α-C-Mannosyltryptophan is not recognized by conventional mannose-binding lectins

Article abstract of DOI:10.1016/j.bmc.2004.02.009

α-C-Mannosyltryptophan (C-Man-Trp) is a novel, naturally occurring C-linked carbohydrate-protein linkage first found in 1994 from human ribonuclease 2. Since then, a number of C-Man-Trp residue have been found from several important proteins such as interleukin 12β, components of complement system, thrombospondin-1, and erythropoietin receptor, however, the biological functions have remained unknown even though its biosynthetic pathway has been revealed. In order to find a clue as to the biological functions, we examined the affinity of C-Man-Trp with conventional mannose lectin such as concanavarin A (Con A) and mannose-binding lectin (MBL). The affinity of C-Man-Trp with Con A, a typical mannose-binding lectin from plant was examined using a Con A-Sepharose column. Unlike p-nitrophenyl-α-O-Man, C-Man-Trp was not retained on the column. MBL-C, a major mannose-binding lectin purified from mouse serum, did not bind with N-biotinylated C-Man-Trp, judging from ELISA based assay. These results imply that C-Man-Trp may be recognized with the other specific proteins associated with its unknown biological functions.

Full text of DOI:10.1016/j.bmc.2004.02.009

Bioorganic & Medicinal Chemistry 12 (2004) 2343–2348
a-C-Mannosyltryptophan is not recognized by conventional
mannose-binding lectins
a
b
c
c
Toshio Nishikawa,a,d, Shigeo Kajii, Chihiro Sato, Zenta Yasukawa, Ken Kitajima
*
and Minoru Isobea
aLaboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa,
Nagoya 464-8601, Japan
bLaboratory of Molecular Bioregulation, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa,
Nagoya 464-8601 Japan
cBioscience and Biotechnology Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan
dPRESTO, Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi, Saitama, Japan
Received 14 January 2004; revised 4 February 2004; accepted 4 February 2004
Abstract—a-C-Mannosyltryptophan (C-Man-Trp) is a novel, naturally occurring C-linked carbohydrate–protein linkage first found
in 1994 from human ribonuclease 2. Since then, a number of C-Man-Trp residue have been found from several important proteins
such as interleukin 12b, components of complement system, thrombospondin-1, and erythropoietin receptor, however, the bio-
logical functions have remained unknown even though its biosynthetic pathway has been revealed. In order to find a clue as to the
biological functions, we examined the affinity of C-Man-Trp with conventional mannose lectin such as concanavarin A (Con A) and
mannose-binding lectin (MBL). The affinity of C-Man-Trp with Con A, a typical mannose-binding lectin from plant was examined
using a Con A-Sepharose column. Unlike p-nitrophenyl-a-O-Man, C-Man-Trp was not retained on the column. MBL-C, a major
mannose-binding lectin purified from mouse serum, did not bind with N-biotinylated C-Man-Trp, judging from ELISA based assay.
These results imply that C-Man-Trp may be recognized with the other specific proteins associated with its unknown biological
functions.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
extensive analysis of the NMR spectra. C-Man-Trp is
the first example of naturally occurring C-glycosyl
amino acid found in proteins, although many synthetic
C-glycosyl amino acids have been reported.6;7 This novel
post-translational modification is catalyzed by micro-
Carbohydrate parts of glycoproteins and glycolipids
have been found to play important roles in a variety of
biological phenomena such as fertilization, embryonic
development, differentiation, neurogenesis, immune
response, and inflammation.1;2 Most of the carbohy-
drate moiety are linked to protein through N-glycosidic
bond with asparagine and O-glycosidic bond with serine
or threonine.3 In 1994, however, Trp-7 residue of ribo-
nuclease 2 isolated from human urine was found to be
a-C-mannosylated at second position of the indole (1 in
Fig. 1).4;5 Interestingly, the mannose moiety of C-Man-
Trp adopt not an usual 4C1, but a mixture of some
conformations including unusual 1C4, judging from
some-associated
enzyme,
ÔC-mannosyltransferaseÕ,
which has not been purified so far.8 This enzyme was
found to recognize an amino acid sequence Trp-x-x-Trp
to glycosylate the first Trp of this motif.9 These studies
imply that this linkage is more common than expected in
the early stage of this study, because the recognition
sequence is contained in thrombospondin type 1 repeat
whose sequence has been found in many proteins such
as extracellular matrix proteins and proteins associated
with complement systems, and in the WS motif whose
sequence has been found in cytokine receptors.10 In fact,
the C-Man-Trp residue has also been found in a variety
of proteins such as recombinant interleukin 12,11 human
complement system,12;13 human platelet thrombospon-
din-114 and recombinant erythropoietin receptor.15
Furthermore, the C-mannosyltransferase activity was
Keywords: C-mannosyltryptophan; Lectin; Mannose-binding lectin;
Con A.
* Corresponding author. Tel.: +81-52-789-4112; fax: +81-52-789-4111;
e-mail: nisikawa@agr.nagoya-u.ac.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.02.009

2344
T. Nishikawa et al. / Bioorg. Med. Chem. 12 (2004) 2343–2348
COR1
H2N
R2HN
HO
COOH
H
OH
OH
O
H
H
O
N
H
OH
H
N
H
H
HO
OH
HO
H
OH
1. R1= Ala-Gln-Trp ---
R2= Thr-Phe-Gln-Pro-Pro-Lys-NH2
2. (C-Man-Trp). R1 = OH, R2 = H
Figure 1. Chemical structure of a-C-mannosyltryptophan.
detected in many organisms such as mammals, nema-
todes, birds, amphibians, fish, but not in Escherichia
coli, insect or yeast.10;16 On the other hand, monomeric
C-Man-Trp (2, in Fig. 1) was isolated not only from
human urine,17;18 but also from marine organisms.19;20
Monomeric C-Man-Trp in blood was reported to be a
potential candidate as a marker molecule for diagnosing
renal function.21 Despite these extensive studies, the
biological function of this novel modification has not
been clarified, although some possibilities were dis-
cussed.10 To clue the biological functions, authentic
C-Man-Trp-containing compounds as well as probes for
these structures are absolutely necessary. However, since
sufficient amount of such compounds are not available
from natural sources or enzymatic synthesis at present,
we as well as others have studied on chemical synthesis
of C-Man-Trp22–24 and its analogs.25 The presence of
D
-mannose in C-Man-Trp residue prompted us to
examine whether C-Man-Trp could be recognized by
conventional a-mannose-binding lectins. The study
might provide some clues not only for the biological
functions of C-Man-Trp in proteins, but also for the
significance of monomeric C-Man-Trp in blood and
urine. In this paper, we describe the affinity of C-Man-
Trp with typical a-mannose-binding lectins using syn-
thetic C-Man-Trp and its derivative.
Figure 2. Affinity chromatograms on Con A-Sepharose column with
O-Man-pNP (A), C-Man-Trp (B), and C-Man-Indole (C). (A) The
concentration of O-Man-pNP was determined by measuring absor-
bance at 405 nm after hydrolysis of the mannoside. O-Man-pNP was
retained in the column and eluted in fractions 18–22. O-Man-pNP was
also eluted in the pass-through fractions because of overloading of the
mannoside. (B) The concentration of C-Man-Trp was determined
spectrophotometrically at 280 nm. In the pass-through fractions, 78%
of the loaded C-Man-Trp were detected. C-Man-Trp was not found in
any fractions eluted with a-MeGlc, a-MeMan or EDTA. (C) The
concentration of C-Man-Indole was determined spectrophotometri-
cally at 280 nm. In the pass-through fractions, 86% of the loaded C-
Man-Indole were detected. C-Man-Indole was not found in any other
fractions.
2. Results and discussion
2.1. C-Man-Trp and C-Man-Ind were not recognized by a
plant lectin Con A
Concanavarin A (Con A) is a plant lectin that can bind
to monomeric and polymeric forms of a-mannosides.26
To examine if C-Man-Trp is recognized by a typical
mannose binding lectin Con A, we employed Con A-
Sepharose column, which was reported to retain
monomeric a-O-mannoside.26 In our experiments, 20%
of the applied O-Man-pNP were recovered in fractions
18–22, which was eluted with 1.0 M a-MeMan and 78%
O-Man-pNP passed through the column, because of
retention capacity of the column (Fig. 2A). On the other
hand, C-Man-Trp was not retained in the column as
shown in Figure 2B. It is thus concluded that C-Man-
Trp is not recognized by Con A.
To obtain a further insight into recognition of the a-C-
mannoside by Con A, the same lectin chromatography
was performed for C-Man-Indole 4, which was readily
prepared from a synthetic intermediate for C-Man-Trp

T. Nishikawa et al. / Bioorg. Med. Chem. 12 (2004) 2343–2348
2345
OBn
OH
H
H
1) n-Bu4NF, THF
O
O
N
N
H
2) H2 (1 atm),
Pd(OH)2-C,
MeOH
Ts
OBn
BnO
HO
OH
OBn
3
OH
4
OH
OH
H
4
5
O
HO
J1-2 = 2 Hz di-equatorial
2
1
3
H
HO
J2-3 = 4 Hz equatorial-axial
J3-4 = 9 Hz di-axial
H
H
H
HN
J4-5 = 9 Hz di-axial
Figure 3. Western blotting of the purified MBL-C using anti-MBL-C.
Mouse serum and purified MBL-C were subjected to SDS-PAGE/
immunoblotting using anti-MBL-C as a primary antibody. Lane 1,
whole serum; lane 2, purified MBL-C; In lane 1, MBL-C is marked by
the asterisk, and the strong bands around 50–60 kDa appear to be
intrinsic immunoglobulins in mouse serum.
4C1 conformation
Scheme 1. Synthesis and conformation of a-C-mannosylindole.
(Scheme 1). As shown in Figure 2C, C-Man-Indole was
not recognized by Con A either.
whose several component proteins were heavily C-
mannosylated.12;13 To examine binding ability of MBL-
C to C-Man-Trp directly, MBL-C was purified from
mouse sera by serial purification steps including the
mannan-Sepharose column chromatography29 (Fig. 3),
and tested for binding activity to C-Man-Trp residues
by the enzyme-linked immunosorbent assay(ELISA)-
based assay,32 by utilizing biotinylated C-Man-Trp
prepared from C-Man-Trp (Scheme 2). No binding was
observed (data not shown), indicating that MBL-C has
no ability to bind the C-Man-Trp residues.
As 1H NMR analysis of C-Man-Indole revealed that the
conformation of the mannose moiety is C1 (diagnostic
4
coupling constants are shown in Scheme 1), which is the
same as that of usual a-O-mannoside, the indole moiety
prevented the interaction with Con A, although some
other a-C-mannosides were reported to bind to Con
A.27;28 Thus, the reason that C-Man-Trp dose not bind
to Con A is the unusual mannose conformation as well
as the structure of aglycon (Trp).
2.2. C-Man-Trp was not recognized by MBL-C
In summary, this study clarifies that the novel C-Man-
Trp cannot be recognized by conventional mannose-
binding lectins such as Con A and MBL-C. These results
led us to suppose that the C-Man-Trp should be rec-
ognized by the other specific proteins, which are
involved in unknown biological functions of C-Man-
Trp. Our preliminary experiments found that several
proteins in mouse serum bound to C-Man-Trp immo-
bilized on Sepharose.33 Efforts to identify the C-Man-
Trp-binding proteins from mammal are currently
underway in our laboratories.
Since C-Man-Trp residue has been found only from
animal, we then examined the affinity of C-Man-Trp
with typical animal lectin. In mouse serum, two C-type
lectins, named as mannose-binding lectins MBL-C and
-A, are known to occur, and can recognize both
monomeric and polymeric forms of a-mannosides.29
Among them, MBL-C is a major serum MBL and 4–7
times more abundant in weight than MBL-A.30 It is
noteworthy that MBL activates complement system,31
O
SO3
HN
NH
O
O
HO
O
H
H
H
OH
N
N
N
O
S
2
5
OH
O
O
O
OH
H
2
HN
NH
O
NH
phosphate buffer
HOOC
S
5
HN
O
Scheme 2. Preparation of biotinyl C-Man-Trp.

2346
T. Nishikawa et al. / Bioorg. Med. Chem. 12 (2004) 2343–2348
3. Experimental section
to give C-mannosylindole 4 (4.3 mg, 74%). 1H NMR
(400 MHz, D2O) d 3.30 (1H, ddd, J ¼ 9, 6, 2 Hz, H-50),
3.73 (1H, t, J ¼ 9 Hz, H-40), 3.76 (1H, dd, J ¼ 12, 6 Hz,
HA-60), 3.86 (1H, dd, J ¼ 12, 2 Hz, HB-60), 3.93 (1H, dd,
J ¼ 9, 4 Hz, H-30), 4.63 (1H, dd, J ¼ 4, 2 Hz, H-20), 5.26
(1H, d, J ¼ 2 Hz, H-10), 6.54 (1H, s, H-3), 7.13 (1H, t,
J ¼ 8 Hz, indole), 7.23 (1H, t, J ¼ 8 Hz, indole), 7.48
(1H, d, J ¼ 8 Hz, indole), 7.64 (1H, d, J ¼ 8 Hz, indole).
13C NMR (100 MHz, D2O) d 62.8, 68.9, 71.5, 73.2, 76.2,
77.3, 102.3, 113.2, 121.6, 122.2, 124.1, 129.2, 136.2,
137.9. HRMS (FAB) calcd for C14H18N1O5S (M+H),
280.1185, found: 280.1177.
3.1. General
Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Brucker ARX-400 (400 MHz) spec-
trometer. Chemical shifts of all compounds were
assigned using t-BuOH (d ¼ 1:22 ppm) as an internal
standard. Data are reported as follows; chemical shift,
integration, multiplicity (s ¼ singlet, d ¼ doublet,
t ¼ triplet, m ¼ multiplet), coupling constant(s), and
assignment. High resolution mass spectra (HRMS) were
recorded on a JEOL JMS-LCmate LC/MS system, and
reported in m=z. Reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.25 mm silica gel
coated glass plates 60F254 (Merck, Art 1.05715) using
UV light as visualizing agent and 7% ethanolic phos-
phomolybdic acid, or p-anisaldehyde solution and
heated as developing agents. Merck silica gel 60 (particle
size 0.063–0.2 mm ASTM) were used for open-column
chromatography. Preparative TLC were carried out on
0.5 mm Merck silica gel plates (60F254, Art 1.05744). All
commercially available reagents were used as received.
3.4. Synthesis of Biotinyl C-Man-Trp
To a solution of Man-Trp 2 (3.3 mg, 9.0 lmol) in
phosphate buffer (2.0 mL, 0.1 M, pH 7.2) was added EZ-
Linke sulfo-NHS-LC-Biotin (15.0 mg, 27.0 lmol,
PIERCE). After stirring at the room temperature for
2 h, the reaction was quenched with 28% ammonia
(1.1 mL). The resulting mixture was stirred at the same
temperature for 1.5 h, concentrated under reduced
pressure. The residue was chromatographed on pre-
parative TLC (CHCl3–MeOH/H2O ¼ 65:65:15). The
product was further purified on a column of Cosmosil
75C18 (gel 200 lL, elution with H2O–MeOH (3:1)) to
give biotinyl Man-Trp 5 (2.6 mg 41%). 1H NMR
(400 MHz, D2O) d 0.71–0.85 (2H, m), 1.14–1.26 (2H,
m), 1.14–1.26 (2H, m), 1.30–1.41 (2H, m), 1.50–1.75
(2H, m), 1.50–1.75 (2H, m), 2.05 (2H, t, J ¼ 7 Hz), 2.22
(2H, t, J ¼ 7 Hz), 2.72 (1H, d, J ¼ 13 Hz), 2.92 (1H, dd,
J ¼ 13, 5 Hz), 2.90–3.00 (2H, m), 3.19 (1H, dd, J ¼ 14,
5, 10.5 Hz, HA-b), 3.22–3.30 (1H, m), 3.42 (1H, dd,
J ¼ 14:5, 5 Hz, HB-b), 3.77 (1H, dd, J ¼ 12, 3 Hz, HA-
60), 3.80–3.86 (1H, m, H-50), 3.96 (1H, dd, J ¼ 6, 2 Hz,
H-40), 4.08 (1H, dd, J ¼ 6, 3 Hz, H-30), 4.17 (1H, dd,
J ¼ 12, 8 Hz, HB-60), 4.36 (1H, dd, J ¼ 7, 5 Hz), 4.41
(1H, dd, J ¼ 7, 3 Hz, H-20), 4.55 (1H, dd, J ¼ 7, 5 Hz),
4.57 (1H, dd, J ¼ 10:5, 5 Hz, H-a), 5.22 (1H, d,
J ¼ 7 Hz, H-10), 7.17 (1H, t, J ¼ 7 Hz, H-6), 7.26 (1H, t,
J ¼ 7 Hz, H-5), 7.47 (1H, d, J ¼ 7 Hz, H-4), 7.73 (1H, d,
J ¼ 7 Hz, H-7). HRMS (FAB) calcd for C33H47N5O10S
(M+H), 706.3122, found: 706.3134.
3.2. Materials
a-C-mannosyltryptophan (C-Man-Trp) was chemically
synthesized according to our method.21;22 Methyl-a-
D-
mannopyranoside (a-MeMan), methyl-a-
anoside (a-MeGlc), and p-nitrophenyl-a-
D
-glucopyr-
-mannopyr-
D
anoside (O-Man-pNP) were purchased from Sigma. Con
A Sepharose gel and BrCN-activated Sepharose gel were
purchased from Amersham Pharmacia Biotech. Yeast
mannan was obtained from Nacalai Tesque Inc. (Kyoto,
Japan). Mannan-Sepharose was prepared by conjugat-
ing yeast mannan with BrCN-activated Sepharose
according to the manufacturerÕs instruction. A mono-
clonal anti-mouse mannose-binding lectin C (MBL-C)
antibody (14D12)27 was provided by Dr. Steffen Thiel
(University of Aarhus, Denmark).
3.3. Synthesis of mannosyl indole
A
solution of N-Ts-mannosylindole
3
(50 mg,
0.06 mmol) and TBAF (1 M in THF, 0.33 mL,
0.33 mmol) in THF (1.6 mL) was heated under reflux for
2.5 h. The reaction was quenched with satd NH4Cl
solution, the mixture was extracted with AcOEt three
times. The combined extract was washed with water and
brine, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatogra-
phy (silica gel 5 g, AcOEt–hexane ¼ 1:5) to give tetra-O-
benzylmannosylindole (18.8 mg, 47%). The product
(13 mg, 0.021 mmol) was dissolved in MeOH (1 mL) and
2 N HCl (5.3 lL) and 20% Pd(OH)2–C (13 mg) were
added. The reaction vessel was filled with N2 gas and the
N2 was replaced with H2 gas. After vigorously stirring at
room temperature for 20 min, the mixture was filtered
through the pad of Hyflo Super-Cel, the filtrate was
concentrated under reduced pressure. The crude product
was purified on preparative TLC (CH2Cl2–MeOH ¼ 4:1)
3.5. Binding assay of Con A to C-Man-Trp
C-Man-Trp and C-Man-Indole: A Con A-Sepharose
column (1 mL, 5 mm · 40 mm) was equilibrated with
10 mM Tris–HCl buffer (pH 7.4) containing 1 mM CaCl2
and 1 mM MnCl2 (Buffer A). An aqueous solution of C-
Man-Trp or C-Man-Indole (2 mM, 0.3 mL) was loaded
on the column. After standing for 10 min, the column
was successively eluted with Buffer A (10 mL), 0.2 M
a-MeGlc in Buffer A (5 mL), 0.2 M a-MeMan in Buffer A
(5 mL), 1.0 M a-MeMan in Buffer A (10 mL), and 5 mM
EDTA in 10 mM Tris–HCl buffer (pH 7.4) (10 mL). The
eluate was collected in 1 mL portions. The concentra-
tions of C-Man-Trp and C-Man-Indole were determined
by absorbance at 280 nm: E1 mM; 1 cm, 3.444 for C-Man-
Trp; E1 mM; 1 cm, 6.978 for C-Man-Indole. Unless other-
wise stated, all procedures were conducted at 4 ꢁC.

T. Nishikawa et al. / Bioorg. Med. Chem. 12 (2004) 2343–2348
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O-Man-pNP: To the same column equilibrated with
€
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Buffer A was charged O-Man-pNP (3 mM, 0.3 mL). The
column was successively eluted as described above. The
eluate was collected in 1 mL portions. To determine the
concentration of O-Man-pNP of each fraction, a 0.1 lL
aliquot was hydrolyzed in 0.1 mL of 1 N HCl at 100 ꢁC
for 30 min. Then 0.1 mL of 1 N NaOH and 1 mL of 1 M
Na2CO3 buffer (pH 9.5) were added, and the absorbance
at 405 nm was measured: E1 mM; 1 cm, 2.458 at pH 9.5 for
p-nitrophenol.
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3.6. Binding assay of MBL-C to C-Man-Trp
Purification of MBL-C: MBL-C was purified from
mouse serum based on the method of Hansen et al.29
Briefly, mouse serum was precipitated by 6.5% (w/v)
polyethylene glycol in BBS/Ca (10 mM barbital-NaOH
(pH 7.4), 0.3 M NaCl, 0.01% Tween 20, 10 mM CaCl2).
The precipitate was dissolved in BBS/Ca and applied to
1 M ethanolamine-treated BrCN-activated Sepharose
column and the pass-through fraction was applied to the
mannan-Sepharose column. After washing the column
with BBS/Ca, the bound MBL-C was eluted with 15 mM
EDTA. The MBL-C thus purified was homogenous on
SDS-PAGE, and amino acid sequence analysis showed
that the MBL-C sample had the N-terminal sequence,
Glu-Thr-Leu-Thr-Glu-Gly-Val, which is completely
identical to the reported sequence of MBL-C.34
ELISA-based method: MBL-C was dialyzed against
PBS. The 96-well plate was coated with 50 lL of MBL-C
at 0–11.5 lg/mL at 37 ꢁC for 2 h. After washing three
times with PBS, the wells were blocked with 1% BSA
(pretreated with sodium periodate to eliminate glycan-
containing impurities) in PBS at 4 ꢁC overnight. After
washing the wells twice with TBS (50 mM Tris–HCl
buffer (pH 7.5), 0.15 M NaCl) and then with TBS con-
taining 0.9 mM CaCl2 (TBS/Ca), biotinylated C-Man-
Trp (1 lg/mL) or biotinylated Trp (1 lg/mL) in TBS/Ca
was added. After incubation at 37 ꢁC for 2 h and
washing four times with TBS/Ca containing 0.05%
Tween 20, color development was performed using
avidin-biotin-peroxidase complex (Vector Laboratories,
CA) under the manufacturerÕs instruction as described
previously.32
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Acknowledgements
We thank Dr. Steffen Thiel for his kind provision of the
antibodies. This research was supported in part by a
Grants-in-Aids for Scientific Research from the Minis-
try of Education, Science, Sports, and Culture of Japan
(MEXT), and PRESTO, Japan Science and Technology
Agency.
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