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J Biol Chem, Vol. 274, Issue 46, 32786-32794, November 12, 1999
From the Friedrich Miescher-Institut, C-Mannosylation is a unique form of
protein glycosylation, involving the C-glycosidic
attachment of a mannosyl residue to the indole moiety of Trp. In the
two examples found so far, human RNase 2 and interleukin-12, only the
first Trp in the recognition motif WXXW is specifically
C-mannosylated. To establish the generality of protein
C-mannosylation, and to learn more about its mechanism, the
terminal components of the human complement system (C6, C7, C8,and C9),
which contain multiple and complex recognition motifs, were examined.
Together with C5b they form the cytolytic agent, the membrane attack
complex. These are the first proteins that are
C-mannosylated on more than one Trp residue as follows: six in C6, four in C7, C8 The modification of proteins by covalent attachment of
carbohydrate is a common feature of both intra- and extracellular
proteins (1, 2). In particular proteins that contain N- or
O-linked oligosaccharides have been known for a relatively
long time (3, 4), and many examples are known (1). Recently, a new type of glycosylation has been discovered in human RNase 2, which differs fundamentally from N- and O-glycosylation with
respect to the protein carbohydrate linkage. It involves the
C-glycosidic attachment of an The terminal complement proteins C6, C7, C8, and C9 are attractive
candidates for studying protein C-mannosylation because of
the following: (i) they contain complex C-mannosylation
motifs (WXXWXXW) as part of their thrombospondin
type 1 repeats (TSR modules); (ii) they possess up to three TSR
modules; and (iii) they have a clearly defined physiological function
(see below). The latter is important, since so far a function for
C-mannosylation of Trp residues has not been established.
The complement, which forms a first line of defense against microbial
infections, can be activated by three different pathways as follows:
the alternative, the classical, and the mannose-binding lectin-mediated
pathway (13, 14). Each of these results in the formation of the
membrane attack complex (MAC), the actual lytic component of complement
(15). The assembly of MAC begins with the formation of C5b by C5
convertase at the target membrane, followed by the sequential addition
of the plasma proteins C6, C7, C8, and C9 (see Reaction 1).
C5b-9n forms a trans-membrane channel, whose exact size
can vary, depending on the value of n. The association
process has been examined in detail both on natural as well as
artificial membranes (16-19). The value of n for the
complete MAC ranges from 12 to 18 with an average of approximately 16 (see Ref. 19 for a discussion), which yields a calculated mass of
1.4 × 106 Da.
Here we have examined the C-mannosylation of Trp residues in
the TSR modules of C6, C7, C8 Materials--
Human complement component C6 was purchased from
Sigma, and components C7, C8, and C9 were from Advanced Research
Technologies, San Diego, CA. Component C9 used in the protein chemical
analyses was a generous gift of Dr. J. Tschopp, University of Lausanne, Switzerland.
Protein Chemistry--
Proteins were reduced and
carboxamidomethylated as described previously (20). Tryptic digestion
of the proteins was performed twice in 50 mM Tris-HCl, pH
8.0, for 2 h at 37 °C at an enzyme to substrate ratio of 1/50
(C7 and C8) or 1/100 (C6 and C9). Subsequently, C7 and C8 were digested
for 4 h with endoproteinase Asp-N (1/50; Roche Molecular
Biochemicals) at 37 °C. The amount of protein per digest was 50-75
µg for C6, C7, and C8, and two digests were made. The total amount of
C9 digested was 7 mg. Cleavage of isolated peptides with 200 ng of
chymotrypsin was performed for 2 h at 37 °C.
Digests were fractionated by C8 reversed phase LC-ESIMS as
described (10). If necessary final purification was achieved by
LC-ESIMS, using 25 mM NH4 acetate, pH 6.0, containing 2 or 80% CH3CN, as buffers A and B, respectively.
Nanospray ESIMSMS and solid-phase Edman degradation were performed
according to published methods (21, 22). The elution position of
PTH-(C2-Man-)Trp was established by sequencing residues
5-10 of human RNase 2 (FT(C2-Man-)WAQW) (5) prior to
analysis of each of the complement peptides.
NMR Spectroscopy--
NMR spectra were measured on a Varian
Unity Plus spectrometer operating at a 1H frequency of 600 MHz. Susceptibility matched NMR tubes (Shygemi) with sample volumes of
220 µl were used. Clean TOCSY spectra were recorded with a mixing
time of 40 and 80 ms. The spectra were folded once in
t1 to increase the resolution. The spectra were recorded with 192 increments and 160 transients. Experiments of samples
in 90% H2O, 10% D2O were recorded using the
Watergate method. Spectra were referenced against acetone ( Peptide Nomenclature--
The numbering of the mature protein
has been used here as follows: C6 (24), C7 (25), and C8 In Vitro C-Mannosylation--
The peptide
Ac-WAKW-NH2, the C6-derived peptide
Ac-LLGDFGPWSDCD-NH2, and the C9-derived peptide
Ac-RMSPWSEWSQCD-NH2 were C-mannosylated in
vitro using salt-washed porcine liver microsomes as the source of
transferase (12). The reaction mixture contained in a total volume of
24 µl the following: 0.9 mM peptide, 22.5 pmol of
Dol-P-[2-3H]Man (5.6 Ci/mmol), porcine liver microsomes
(77 µg of protein), 20 mM Hepes-NaOH, pH 7.2, 0.2% (w/v)
Triton X-100, 2 µg/ml benzamidine, 5 µg/ml pepstatin, 5 µg/ml
leupeptin, 2 mM EGTA. The reaction was performed for 30 min
at 37 °C and stopped by the addition of 2 ml chloroform/methanol 3:2
(v/v) and 0.48 ml of water. After centrifugation the upper, aqueous,
phase contained the peptide. The radioactivity in 0.2 ml of the upper
phase was determined by scintillation counting (8). For preparative
purposes, 10 reactions were performed for 42 h at room temperature
(12). The radiolabeled peptide was reduced, carboxamidomethylated, and isolated by C18 reversed phase HPLC as described (5). Final purification was achieved after tryptic cleavage and fractionation by
C8 reversed phase LC-MS (12).
Component C9--
The fractionation of the tryptic peptides from
carboxamidomethylated C9 by reversed phase LC-ESIMS is shown in Fig.
1A. The MS data were extracted
for the theoretical mass of the peptide(s) containing a
C-mannosylation motif, with or without the mass of a hexosyl
residue (162 Da) added. Two forms of peptide T2, comprising residues
24-38 of the mature protein (for nomenclature of peptides and
numbering system see "Experimental Procedures"), were detected. Peptide T2-1, eluting at 41.1 min, had a molecular mass of 2101 Da
(Fig. 1B, upper panel), which was 162 Da heavier than
expected from the cDNA sequence (28, 29). This suggested the
presence of one hexosyl residue. ESIMSMS of this fraction yielded a
nearly complete series of y ions, confirming its sequence and
localizing the hexosyl residue to Trp-27 (Fig.
2A and Table
I). The spectrum showed secondary neutral
losses of 120 Da from [M+2H]2+ and y132+.
This fragmentation has typically been observed with aromatic C-glycosides (30, 31) and has previously been found with
(C2-Man-)Trp in RNase 2 (5) and IL-12 (12). Edman
degradation of this peptide yielded at the fourth cycle a
PTH-derivative that comigrated with PTH-(C2-Man-)Trp
obtained from human RNase 2 (Fig. 1C), a protein whose C-mannosylation has previously been extensively
characterized (5-7).
To confirm the identity of the mannosyl residue, the peptide was
analyzed by NMR spectroscopy. The region of the spectrum that is just
upfield from the water resonance is expected to be empty for a random
coil 15-mer peptide. It would only show signals of upfield shifted H
The identification of a second form of peptide T2 (T2-2 in Fig.
1A) was surprising, since its mass (2263 Da, Fig.
1B) indicated the presence of two hexosyl residues. However,
the peptide only contained a single, simple C-mannosylation
motif (-WSEW-). Its MS spectrum did not show the conspicuous loss of
162 Da which would indicate the presence of an O-linked
hexose (Fig. 1B). In agreement with this, ESIMSMS
demonstrated unequivocally that in this peptide Trp-27 and -30 were
both C-hexosylated (Fig. 2B and Table I). The
PTH-derivatives observed at cycles 4 and 7 during Edman degradation of
this peptide comigrated exactly with each other and with
PTH-(C2-Man-)Trp from RNase 2 (Fig. 1D).
The anomeric part of the 1H NMR spectrum of peptide T2-2
is shown in Fig. 3C. Interestingly, in comparison with
sample T2-1, an additional doublet resonance was observed. The
resonances at 5.20 and 5.18 ppm were split by J-couplings of
7.1 and 7.4 Hz, respectively. Fig. 3D shows part of the
TOCSY spectrum, recorded with a mixing time of 80 ms. It is evident
from this spectrum that these anomeric resonances are part of similar
spin systems with approximately the same chemical shifts (Table II). In
fact the TOCSY spectra are similar to those recorded of the peptides with only one glycosylated Trp. In the case of peptide T2-2 two indole
H2 resonances were missing. Thus, it is concluded that in this
particular case both Trp residues in the WXXW motif were C2-mannosylated. The signal at 5.27 ppm with an
intensity of about 15%, caused by the
cis-trans-isomerization of the Ser-Pro bond appeared in this
spectrum as well.
Examination of the LC-ESIMS data did not reveal the presence of
unmodified peptide T2. Furthermore, the other two Trp residues in
component C9, at positions 176 and 436, were observed in the unmodified
form only.
Component C8--
Component C8 is a heterotrimeric protein
consisting of a disulfide-linked
Peptide
From the C8
These results extend the unexpected finding in C9 that Trp residues
without a Trp or another aromatic residue at position +3 can be
C-mannosylated.
Component C7--
Of the two TSR modules in component C7 only the
second one contains WXXW motifs. Nevertheless, a
tryptic/Asp-N peptide from the first repeat was found that had a mass
that was 162 Da larger than expected from the cDNA sequence (T1-D,
1897 Da, residues 9-22). ESIMSMS of this peptide (Fig.
4A) positioned the substituent at Trp-14, and a C-glycosidic linkage was concluded from the
120-Da loss from the parent, the y10, and y11 ions. Further evidence was obtained by Edman degradation of the peptide that had been purified
by reversed phase LC-ESIMS at pH 6.0. The PTH-derivative produced in
cycle 6 comigrated with PTH-(C2-Man-)Trp from RNase 2 (Table I).
From the second repeat four different C-glycosylated
peptides were obtained. Approximately equal amounts of unmodified
peptide (T50-D-1, 3193 Da, residues 465-494) and
mono-hexosylated peptide (T50-D-2, 3355 Da) were detected. A nearly
complete y and b ion series in the ESIMSMS analysis of T50-D-1
confirmed its sequence and the absence of modification (Table I).
Similarly, the spectrum of T50-D-2 confirmed the structure and
established the site of modification at Trp-484, with 120-Da losses
from the parent and the doubly charged y13, y17, and y23 and y24 ions
(Table I). Two peptides containing two modified Trp residues were found
as minor fractions, T50-D-3 and T50-D-4 with a mass of 3517 Da. ESIMSMS analysis established that in T50-D-3 Trp-481 and -484 were modified, whereas T50-D-4 contained modified Trp-484 and -487 (Table I). The
amount of these two peptides was too small for further purification and
analysis by Edman degradation.
Component C6--
C6 contains three TSRs, with WXXW
motifs present in the first and third repeat (residues 8-11 and
547-553). In the second repeat a single Trp is present but no
C-mannosylation motif. The C-mannosylation of
Trp-14 in C7, however, warranted the analysis of this region of C6.
Fractionation of the tryptic digest of C6 yielded a variety of peptides
that originated from the TSR modules. This complexity was caused by the
partial modification of most Trp residues. Peptide T1 (residues 1-16)
from the first repeat was detected in two chromatographically separated
fractions. Each of these was digested with chymotrypsin and purified to
apparent homogeneity by reversed phase LC-ESIMS. The ions observed in
the collision-induced dissociation spectrum of T1-Ch-1 (853 Da;
residues 7-11 containing 1 hexosyl residue) demonstrated Trp-8 to
contain the hexosyl residue. Since the parent, and the b3 and b4 ions
showed the typical 120 Da loss, the presence of a
C-glycosidic linkage was concluded. These results were
confirmed by Edman degradation (Table I). The ESIMSMS analysis of
T1-Ch-2 (1578 Da, residues 7-16 containing 2 hexosyl residues) is
shown in Fig. 4B. The spectrum illustrates the complexity
that may arise due to the presence of more than one
(C2-Man)-Trp residue. Loss of 120 Da, as well as multiple
losses of H2O, were observed twice from the doubly charged
parent ion. Furthermore, the data clearly show that both Trp-8 and -11 were modified with a hexosyl residue. Edman degradation confirmed the sequence of the peptide and provided chromatographic evidence for the
presence of (C2-Man-)Trp at cycles 2 and 5 (Table I).
Peptide T8, which contains Trp-69 but no recognition motif, was
detected mainly in the unmodified form (2653 Da, residues 57-78).
Since the mass of this peptide as well as its ESIMSMS spectrum fully
established its structure, no Edman degradation was carried out. The
peptide with modified Trp-69 was a minor fraction. ESIMSMS of the
unpurified chymotryptic fragment (T8-Ch, 2058 Da, residues 63-78)
confirmed its identity and provided clear evidence for its
C-hexosylation. The observations that its mass and that of
the y11 and y12 ions was 162 Da higher than expected from the cDNA
showed that Trp-69 contains the hexosyl residue (Table I). The loss of
120 Da typical for aromatic C-glycosides was observed for
the parent, the y11, and y12 ions. Because the peptide was obtained in
very low amounts and impure, no attempt was made to analyze it by Edman degradation.
Peptide T69, which contains Trp-547, -550, and -553, was detected
in four separate chromatographic fractions. The major one contained
modified Trp-547 and -550, as shown by ESIMSMS analysis and Edman
degradation (Table I) of its purified chymotryptic fragment (T69-Ch,
1964 Da, residues 540-553). Minor amounts of T69 with Trp-550 and 553 (T69-2; 2890 Da) or all three Trp residues modified (T69-3, 3052 Da)
were detected. The amount of these peptides did not allow further
purification and characterization by Edman degradation. Nevertheless,
their analysis by ESIMSMS was consistent with C-hexosylation
as summarized in Table I.
A small amount of completely unmodified T69 was detected as well
(T69-1, 2566 Da). Since its primary structure was fully established by
ESIMSMS, no Edman degradation was done.
Component C6 contains six further Trp residues. The LC-ESIMS data of
the first fractionation of the digest was extracted for the masses of
each of the expected peptides assuming either the presence of Trp or
(C2-Man-)Trp. All peptides were found in the unmodified
form only.
A summary of the data obtained for all four terminal components,
including an estimate of the stoichiometry of modification, is
shown in Fig. 5.
In Vitro C-Mannosylation--
C-Mannosylation in human
RNase 2 and IL-12 can be reproduced in vitro using peptides
containing the motif WXXW and a microsome-associated transferase (8, 12). The modification in these cases is completely restricted to the first Trp, and replacement of the second by Ala
completely abolishes the modification in vitro and in
vivo (8, 9). In contrast, the results summarized in Fig. 5 show that all terminal components of complement contain one or more C-mannosylated Trp residues with a Cys rather than Trp or
another aromatic residue at the +3 position. It was therefore of
interest to examine the in vitro C-mannosylation
of such peptides.
The peptide Ac-LLGDFGPWSDCD-NH2 derived from C6 and
Ac-RMSPWSEWSQCD-NH2 derived from C9 were incubated with
porcine liver microsomes and Dol-P-[2-3H]Man. The general
acceptor peptide Ac-WAKW-NH2 was used as a positive control
(12). As shown in Fig. 6A no
C-mannosylation could be detected in the peptide from C6,
whereas the one from C9 was nearly as effective an acceptor as the
control peptide. To determine the site(s) of modification the
radiolabeled C9 peptide was isolated from the reaction mixture by
C18 reversed phase HPLC (Fig. 6B). This yielded
a major peak of radioactivity eluting at 19.5 min which contained a
peptide with a mass expected for the mono-hexosylated peptide from C9
(1771 Da). The minor peak eluting at 18.5 min contained 8% of the
recovered radioactivity. ESIMSMS analysis demonstrated it to be the
same peptide, but with oxidized Met at position 2. The major peptide
was cleaved with trypsin to remove Ac-Arg and yielded a single
radioactive peak upon purification by rechromatography on a
C8 HPLC column (data not shown). The purified peptide had a
mass of 1574 Da, corresponding to
MSPWSEWSQ(carboxamidomethyl)CD-NH2 plus an additional
hexosyl residue. The ESIMSMS spectrum of this peptide is depicted in
Fig. 6C. The m/z values of the b4, b5, and b6
ions identified the first Trp residue as the attachment site of the
mannosyl residue. Furthermore, the values of the y5, y6, and y7 ions
provided evidence for the unmodified second Trp. Importantly, y ions at
m/z 856, 985, and 1072, which would have indicated
C-mannosylation of the second Trp, were conspicuously absent
in the spectrum (arrows in Fig. 6C). These
results were confirmed by Edman degradation of the radiolabeled
peptide. A peak of radioactivity was observed in the 4th cycle (the
position of the first Trp) but not in cycle 7 (data not shown). Thus,
in vitro C-mannosylation of the peptide from C9
was restricted to the first Trp in the WXXW motif.
The results presented here demonstrate that
C-mannosylation of Trp residues is not a rare event
restricted to RNase 2 and IL-12. Nearly all Trp residues in the TSR
modules of the four terminal components of complement can be
C-mannosylated (Fig. 5). The most complete analysis was
carried out on component C9, where the presence of the
(C2-Man-)Trp residues at positions 27 and 30 was
established by MS, Edman degradation, and NMR. The identity of the
hexosyl residue was established by comparing chemical shifts and
J-coupling data of the peptides from C9 with those of
peptides obtained from RNase 2 and IL-12. The close agreement of these
parameters showed that the chemical structure of residues 27 and 30 of
C9 is identical to that of (C2-Man-)Trp present in RNase 2 and IL-12 (5, 6, 12). No NMR data are available for C-linked
hexoses other than mannose. However, they are expected to have
significantly different chemical shifts, connectivity patterns, and
coupling constants (6). For the other three terminal components,
(C2-Man-)Trp in the major peptides (modified fraction >0.1
mol/mol, Table I) was established by MS and comigration of its
PTH-derivative with PTH-(C2-Man-)Trp from human RNase 2. No
further NMR analysis was done for these, because of lack of sufficient
amounts of protein. Furthermore, we feel confident that the MS and
Edman degradation studies are sufficiently supported by NMR in the past
(5-7, 12) and by the analysis of C9 (this paper). Therefore, NMR is
only required in unusual new cases. Exact comigration of the
PTH-derivatives of the modified Trp residues with authentic
PTH-(C2-Man-)Trp was always observed (Table I). Due to the
lack of synthetic standards and natural examples, the elution position
of PTH-(C2-hexose)-Trp with hexoses other than mannose has
not been established. It seems unlikely, however, that these would
comigrate with PTH-(C2-Man-)Trp. The small peak eluting
shortly after the main peak of PTH-(C2-Man-)Trp (* in Fig.
1, C and D) is a diastereomer (5). The good
separation between these peaks suggests that the resolution of the
system is sufficient to separate other hexose derivatives as well. The
recent publication of a synthetic route for (C2-Man-)Trp
will possibly also lead to the synthesis of the other hexosyl
derivatives and provide a solution to this matter (33).
In Fig. 5 an estimate of the stoichiometry of modification has been
indicated. These values were calculated as a fraction of the total area
of the [M + nH]n+ ions
(n = 2 and 3) of a particular peptide in the original
chromatogram of a digest. Differences in the efficiency of ionization
of the differently modified forms of a peptide could affect the
accuracy of intermediate values. The extreme values of 0 (no modified
peptide found) and 1 (no unmodified peptide found) are unequivocal,
however. Furthermore, in the case of C9 shown in Fig. 1A,
where the two modified peptides were obtained in pure form from the
original chromatogram, a good correlation between integration of the UV and MS data was observed.
C-Mannosylation is a significant feature of the MAC. The
mature complex, C5b-C916 (19), has a calculated mass of
approximately 1.4 × 106 Da and contains a total of
113 Trp residues. Of these, 50 have been found to undergo
C-mannosylation. Taking into account the degree of
modification of each of them, and assuming a random distribution in MAC
particles, we calculated that on average a MAC contains 34 (C2-Man-)Trp residues. It has been hypothesized, but not
proven, that the TSR modules in the complement proteins are involved in adhesion to each other and perhaps in the assembly of the MAC (34).
Some support for this has been obtained from the inhibition of the
assembly process by a monoclonal antibody specific for the third TSR
module in C6 (35). Whether the (C2-Man-)Trp residues play a
role in facilitating complex formation on the surface of the pathogen
remains to be determined. Although interaction of
(C2-Man-)Trp in the MAC with the multivalent
mannose-binding lectin from serum has not been demonstrated yet, it
could play a role, since this lectin has been shown to bind to a
variety of microorganisms (for an overview see Ref. 14). Furthermore,
many pathogenic microorganisms are known to carry mannose-binding
proteins on their surface (36).
The C-mannosylation of the terminal components of complement
reveals a number of novel features of protein
C-mannosylation and the properties of TSR modules in general.
A new finding is that the TSR modules in the terminal complement
components contain more than one modified Trp residue. Unexpectedly, Trp residues that do not have a Trp or another aromatic residue at the
+3 position were found to be C-mannosylated. The most
extreme examples being Trp-69 in C6 and Trp-14 in C7, which are not
even part of a WXXW motif (Fig. 5). This is in contrast with
human RNase 2 and IL-12 (5, 12). Reexamination of their LC-ESIMS data,
in the light of these new findings, confirmed that in these proteins
C-mannosylation takes place exclusively on the first Trp.
The case of RNase 2 is particularly compelling, because it holds for
the enzyme from urine and HL-60 cells (5, 10), as well as for
recombinant RNase 2 and the hybrid RNase 2.4 from HEK-293 cells (9).
Furthermore, in vitro studies with the microsome-associated transferase from liver and IL-12-secreting B-lymphoblastoid cells confirm these findings (12). The most likely explanation for the
C-mannosylation of Trp residues that do not have a Trp or another aromatic residue at the +3 position is that the terminal complement components contain, in addition to the WXXW
motif, another signal for C-mannosylation of these Trp
residues. In all cases such Trp residues are part of the sequence
W(S/T)XC (Fig. 5). If this sequence, or a segment of it,
would form the recognition motif, it seems likely that another
transferase than the one recognizing WXXW is involved.
Support for this was provided by the in vitro C-mannosylation experiments (Fig. 6). No
C-mannosylation of the peptide from C6 was observed, and
C-mannosylation of the one from C9 was restricted to the
first Trp residue. An alternative explanation would be that the extra
signal is located outside this sequence or that it is formed by a
three-dimensional "signal patch" akin to substrates for
UDP-GlcNAc:lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase. These proteins
contain a conformation-dependent protein determinant for
interaction with the transferase that is formed by amino acid residues
that are separated in the primary structure (37).
The data obtained here (Fig. 6), together with those obtained
previously (5, 12), allow a partial definition of interspersing residues that are compatible with C-mannosylation of the
WXXW motif. Whereas Thr, Ala, Gly, and Ser may occur at
position +1, Cys, Ala, Asn, Ser, Glu, and Gln are allowed at position
+2. Clearly, further studies are needed to complete this analysis.
There is no three-dimensional structure available for a TSR module, but
the results presented here allow the definition of some of its spatial
properties in the complement components. The hydrophilic nature of a
mannosyl residue makes it unlikely that it is buried in the molecule.
Consequently, the Trp residues that become C-mannosylated
probably occur on the surface of the module. This proposal is
consistent with the position of Trp-7 in the three-dimensional
structure of recombinant human RNase 2 (38) and model building of the
mannosyl residue (39). Furthermore, the occurrence of more than one
(C2-Man-)Trp residue within a motif poses sterical
restrictions on possible backbone conformations. Model building shows
that in such a case the polypeptide chain cannot adopt a helical
conformation like it does in RNase 2, which contains only a single
(C2-Man-)Trp residue.
In addition to TSP-1 and the terminal components of complement, TSR
modules have been found in a number of other proteins as follows:
properdin (40), F-, M-, and SCO-spondin (41-44), semaphorin F and -G
(45), brain-specific angiogenin inhibitor-1, -2, and -3 (46, 47), a
disintegrin and metalloproteinase with thrombospondin repeat-1 (48),
aggrecanase (49), UNC-5 (50), and thrombspondin-related anonymous
protein (51). Since all of them contain at least one WXXW
motif, it will be of interest to examine these proteins for
C-mannosylation as well. TSR modules have been classified
into three groups based on length, position of cysteine residues, and
net charge (52). The terminal components contain examples of each of
these groups, and the present results show that all of them can be
C-mannosylated (Fig. 5). The exact function of the TSR
module, and therefore that of (C2-Man-)Trp, remains
unknown. By far the most information has been obtained for TSP-1. TSP
is a modular cellular adhesion molecule that has been proposed to
interact with a variety of cells and macromolecules (for an overview
see Refs. 53 and 54). Its TSR modules, and more importantly the
WXXW motif and the neighboring sequence CSVTC, have been
implicated in the adhesive process with a number of cells (55) and in
protein-protein (56-58) and protein-glycosaminoglycan interactions
(59). It is of interest to note that C6, C7, C8, and C9, as well as
most of the above-mentioned proteins, act at the cell surface through
protein-protein and protein-membrane interaction.
The results obtained here may also bear upon proteins that are not
related to TSP. One of the best conserved portions of the sequence of
the TSR module, WSXWS (52), occurs in nearly all type 1 cytokine receptors (60). Their analysis for C-mannosylation would be of interest because mutagenesis studies have revealed the
functional importance of this so-called "WSXWS box" (61, 62).
In conclusion, we have shown that C-mannosylation can occur
on multiple Trp residues in a protein. As exemplified by the membrane attack complex, this modification may alter a large proportion of the
Trp residues. The results further demonstrate that this post-translational modification is more widespread than initially seemed to be the case. In particular proteins that contain TSR modules,
and the class 1 cytokine receptors appear to be highly probable
candidates for C-mannosylation. New mechanistic insights were obtained by the discovery of C-mannosylation of Trp
residues outside or at the C-terminal end of WXXW
motifs. Finally, the C-mannosylation of four complement
proteins with clearly defined physiological roles provides the
structural basis for studies on the function of
(C2-Man-)Trp residues.
We thank Dr. Jürg Tschopp,
University of Lausanne, for the generous gift of component C9;
Renate Matthies for amino acid sequencing; and Drs. Brian Hemmings and
Jack Rohrer for reading the manuscript.
*
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.
The abbreviations used are:
IL-12, interleukin
12;
Dol-P-Man, dolichyl-phosphate-mannose;
ESIMS, electrospray
ionization MS;
LC-ESIMS, high performance liquid chromatography
interfaced with ESIMS;
MAC, membrane attack complex;
MS, mass
spectrometry;
PTH-, phenylthiohydantoin-;
TSP, thrombospondin;
TSR, TSP
type 1 repeat;
HPLC, high pressure liquid chromatography.
The Four Terminal Components of the Complement System Are
C-Mannosylated on Multiple Tryptophan Residues*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and C8
, and two in C9. Thus, from the 113 Trp residues in the complete membrane attack complex, 50 were found to
undergo C-mannosylation. The other important finding is
that in C6, C7, C8, and C9 Trp residues without a second Trp (or
another aromatic residue) at the +3 position can be
C-mannosylated. This shows that they must contain an
additional C-mannosylation signal. Whether this is encoded
in the primary or tertiary structure is presently unknown. Finally, all
modified Trp residues are part of the highly conserved core of the
thrombospondin type 1 repeats present in these proteins. Since this
module has been found in a large number of other proteins, the results
suggest further candidates for C-mannosylation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannopyranosyl residue to
the C-2 atom of the tryptophan side chain (5-7)
(Scheme 1). This modification has been
shown to be catalyzed by a microsome-associated transferase, which
C-mannosylates the first Trp residue in the recognition
sequence -WXXW- (8, 9). The enzyme uses dolichylphosphate
mannose as the sugar donor, and its activity has been found in mammals,
birds, amphibians, and fish. Furthermore, it could also be detected in
most mouse organs that were examined (10, 11). In addition to the
widespread distribution of the C-mannosyltransferase, its
recognition motif, WXXW, has been found in over 300 secreted mammalian proteins (9). This strongly suggests that
C- mannosylation is a more common form of glycosylation
than indicated by the fact that only two C-mannosylated
proteins have been characterized so far, i.e. human RNase 2 (5, 6) and IL-121 (12). These
two proteins contain a single C-mannosylation motif of the
simple structure WXXW. To further our insight into the process of protein C-mannosylation, we examine here proteins
that contain multiple and more complex C-mannosylation
motifs.
View larger version (17K):
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Scheme 1.
Structure of
C2- -mannosyltryptophan.
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[in a new window]
Reaction 1.
, C8, and C9 by analyzing peptides by ESIMS, Edman degradation, and NMR. It was found that
C-mannosylation can occur at 50 out of 113 Trp residues in
the MAC and that also Trp residues without a second Trp at position +3
can undergo this modification.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 2.225 ppm (23)).
(26) as
corrected in Swiss-Prot entry P07357 and C8 (27) and C9 (28).
Tryptic peptides have been numbered according to their occurrence in
the mature polypeptide. Peptides that resulted from tryptic digestion
have been labeled with the prefix "T," whereas subsequent digestion with endoproteinase Asp-N and chymotrypsin have been indicated with the
affix "D" and "Ch," respectively.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
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Fig. 1.
Isolation and characterization of
C-mannosylated peptides from component C9.
A, tryptic peptides from reduced and carboxamidomethylated
C9 were fractionated by reversed phase LC-ESIMS using a C8
column. Peaks T2-1 and T2-2 contained the peptide comprising residues
24-38 with one (B, upper panel) and two hexosyl residues
(B, lower panel) attached. The calculated mass of the
unmodified peptide is 1939 Da. C, peptides T2-1 and T2-2
were analyzed by Edman degradation. The elution pattern of the
PTH-derivative observed at cycle 4 of peptide T2-1, and cycles 4 and 7 of peptide T2-2 (solid lines) (D) is compared
with that of PTH-(C2-Man-)Trp from human RNase 2 (dotted line). The asterisk indicates a
diastereomer of the main PTH-derivative of (C2-Man-)Trp
(5). amu, atomic mass units.
View larger version (30K):
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Fig. 2.
Tandem MS analysis of
C-mannosylated peptides from C9. A,
nanospray ESIMSMS of the peptide T2-1 from C9 (2102 Da; residues
24-38), where W* indicates (C2-Man-Trp).
CamC, carboxamidomethylcysteine. B, nanospray
ESIMSMS of the peptide T2-2 from C9 (2263 Da; residues 24-38). The
loss of 120 Da from singly and doubly charged ions has been indicated
with 120 and 60, respectively. Loss of
H2O has been indicated with asterisks.
Summary of the evidence for the C-mannosylation of Trp residues in the
TSRs of the terminal complement components
resonances in case the peptide would obtain stable turns or a -sheet
structure. However, the amide resonances exchange very fast with
D2O, and the observed JHNH
couplings are all around the random coil value of 6-7 Hz, indicating
that the peptide was not structured (data not shown). This allows the unequivocal identification of anomeric sugar protons, which resonate in
this region. The anomeric part of the spectrum of the pentadecapeptide T2-1 is shown in Fig. 3B and
is compared with that of the C-mannosylated peptide isolated
from IL-12 (Fig. 3A (12)). It is evident from this
comparison that the doublet signal that resonates at 5.18 ppm and that
was split by a J coupling of 7.4 Hz is the anomeric H1'
resonance of (C2-Man-)Trp. This interpretation was
confirmed by the disappearance of one of the H2 resonances of Trp and
by the assignments of the other resonances of the sugar moiety and of
the Trp residue with the help of TOCSY experiments (Table
II). An additional doublet signal at 5.27 ppm which was split by a J coupling of 6.5 Hz was observed.
This signal has an intensity of only 15%. Minor components of the same
magnitude were also observed for the aromatic protons of
(C2-Man-)Trp. We therefore concluded that the extra
anomeric resonances were caused by the
cis-trans-isomerization of the peptide bond between Ser and
Pro preceding the mannosylated Trp (32), rather than by a heterogeneity
in the sugar moiety.
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Fig. 3.
NMR spectra of the pentadecapeptides T2-1
and T2-2 derived from component C9. A, anomeric part
of the 1H spectrum of an IL-12-derived heptapeptide with a
C2-Man Trp (12); B, peptide T2-1; and
C, peptide T2-2. D, part of the TOCSY spectrum
of T2-2, recorded with a mixing time of 80 ms. Correlations involving
the two spin systems identified as C2-linked mannosyl residues with
cross-peaks with the anomeric protons are shown.
Chemical shifts of the C2-mannosylated Trp residues at 300 K in
component C9, IL-12 (12), and RNase 2 (5)
-
dimer and a non-covalently
associated subunit. Both the and subunit contain two TSR
modules. To obtain peptides suitable for MS and sequence analysis,
carboxamidomethylated C8 was digested first with trypsin and
subsequently with endoproteinase Asp-N. Fractionation by reversed phase
LC-ESIMS yielded two C-glycosylated peptides originating
from the -subunit, T1-D (2155 Da; residues 1-26 with one hexosyl
residue) and T57 (2317 Da; residues 508-522 with three hexosyl
residues). The fraction containing T1-D was further cleaved with
chymotrypsin, yielding pure T1-D-Ch (residues 10-18) upon LC-ESIMS
purification. Nanospray ESIMSMS of this peptide established that the
sugar moiety was attached to Trp-14, whereas Trp-17 occurred in the
unmodified form (Table I). The peptide was sequenced by Edman
degradation, showing exact comigration of the PTH-derivative at cycle 5 with PTH-(C2-Man-)Trp from RNase 2 (Table I).
T57 contained three hexosyl residues and was purified to
apparent homogeneity by LC-ESIMS at pH 6.0 (data not shown). Its
nanospray ESIMSMS spectrum and Edman degradation (Table I) showed
unequivocally that Trp-512, -515, and -518 were
C-mannosylated. Consistent with this, no loss of 162 Da was
observed in MSMS experiments with either T1-D-Ch or T57, which
would have been evidence for O-linked hexosyl residues.
Since only C-mannosylated forms of T1-D and T57 were
detected in the chromatogram of the starting digest, it is concluded
that these four Trp residues were fully modified.
chain two peptides, T1-D (1942 Da, residues 10-22
with two hexosyl residues) and T59 (2010 Da, residues 494-506 with
two hexosyl residues) were detected and purified further by LC-ESIMS at
pH 6.0 (data not shown). Both Trp-16 and -19 in T1-D were modified,
as was demonstrated by nano-ESIMSMS, which showed a continuous series
of b and y ions covering both Trp residues, as well as 120-Da loss from
a number of ions (Table I). These results were confirmed by Edman
degradation (Table I). Peptide T59 still contained a contaminating
peptide (YILNTR), even after rechromatography. Its presence did not,
however, interfere with further analysis. ESIMSMS localized the hexosyl
residues on Trp-497 and -500, and 120-Da losses were observed from the
parent as well as the y7 and y9 ions (Table I). Edman degradation
confirmed Trp-494 to be unmodified and yielded
PTH-(C2-Man-)Trp in cycles 4 and 7 (Table I). With neither
of these peptides was any evidence for O-linked hexosyl
residues obtained.
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Fig. 4.
Tandem MS analysis of
C-mannosylated peptides from components C6 and
C7. A, nanospray ESIMSMS of the peptide T1-D from C7
(1897 Da; residues 9-22). B, nanospray ESIMSMS of the
peptide T1-Ch-2 from C6 (1578 Da; residues 7-16). The loss of 120 Da
from singly and doubly charged ions has been indicated with
120 and 60, respectively. Loss of H2O
has been indicated with asterisks. amu, atomic mass
units.
View larger version (29K):
[in a new window]
Fig. 5.
Summary of the
C-mannosylation of the terminal components of
complement. The approximate degree of modification (see
"Discussion") of a particular Trp residue has been indicated
above the sequence, where a solid circle
indicates complete modification and an open circle indicates
no modification at all. The three different types of TSR module have
been indicated as follows: 
, group 1; 
, group 2; and 
, group 3 TSR, as defined in Ref. 52.
View larger version (27K):
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Fig. 6.
In vitro
C-mannosylation of peptides derived from C6 and
C9. A, peptides (0.9 mM) were incubated
with 0.9 µM Dol-P-[2-3H]Man and purified,
salt-washed porcine liver microsomes (77 µg of protein). After 30 min
at 37 °C the reaction mixture was extracted with
chloroform/methanol, and the radioactivity in the aqueous phase was
determined. Lane 1, positive control peptide; lane
2, peptide derived from C9; lane 3, peptide derived
from C6; lane 4, control without acceptor peptide.
B, the radiolabeled C9-derived peptide was isolated by
C18 reversed phase HPLC. the eluate was monitored at 214 nm
(upper panel). The lower panel shows the
radioactivity in each fraction. The total recovery of radioactivity was
48%. C, the ESIMSMS spectrum of the purified radiolabeled
C9-derived peptide. The loss of 120 Da from fragment ions and [M + 2H]2+ has been indicated with 120 and
60, respectively. The arrows indicate the
position of y ions that would have appeared if the second Trp, rather
than the first one, were C-mannosylated. amu,
atomic mass units.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
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.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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  E. Muroi, S. Manabe, M. Ikezaki, Y. Urata, S. Sato, T. Kondo, Y. Ito, and Y. Ihara C-Mannosylated peptides derived from the thrombospondin type 1 repeat enhance lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells Glycobiology, September 1, 2007; 17(9): 1015 - 1028. [Abstract] [Full Text] [PDF]
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K. Julenius NetCGlyc 1.0: prediction of mammalian C-mannosylation sites Glycobiology, August 1, 2007; 17(8): 868 - 876. [Abstract] [Full Text] [PDF]
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 J. S. Li, L. Cui, D. L. Rock, and J. Li Novel Glycosidic Linkage in Aedes aegypti Chorion Peroxidase: N-MANNOSYL TRYPTOPHAN J. Biol. Chem., November 18, 2005; 280(46): 38513 - 38521. [Abstract] [Full Text] [PDF]
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E. V. Bocharov, D. M. Korzhnev, M. J. J. Blommers, T. Arvinte, V. Yu. Orekhov, M. Billeter, and A. S. Arseniev Dynamics-modulated Biological Activity of Transforming Growth Factor beta 3 J. Biol. Chem., November 22, 2002; 277(48): 46273 - 46279. [Abstract] [Full Text] [PDF]
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 K. Tan, M. Duquette, J.-h. Liu, Y. Dong, R. Zhang, A. Joachimiak, J. Lawler, and J.-h. Wang Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication J. Cell Biol., October 28, 2002; 159(2): 373 - 382. [Abstract] [Full Text] [PDF]
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 A. Gonzalez de Peredo, D. Klein, B. Macek, D. Hess, J. Peter-Katalinic, and J. Hofsteenge C-Mannosylation and O-Fucosylation of Thrombospondin Type 1 Repeats Mol. Cell. Proteomics, January 1, 2002; 1(1): 11 - 18. [Abstract] [Full Text] [PDF]
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 M. Anand, J. S. Rush, S. Ray, M.-A. Doucey, J. Weik, F. E. Ware, J. Hofsteenge, C. J. Waechter, and M. A. Lehrman Requirement of the Lec35 Gene for All Known Classes of Monosaccharide-P-Dolichol-dependent Glycosyltransferase Reactions in Mammals Mol. Biol. Cell, February 1, 2001; 12(2): 487 - 501. [Abstract] [Full Text]
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S. Hartmann and J. Hofsteenge Properdin, the Positive Regulator of Complement, Is Highly C-Mannosylated J. Biol. Chem., September 8, 2000; 275(37): 28569 - 28574. [Abstract] [Full Text] [PDF]
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J. Hofsteenge, K. G. Huwiler, B. Macek, D. Hess, J. Lawler, D. F. Mosher, and J. Peter-Katalinic C-Mannosylation and O-Fucosylation of the Thrombospondin Type 1 Module J. Biol. Chem., February 23, 2001; 276(9): 6485 - 6498. [Abstract] [Full Text] [PDF]
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