C-terminal
portions of these peptides, however, occurred in two forms, one having
a mass that agreed with the cDNA sequence and one that was 146 Da
heavier. The heavier peptides showed facile dissociation of the
substituent in the LC-ESIMS experiment (Fig. 8, B-D), as
was observed with the O-glycosylated peptides described
above. The primary structure of these peptides was verified by
nanospray ESIMSMS (Table IV), and the ratio of modified/unmodified
peptides was estimated by integrating the area under their peaks in
Fig. 8, B-D. Given the location of the O-linked
sugars in platelet TSP-1 and recombinant TSR3-Sf9 and TSR3-H5,
we assume that the dHex residues are located on Ser-377, Thr-432, and
Thr-489. These results have been summarized in Fig. 4.
By using the same strategy, TSR123 from High-Five cells was examined as
well (data not shown). In this case the first TSR was appreciably
C-hexosylated, whereas the other two were less than 5%
modified on Trp residues (Fig. 4). Small amounts of O-linked dHex and Hex-dHex were found in TSR1 and TSR2, whereas only the disaccharide was found on 30% of TSR3.
The results presented in this paper show that two sequences that
have been implicated in several activities of human platelet TSP-1 do
not occur as such but are covalently modified.
O-Glycosylation--
By using a novel, potent mass spectrometric
approach that will be described in detail
elsewhere,2 we demonstrated
that the three TSRs in platelet TSP-1 carry the O-linked
disaccharide Glc-Fuc-O-Ser/Thr in the sequence
CSX(S/T)CG. This sequence is common to many
TSR-containing proteins. In fact out of 88 TSRs examined, 63 had a Ser
or Thr at the position preceding the Cys. Numerous functions have been
assigned to this sequence, including cell adhesion (16, 33) and binding
to the cell surface receptor CD36, an 88-kDa membrane protein that is
expressed on many cells (18). This receptor mediates the in
vitro inhibition of basic fibroblast growth factor-induced
endothelial cell migration by TSP-1 (34) and is part of the signaling
pathway utilized by TSP-1 in the inhibition of neovascularization
in vivo (35). Direct binding between native TSRs and CD36
remains to be demonstrated, as does any effect of the
O-linked disaccharide.
O-Linked fucose has been found in the EGF-like modules from
a number of proteins (Table V) and has
been assumed to be exclusive for this type of structure (36). The
presence of the disaccharide Glc-Fuc-O-Ser/Thr in human
platelet TSP-1 and the O-fucosylation of the proteinase
inhibitor PMP-C from Locusta migratoria (37) show that this
modification is more general. TSP-1 is the first identified protein
that carries the O-linked disaccharide
Glc-Fuc-O-Ser/Thr. Labeling experiments had provided
evidence that Glc-
The importance of the O-linked, fucose-based
tetrasaccharide has recently been shown in the Notch receptor system.
Drosophila Notch plays a role in intercellular signaling
through interactions with ligands like Delta and Serrate/Jagged. These
interactions are positively or negatively regulated, respectively, by
the secreted protein Fringe. Moloney et al. (46) and
Brückner et al. (47) have recently demonstrated that
Drosophila and mammalian Fringe proteins are
O-fucose-specific
TSP-1 has three EGF-like modules, none of which has the
CXXGG(S/T)C recognition sequence. The first
module, however, has the recognition sequence for
O-glucosylation, CXSXPC, and
O-linked disaccharide Xyl-Glc has been described in bovine platelet TSP (49).
C-Mannosylation--
Several roles for the
WXXWXXW sequences in the TSRs of TSP-1 have been
suggested. Fibronectin has been shown to bind to the synthetic peptide
GGWSHW (50). The same peptide binds to latent TGF-
C-Mannosylation of Trp has only been documented for
vertebrate tissues and cells, although the earliest evidence for
hexosylation of Trp was found in a neuropeptide from stick insect (25).
Previously, it has been reported that Spodoptera
frugiperda cells (Sf9) or Drosophila
melanogaster Schneider 2 (S2) cells do not
C-mannosylate RNase 2 (23). For S2 cells, these findings
were confirmed by the results obtained with TSR123. However, Sf9
cells, and in addition Trichoplusia ni cells (High-Five),
did C-mannosylate TSRs (Fig. 4). The reason for this
difference remains unclear. The only relevant distinction between
the two studies may be the use of different secretion signals.
Nevertheless, our results show conclusively that insects are capable of
C-mannosylation and that the modification is widespread in
the animal kingdom.
Not all TSRs have the same C-mannosylation pattern. In
TSP-1 the first Trp in the sequence
WXXWXXWXXC is only modified in TSR2
(Fig. 4). Furthermore, the third Trp in this sequence in properdin, and
the terminal components of complement are modified in 8 out of 9 cases
(21, 22). This led to the hypothesis that an additional signal,
different from the sequence WXXW, is present in these
proteins (22). In contrast, in TSP-1 the third Trp was never
C-mannosylated (Fig. 4). Either the TSRs in these proteins differ structurally or megakaryocytes, the site of synthesis of platelet TSP-1, are unable to perform this kind of
C-mannosylation.
C- and O-glycosylations of platelet TSP-1
were stoichiometric in TSRs1 and -2, whereas the degree of modification
was higher than 90% in TSR3. In contrast, recombinant modules were
generally modified less, and consequently were heterogeneous in mass
(Fig. 4). O-Fucosylation of TSR1 and -2 in TSR123-S2 was
much lower than in TSP-1, and in all three modules elongation with
hexose was completely absent. It is possible that Schneider 2 cells do not contain the glucosyltransferase, although the activity has been
reported recently to occur in Drosophila (36). The level of
C-mannosylation in TSR123-H5 was different for different
residues, and always lower than in platelet TSP-1 (Fig. 4).
Modification of Trp-480 in TSR123-H5 was also lower compared with
TSR3-H5, which itself was less C-mannosylated than platelet
TSP-1. These differences may result from overexpression of these
proteins in the High-Five cells, if the amount of
C-mannosyltransferase or dolichyl-P-Man were
limiting. This would agree with the 5-fold higher level of expression
of TSR123-H5 compared with
TSR3-H5.4
N-Glycosylation and mucin-type
O-glycosylation of recombinant proteins in Sf9 and
High-Five cells has recently been reviewed (55, 56), and a direct
comparison of these cells has been made (57). In contrast, nothing was
known about O-linked fucose and its elongation in these
cells. Given the good expression of TSRs in insect cells and the large
number of biologically interesting proteins that contain these modules,
further studies on these glycosylation reactions in this type of cells
seem warranted.
-mannosyl residue to the C-2 atom of the first
tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368,
-420, -423, and -480 are C-mannosylated. Mannosylation also
occurred in recombinant, baculovirally expressed TSR modules from
Sf9 and "High Five" cells, contradictory to earlier reports
that such cells do not carry out this reaction. In the course of these
studies it was appreciated that the TSRs in TSP-1 undergo a second form
of unusual glycosylation. By using a novel mass spectrometric approach,
it was found that Ser-377, Thr-432, and Thr-489 in the motif
CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such
a disaccharide has been identified, although protein
O-fucosylation is well described in epidermal growth
factor-like modules. Both C- and
O-glycosylations take place on residues that have been
implicated in the interaction of TSP-1 with glycosaminoglycans or other
cellular receptors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-granules of
platelets (1). The 150-kDa monomer has a modular structure, with (from
the N to C terminus) a heparin-binding domain, oligomerization domain,
procollagen module, three type 1 repeats (TSRs, also called properdin
modules), three EGF-like repeats, a calcium binding domain, and a
globular C-terminal domain (2). It belongs to a protein family, which
contains at least four other members (3). Upon activation with
thrombin, for example, TSP-1 is secreted and binds to the surface of
activated platelets, thereby promoting their aggregation (4). In
addition, TSP-1 is incorporated into the fibrin network of the clot,
where it influences the structure of the fibrin filaments (5, 6) and
possibly their cleavage during fibrinolysis (7). TSP-1 is also secreted
by growing cells and can interact with cellular surfaces, extracellular
matrix, and soluble macromolecules. Roles for TSP-1 in a variety of
processes, such as embryonic development, response to injury, tumor
growth, and metastasis, angiogenesis, inflammation, growth factor
activation, and neurite outgrowth, have been postulated (for reviews
see Refs. 8-11).
,
laminin, and collagen VII (see Ref. 9 for a review). By using synthetic
peptides, the amino acid sequence WSXW, which is present in
all three TSRs, has been proposed to be involved in the binding of
heparin as well as TGF-
(12, 13). However, in studies using
proteolytic fragments obtained from platelet TSP-1 (14), or recombinant
TSRs from Sf9 cells (15), no heparin binding was observed at
physiologic ionic strength. The amino acid sequence CSVTCG has been
suggested to be a cell-binding site in the second and third TSR of
TSP-1 (16-18). Although direct binding between the peptide YCSVTCG and Jurkat cells bearing CD36 has been reported (18), no increased binding
of TSP-1 to COS cells expressing the receptor could be observed (19).
At present the reasons for the discrepancies between different
approaches remain unclear.
-mannopyranosyl residue to
the C-2 position of the first Trp (20). We have shown that other
proteins that contain TSRs, i.e. C6, C7, C8
, C8
, C9,
and properdin, are C-mannosylated on multiple Trp residues
(21, 22). This form of protein glycosylation appears to be widespread in biology, since the C-mannosyltransferase activity has
been found in mammals, birds, amphibians, and fish (23, 24), and tryptophan hexosylation has been found in insect cells (25).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Nano-electrospray mass spectrometric
analyses were performed on a quadrupole-time-of-flight (Q-TOF) mass
spectrometer (Micromass, Manchester, UK) in the positive ion mode.
Purified glycopeptides (~4-8 pmol/µl dissolved in methanol, 0.4%
trifluoroacetic acid (1:1, v/v)), were loaded into homemade nanospray
capillaries. In the CID tandem MS studies a precursor ion was selected
in the quadrupole analyzer and partially fragmented in the hexapole
collision cell, with the pressure of argon at 2.7 × 10
5 millibar and collision energy 20 eV. All
masses were analyzed with an orthogonal time-of-flight analyzer
equipped with a reflector, a microchannel plate detector, and a
time-to-digital converter. Data acquisition was optimized to give the
highest possible resolution and signal-to-noise ratio, even in the case
of very low abundant signals. Analysis of the data was done with the
Masslynx Windows NT PC data system. NaI and/or deglycosylated b and y
ion series of the analyzed peptide were used for calibration. The mass
accuracy of all measurements was within 0.2 m/z units.
0.15 V. The GP40 pump was
run in isocratic mode, using 15% NaOH as eluent and flow rate of 1 ml/min. Monosaccharide standards (Fuc, Rha, Gal, Glc, and Man) were
used for optimization and calibration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Summary of the main proteolytic fragments of
platelet TSP-1 and recombinant TSR modules used to study
post-translational modifications. For the sake of clarity, the
numbering of peptides and amino acid residues in TSP-1 has also been
used for the recombinant proteins. Trp residues that are part of the
C-mannosylation motif WXXW have been
underlined. Sequences that differ between recombinant
proteins and platelet TSP have been shown in italics. Parts
of the molecules that are not relevant to this study have been
indicated with dotted lines. The domains in TSP-1 have been
indicated as follows: circled N, N-terminal domain; ,
procollagen homology domain;
, type 1 repeat;
, type 2 repeat (EGF-like);
, type 3 repeat (calcium-binding); circled
C, globular C-terminal domain.
View larger version (26K):
[in a new window]
Fig. 2.
Peptide mapping of TSP-1 from human
platelets. A, reduced and carboxamidomethylated TSP-1
was cleaved with trypsin. The resulting peptides were fractionated by
reversed phase LC-ESIMS, using a C8 column. Peptides T42 and T49 were
found in modified form only, whereas peptide T55 occurred both in the
modified (T55a) and unmodified form (T55b). B-D, elution
profile of the chymotryptic digests of T42 (B), T49
(C), and T55a (D), obtained from the experiment
shown in A. In all cases the affixed "-Ch-1" and
"Ch-2" indicates the N- and C-terminal fragments, respectively (see
Fig. 1 for peptide nomenclature). The insets show the ESIMS
spectra of the C-terminal fragments, illustrating the dissociation of a
308-Da substituent.
View larger version (67K):
[in a new window]
Fig. 3.
Low energy CID tandem-ESIMS of the
C-mannosylated peptides from platelet TSP-1.
Purified tryptic/chymotryptic peptides from the TSR modules of platelet
TSP-1 were analyzed. A, T42-Ch-1; B, T49-Ch-1;
C, T55a-Ch-1. The 120-Da loss, typical for aromatic
C-glycosides, has been indicated with 120 and
60 for singly and doubly charged ions, respectively.
Multiple losses of H2O have been indicated with asterisks.
W#, C-mannosylated tryptophan;
(Cam-)C, carboxamidomethylcysteine.
Characterization of C-mannosylated (C-Man) and O-glycosylated peptides
from TSP-1
View larger version (18K):
[in a new window]
Fig. 4.
Summary of the C- and
O-glycosylations of the TSR modules of platelet and
recombinant TSP. C-Mannosylation has been indicated
with closed hexagons, with the approximate percentage of
modification below. Note that in TSR123-H5 C-hexosylation
was demonstrated by ESIMS only, which does not establish the identity
of the monosaccharide. In the other cases independent evidence for
C-mannosylation was obtained by Edman degradation. The exact
site of O-glycosylation and the identity of the sugars in
TSR123-S2 and -H5 was not determined but was assumed to be the same as
in the other proteins. The percentage modification was estimated by
determining the area of relevant peaks in the UV trace at 214 nm of the
LC-ESIMS separation (TSR123-S2, TSR123-H5, TSR3-Sf9, and
TSR3-H5). Since in the case of the third TSR of TSP-1 the peak
contained more than one peptide, the percentage modification could only
be estimated from the areas of the [M + 2H]2+ and [M + 3H]3+ peaks in the total ion current trace of the LC-ESIMS
separation.
View larger version (91K):
[in a new window]
Fig. 5.
Low energy CID nano-ESI Q-TOF tandem MS
sequencing of the glycopeptides T42-Ch-2-AMP (A),
T49-Ch-2 (B), and T55-Ch-2 (C).
For experimental conditions and assignment see the text.
Molar ratios of monosaccharides in O-linked glycans in TSP1 and
TSR3
View larger version (15K):
[in a new window]
Fig. 6.
Typical HPAE-PAD chromatograms of
monosaccharide standards (a) and monosaccharides
(b) released from investigated
O-glycopeptides, in this case
TSR3-Sf9-T55-D-2a. Peaks corresponding to Fuc (peak
1) and Glc (peak 2) in the experimental sample are
clearly visible. The origin of the 3rd peak is discussed in
the text.
View larger version (11K):
[in a new window]
Fig. 7.
Peptide mapping of recombinant TSR3 from
Sf9 cells. TSR3-Sf9 was cleaved with trypsin and
subsequently with endoproteinase AspN. The peptides were fractionated
by reversed phase LC-ESIMS, using a C8 column (see "Experimental
Procedures"). Relevant fractions have been labeled with the name of
the peptide (for nomenclature, see Fig. 1), whereas the remainder has
been numbered in italics only.
Characterization of C-mannosylated (C-Man) and O-glycosylated peptides
from recombinant module TSR3 from Sf9 cells
View larger version (18K):
[in a new window]
Fig. 8.
Peptide mapping of recombinant TSR123 from
Drosophila S2 cells. A, the tryptic
digest of TSR123-S2 was fractionated by LC-ESIMS on a C8 column. The
part of the chromatogram containing the relevant peptides has been
shown only. The arrows indicate the elution positions of the
peptides that were 146 Da heavier than predicted, as detected by
extraction of the MS data for their appropriate masses. The
nomenclature of the peptides is indicated in Fig. 1. B-D,
chymotryptic fragments obtained from peptide TSR123-S2-T42
(B), TSR123-S2-T49 (C), and TSR123-S2-T5
(D). Peptides whose masses agreed with those predicted from
the cDNA sequence have been numbered in
italics only. Those that were heterogeneous in mass have
been indicated with their name in addition. The insets show
the spectra of the chymotryptic fragments that were 146 Da heavier than
expected from the cDNA sequence ([M + 2H]2+ ions).
The dissociation of this substituent has been indicated with the
difference in m/z value (dotted line).
Characterization of O-glycosylated peptides from recombinant TSR123
from Drosophila S2 cells
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,3-Fuc-
1-O-Ser/Thr occurs in
unidentified proteins from Chinese hamster ovary cells (38). In
addition, it had been isolated as the amino acid fucoside from human
urine (39, 40). O-Fucosylation of EGF-like modules occurs
most likely in the Golgi (41), and the fucosyltransferase has been
isolated (42). Based on primary structure comparisons, a consensus
sequence for this modification in EGF-like modules (CXXGG(S/T)C) has been proposed (43). However,
the O-fucosylation sites in TSP do not conform to this
sequence (Table V). This raises the question as to whether the same
fucosyltransferase is involved or whether more than one transferase
exists. O-Linked fucose monosaccharide can be elongated into
a di- or a tetrasaccharide in a protein-specific manner (Table V) (38).
Supposedly, the elongating transferases recognize the fucose and as yet
unidentified signals in the protein. Until now, the tetrasaccharide has
exclusively been found in the EGF-like modules of factor IX and Notch
(36, 44), whereas disaccharide has only been observed in TSRs (this paper).3 This raises the
possibility that structural features specific for each of these modules
lead to elongation of O-fucose into disaccharide and
tetrasaccharide, respectively. Whether this is determined by the
primary or the tertiary structure, as for other elongating
glycosyltransferases (45), remains to be determined. In this
context it should be noted that proper folding of the EGF-like module
is required for fucosylation (42), strongly indicating that elongation
also must take place after attainment of the three-dimensional
structure.
Comparison of sequences around glycosylation sites involving O-linked
fucose
1,3N-acetylglucosaminyltransferases that perform the
first step in the synthesis of the tetrasaccharide. Importantly, this
modification modulates the ligand-receptor interaction in
vivo. Because TSP-1 also functions in protein-protein interactions at the cell surface (9), it may be worthwhile to search for proteins
that modulate these, analogous to Fringe in the Notch system. An enzyme
activity that elongates O-linked
-fucose to the
disaccharide has been identified in Chinese hamster ovaryl cells (48).
The enzyme is widespread in biology, suggesting that the disaccharide
has an important biological function (36, 48).
and inhibits its
activation by TSP-1 (12). By using the yeast two-hybrid system, Aho and
Uitto (51) found laminin and collagen VII to bind to this region of the
TSR as well. It remains to be demonstrated conclusively, however, that
native TSRs from TSP-1 bind to these molecules. Therefore, any effect
that C-mannosylation has on these interactions is unknown.
The situation concerning heparin binding to these sequences is
inconsistent. The peptide SHWSPWSS has been found to inhibit TSP-1
binding to heparin (13), and peptides containing this sequence in
addition to conserved basic residues display high affinity heparin
binding (52). Importantly, the two Trp residues are essential for this
interaction. On the one hand, these data are supported by the
observation that heparin binding to midkine, a protein that is now
recognized to be a TSR homologue (53), affects the NMR resonances of
the Trp residue corresponding to the second Trp in the above-mentioned
peptide (54). On the other hand, recombinant TSRs, or a proteolytic fragment from TSP-1, which are both folded molecules, do not bind heparin (see Introduction). It is difficult to reconcile these results
at present, but purification of the various glycoforms of recombinant
TSRs identified in this paper and comparing their heparin-binding
properties may resolve some of these discrepancies.
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ACKNOWLEDGEMENTS |
---|
We thank Renate Matthies for help with amino acid sequencing and Drs. Tim Smilda and Anne Gonzalez for reading the manuscript. The ESI-quadrupole time-of-flight instrument was purchased with a grant from the "Hochschulbauförderungsgesetz" of the University of Münster (to J. P. K.).
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FOOTNOTES |
---|
* This work was supported in part by the Novartis Research Foundation (to J. H.) and by NHLBI Grants HL54462 (to D. F. M.) and HL28749 (to J. L.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Friedrich
Miescher-Institut, P. O. Box 2543, CH-4002 Basel, Switzerland. Tel.: 41 61 697 4722; Fax: 41 61 697 3976; E-mail: hofsteen@fmi.ch.
Supported by the Interdisciplinary Center for Clinical
Research, University of Münster.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M008073200
2 B. Macek, J. Hofsteenge and J. Peter-Katalinic, manuscript in preparation.
3 S. Hartmann and J. Hofsteenge, unpublished results.
4 K. G. Huwiler and D. F. Mosher, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
TSP, thrombospondin;
Cam, carboxamidomethyl;
CID, collision-induced dissociation;
CPase, carboxypeptidase;
dHex, deoxyhexosyl;
ESIMS, electrospray ionization
mass spectrometry;
Hex, hexosyl;
HPAE-PAD, high pH anion exchange
chromatography with pulsed amperometric detection;
LC, liquid
chromatography;
PTH, phenylthiohydantoin;
TSR, TSP type 1 repeat;
HPLC, high pressure liquid chromatography;
TGF-, transforming growth
factor-
;
EGF, epidermal growth factor.
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