Thermodynamic analysis of interactions between N-linked sugar chains and F-box protein Fbs1

Article abstract of DOI:10.1021/jm0489511

Fbs1 is a recently discovered F-box protein that was proposed to recognize high-mannose-type asparagine-linked glycoprotein sugar chains. To reveal the specificity of Fbs1, Manα1→6Manβ1→4GlcNAc₂, Manα1→3Manβ1→4GlcNAc₂, and Manα1→ 3(M

Full text of DOI:10.1021/jm0489511

3126
J. Med. Chem. 2005, 48, 3126-3129
Thermodynamic Analysis of Interactions
between N-Linked Sugar Chains and
F-Box Protein Fbs1
Shinya Hagihara, Kiichiro Totani, Ichiro Matsuo, and
Yukishige Ito*
RIKEN (The Institute of Physical and Chemical Research),
2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, and
CREST, Japan Science and Technology Agency,
Kawaguchi, Saitama 332-1102, Japan
Received December 27, 2004
Abstract: Fbs1 is a recently discovered F-box protein that was
proposed to recognize high-mannose-type asparagine-linked
glycoprotein sugar chains. To reveal the specificity of Fbs1,
ManR1f6Manâ1f4GlcNAc2, ManR1f3Manâ1f4GlcNAc2, and
ManR1f3(ManR1f 6)Manâ1f4GlcNAc2 were synthesized
and their affinities for Fbs1 were evaluated in comparison with
previously synthesized Man9GlcNAc2 and Man8GlcNAc2. These
analyses revealed that Man3GlcNAc2 had the strongest affinity
and the chitobiose and R1f6 linked Man residue are necessary
for Fbs1 to recognize a sugar.
Figure 1. Glycoprotein processing and quality control in ER.
Ubiquitination of proteins is a signal for the protea-
some-mediated protein degradation pathway.1 This
process is mediated by a combination of ubiquitin (Ub)
activating enzyme (E1), Ub conjugating enzyme (E2),
and Ub ligase (E3). Numerous types of Ub ligase exist
in the cytosol and play an important role in the selective
ubiquitination of target proteins.2 SCF complex is one
of the most extensively studied families of Ub ligases.3
It consists of Skp1, Cul1, Roc1, and an F-box protein
that recognizes the target protein (Figure 1). F-box
proteins comprise an F-box domain that binds to Skp1
and various C-terminal domains for target recognition.4
Fbs15 and Fbs26 are recently discovered F-box proteins
that were proposed to recognize high-mannose-type
asparagine (Asn)-linked glycoprotein sugar chains (N-
glycans).
Asn-linked sugar chains are introduced cotransla-
tionally in the endoplasmic reticulum (ER) as a tetradeca-
saccharide (Glc3Man9GlcNAc2, Figure 1).7 Recent re-
ports strongly suggest that misfolded glycoproteins
destined for degradation are trimmed to Man8GlcNAc2,
which in turn is recognized by the R-mannosidase I-like
protein (MLP; EDEM or Htm1p).8 The latter partici-
pates in ER-associated degradation (ERAD) as an
acceptor of terminally misfolded glycoproteins, which
are transported from the ER to cytosol and degradated
by the Ub-proteasome system.9 Hence, Fbs1 and Fbs2
are likely to recognize the Man8GlcNAc2 form of N-
glycans. The accumulation of misfolded proteins in
neurons is now considered the origin of a growing
number of neurodegradative disorders.10 The expression
of Fbs1 is restricted to the adult brain and testis,
implying that Fbs1 has a role in the mechanism of such
disorders.
A pull-down analysis of the interaction between
Fbs1 and N-linked glycoproteins revealed that Fbs1
recognizes N-glycans containing a chitobiose (GlcNAcâ1f
4GlcNAc) structure with pendent mannose residues.5
In addition, NMR-based experiments and an X-ray
analysis of Fbs1 in complex with chitobiose have clari-
fied the sugar recognition site of Fbs1.11 However, a
quantitative analysis of the binding of N-linked sugar
chains to Fbs1 has yet to be conducted. Herein, we
report the thermodynamic analysis of interactions
between Fbs1 and a series of high-mannose- and
complex-type oligosaccharides and their partial struc-
tures using isothermal titration calorimetry (ITC).12
These experiments provided detailed information on the
mechanism by which Fbs1 recognizes sugars.
To quantify the binding of Fbs1 to N-linked oligosac-
charide, we prepared oligosaccharide probes (Figure 2).
The synthesis of Man9GlcNAc2 (1) and Man8GlcNAc2 (2)
were conducted on the basis of the reported proce-
dure.13,14 To reveal the specificity of Fbs1, ManR1f
6Manâ1f4GlcNAc2 (5), ManR1f3Manâ1f4GlcNAc2
(6), and ManR1f3(ManR1f6)Manâ1f4GlcNAc2 (4)
were synthesized and their affinities for Fbs1 were
evaluated in comparison with 1 and 2. As depicted in
Scheme 1, preparation of 4-6 was conducted in a
divergent manner from triol 16. Thus, thioglycoside 1115
was used as the donor, and a reaction with 10,16 under
activation by N-iodosuccinimide (NIS) and triflic acid
(TfOH),17 afforded the chitobiose component 12. 12 was
deacetylated to 13, which was subjected to â-mannno-
sylation via intramolecular aglycon delivery using 14
as the donor.18 The obtained trisaccharide 15 was
acetylated, deacetalized, and desilylated under high-
pressure conditions19 to afford the acceptor 16. R-
Mannosylation using the chloride 1720 (1.5 equiv) si-
multaneously provided R1f6 linked (19) and R1f3
* To whom correspondence should be addressed. Phone: 81-48-467-
9430. Fax: 81-48-462-4680. E-mail: yukito@riken.jp.
10.1021/jm0489511 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/08/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3127
Figure 3. ITC profiles for the binding of synthetic oligosac-
charides to Fbs1 at 20 °C. (a) Man8GlcNAc2, (b) Man8GlcNAc,
and (c) Man3GlcNAc2 were injected every 4 min into buffer
solution containing Fbs1 (20 µM). Upper traces show the raw
ITC data. Lower traces show the molar heat values plotted as
a function of the molar ratio ([oligosaccharide]/[Fbs1]). The
solid line represents the best-fit binding isotherm. The data
were fitted using a single site model.
Table 1. Thermodynamic Parameters of Interactions between
Fbs1 and Synthetic Oligosaccharides
Ka × 10-5
∆H
∆S
entry
oligosaccharide
1 (Man9GlcNAc2)
2 (Man8GlcNAc2)
3 (Man8GlcNAc)
4 (Man3GlcNAc2)
5 (ManR1f6ManGlcNAc2)
6 (ManR1f3ManGlcNAc2)
7 (GlcNAc2)
(M-1
)
(kcal mol-1
)
(cal mol-1 K-1
)
1
2
3
4
5
6
7
8
9
3.3
4.1
-7.8
-5.9
-1.4
5.6
NDa
8.5
-11.7
-7.2
-7.0
-12.9
0.7
-1.8
Figure 2. Oligosaccharides used in this study.
3.3
0.63
NDa
NDa
0.24
Scheme 1a
8 (Man3)
9 (Complex-type)
-10.3
-14.9
a No detectable binding.
separated by column chromatography on silica gel and
were deprotected under standard conditions. Depro-
tected oligosaccharides 4-6 were identified by compari-
son with previously reported NMR spectra.
We first evaluated the affinity of Fbs1 for full-length
high-mannose-type glycans 1 and 2 in comparison with
Man3GlcNAc2 4. To obtain thermodynamic parameters
for the binding of these synthetic oligosaccharides to
Fbs1, the binding was analyzed by ITC.12 Titration was
conducted by adding 6 µL of synthetic oligosaccharide
(300 µM) every 4 min into a buffer solution (10 mM
sodium phosphate, pH 7.2, and 100 mM NaCl) contain-
ing Fbs1 (20 µM). Molar heat values for the binding of
2 plotted as a function of the [2]/[Fbs1] molar ratio are
shown in the lower panel (Figure 3a). The solid line
shows the binding isotherm obtained by the fitting of
the data to a binding model involving a single set of
identical sites. The thermodynamic parameters obtained
by the fitting are summarized in Table 1. The binding
constant (Ka) of 2 (entry 2) was determined as 4.1 ×
105 M-1, which was similar to that of 1 (entry 1). In
contrast, the binding of Man8GlcNAc (3)14 was below
the detection limit of ITC under these conditions (Figure
3b). This result underscores the strict requirement of
the chitobiose (GlcNAc2) portion for glycan recognition
and corroborates well the result of a crystallographic
study.11 On the other hand, pentasaccharide 4 (entry
4) showed substantially stronger affinity (Ka ) 8.5 ×
105 M-1). These results suggest that Fbs1 mainly
recognizes the core region of N-glycans, and extended
outer mannose residues of full-length chains cause steric
repulsion resulting in reduced affinity.
a Reagents and conditions: (a) NIS, TfOH, CH2Cl2, 98%; (b) 30%
H2O2(aq), LiOH, THF, 90%; (c) DDQ, CH2Cl2; (d) MeOTf, DTBMP,
CH2ClCH2Cl, 87% (two steps); (e) (i) Ac2O, pyridine; (ii) PPTS,
CH3CN; (iii) HF/pyridine, DMF, 1 Gpa, 70% (three steps); (f)
AgOTf, CH2ClCH2Cl; (g) (i) ethylenediamine, n-BuOH, 80 °C; (ii)
Ac2O, pyridine; (iii) NaOMe, MeOH; (iv) H2, 10% Pd/C, H2O-
MeOH.
linked (20) tetrasacchaides and Man3GlcNAc2 (18) in
23%, 16%, and 34% yield, respectively, with 22%
recovery of the acceptor 16. These products were easily

3128 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Letters
for valuable discussion, and Ms. Akemi Takahashi for
technical assistance.
Supporting Information Available: Experimental pro-
cedures, NMR data, and ITC profiles. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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Subsequently, the affinities of the partial structures
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R1f6 linked tetrasaccharide 5 (entry 5) had an affinity
marginally weaker than that of 4, while the R1f3
linked congener 6 had a drastically reduced affinity
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JM0489511