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Ordered self-assembly of the collagenous domain of

adiponectin with noncovalent interactions via

glycosylated lysine residues
Ayako Takuwa1, Takuya Yoshida1, Takahiro Maruno2, Kazuki Kawahara1, Masayoshi Mochizuki3,
Yuji Nishiuchi3,*, Yuji Kobayashi2 and Tadayasu Ohkubo1
1 Graduate School of Pharmaceutical Sciences, Osaka University, Japan
2 Department of Biotechnology, Graduate School of Engineering, Osaka University, Japan
3 Peptide Institute Inc., SAITO Res Ctr, Ibaraki, Osaka, Japan

T. Yoshida and T. Ohkubo, Graduate School
of Pharmaceutical Sciences,
Osaka University, 1-6 Yamadaoka, Suita,
Osaka 565-0871, Japan
Fax: +81 6 6879 8222
Tel.: +81 6 6879 8220
E-mails: (T. Y.); (T. O.)
*Present address:
GlyTech, Inc., Shimogyo-ku, Kyoto,
600-8813, Japan
(Received 7 September 2015, revised 18
November 2015, accepted 23 November
2015, available online 28 January 2016)

Adiponectin, an anti-atherogenic and insulin-sensitizing adipokine, forms

multiple isoforms including a trimer, a hexamer and heavier oligomers
(mainly octadecamer) that determine their biological activities. We designed
89-residue peptides containing modifications found in the collagenous domain of
native adiponectin. Circular dichroism and analytical ultracentrifugation
measurements showed that the peptide bearing glucosyl-galactosyl-hydroxylysine
residues forms a stable collagen-like triple helical structure and spontaneously
assembled into an octadecamer. An assembly model mediated by noncovalent
interactions via glycosylated lysine residues for the octadecamer was constructed. Our findings clarified an essential role of glycosyl modifications to
coordinate the ordered self-assembly of adiponectin.
Keywords: adiponectin; collagen; glycosylation; octadecamer; self-assembly

Edited by Laszlo Nagy

Adiponectin is an anti-atherogenic and insulinsensitizing hormone secreted from adipose tissue. The
circulating level of adiponectin decreases not only in
type 2 diabetes and cardiovascular disease, but also in
obesity, which causes various pathological conditions
[1,2]. For the last few decades, the number of patients
diagnosed with metabolic syndrome, type 2 diabetes,
and cardiovascular disease has been increasing. Thus,
adiponectin represents an attractive potential therapy
target of metabolic syndrome [3].
A distinctive feature of adiponectin is that it exists in
various oligomeric forms including a trimer, a hexamer

and heavier multimer forms called high molecular

weight multimers (HMW) [4]. It is considered that these
oligomers differ in their physiological activities, stabilities, and correlations with clinical conditions. In particular, HMW has been known as the most active form on
the basis of a report that HMW stimulates the AMPK
pathway more effectively and exerts a higher curative
activity against insulin resistance than other adiponectin
isoforms [5]. Therefore, it is important to understand the
mechanism of oligomerization. The primary sequence of
adiponectin is constructed from three domains: the Nterminal variable region, the collagenous domain, and

AUC, analytical ultra centrifugation; CD, circular dichroism; HMW, molecular weight multimers; SV-AUC, sedimentation velocity analytical
ultracentrifugation; TCEP, tris-(2-carboxyethyl)-phosphine.

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Assembly of adiponectin collagenous domain

Fig. 1. Amino acid sequence of the adiponectin variable region

(purple) and the collagenous domain (black). Four proline residues
(blue) are substituted by 4(R)-hydroxyproline in VC-Hyp4 and
VC-Hyp4/Ghl4. In VC-Hyp4/Ghl4, four lysine residues (red) are also
substituted by glucosyl-galactosyl-hydroxylysine, whose structural
formula is shown at the bottom.

phase synthesis method and the native chemical ligation.

The details of this synthesis have been previously reported
[16]. The peptide without modifications was prepared using
an E. coli expression system. BL21(DE3) was transformed
with an expression vector for the human adiponectin variable region and collagenous domain with a polyhistidine
tag and then cultured in LB medium with 100 mgL1
ampicillin. Protein expression was induced with 0.2 mM
IPTG over night at 25 C. Cells were harvested and sonicated in 10 mM Tris-HCl pH 7.5, 500 mM NaCl. The supernatant was purified with a Ni chelating column. After the
polyhistidine tag was enzymatically removed, the VC peptide was further purified using gel filtration. All peptides
were dissolved in 10 mM Tris-HCl pH 7.5, 150 mM NaCl at
a concentration of 100 lM for circular dichroism (CD)
measurements and 200 lM for analytical ultra centrifugation (AUC) experiments. These concentrations were determined using the absorbance at 280 nm. To ensure the
formation of collagen triple helices, peptides were heatdenatured at 50 C and then annealed at 4 C for 11 days.

the C terminal globular domain [69] (Fig. 1). The trimer is formed through hydrophobic interactions among
the globular domains and a propensity of the collagenous domains to take the triple helical structure [10,11].
The hexamer is formed by a disulphide bond at a conserved Cys residue in the variable region between two
trimers [12]. However, the oligomerization mechanism
for HMW remains to be elucidated. The recombinant
adiponectin expressed in Escherichia coli could only
assemble into the trimeric and hexameric forms, but not
the HMW form, supporting the report that post-translational modifications are necessary for the formation of
HMW [13,14]. As post-translational modifications,
human adiponectin undergoes hydroxylation on several
Pro residues. Furthermore, the collagenous domain
includes four conserved Lys residues (Lys65, 68, 77 and
101), and it has been reported that all of them are glucosyl-galactosyl hydroxylated. Although it was shown that
the replacement of the conserved Lys residues by Arg
residues and inhibition of modification enzymes with
chemical reagents impaired the formation of HMW
in vivo [14,15], the contribution of glycosylation of Lys
residues in the self-assembly of adiponectin has not yet
been clarified. In this study, to explore the molecular
basis of the high order multimerization of adiponectin,
physico-chemical analyses were carried out using synthetic peptides.

Experiments of sedimentation velocity method were carried

out with an analytical ultracentrifuge XL-I (Beckman Coulter, Brea, CA, USA) with an An-50Ti rotor. Absorbance
scans at 280 nm were performed 400 times with an interval
of 2 min at 42 000 rpm at either 4 or 20 C. Collected data
were analysed using the Sedfit program with the c(s) distribution analysis method [17]. All sedimentation coefficients
(s values) were corrected as the condition of water at
20 C. For sedimentation equilibrium analysis of VC-Hyp4/
Ghl4, absorbance profiles at 295 nm were measured with
the rotor speed of 9000 rpm at 20 C.

Materials and methods

Model building

Preparation of peptides
Two VC series peptides, VC-Hyp4 and VC-Hyp4/Ghl4,
were prepared with fragments synthesis using the solid-


Measurements of CD
Circular dichroism measurements were performed with a
spectropolarimeter J-720W (JASCO, Tokyo, Japan) and a
quartz cuvette with 1 mm light path. Temperature was controlled with a temperature control unit PTC-348W
(JASCO). CD spectra at a constant temperature were collected in the wavelength range from 260 to 210 nm. The
thermal melting profiles were obtained by monitoring CD
value at a fixed wavelength of 222 nm, as the temperature
was increased from 4 C to 50 C. The temperature where
the helix-formed and unfolded peptide fractions are equally
populated is defined as the transition temperature (Tm).

Measurements of AUC

The molecular model of adiponectin 18-mer was

constructed using GROMACS 5.0 [18]. To construct the
glycosylated lysine residue, a-D-glucopyranosyl-(1?2)-b-Dgalactopyranosyl moiety was composed by the Glycam

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A. Takuwa et al.

Assembly of adiponectin collagenous domain

Carbohydrate Builder (, Univ. of Georgia)

and manually incorporated into amino acid topologies. A
peptide fragment corresponding to the collagenous domain
of human adiponectin was constructed with dihedral angles
of collagen model peptides and packed into a triple helical
structure. The relative arrangement of two triple helices,
which is consistent with a hexagonal arrangement of triple
helices, was searched by simulated annealing calculations
with a inter helices distance restraint. An optimized packing
of two triple helices was extended to a hexagonal arrangement of six triple helices by successive symmetry rotation
operations and the resulting sextuple trimer bundle structure was energy-minimized in water.

Results and discussion

To investigate the molecular basis of higher order
oligomerization of adiponectin in this study, we have
designed two peptides, VC-Hyp4 and VC-Hyp4/Ghl4,
derived from the adiponectin variable region and collagenous domain (Glu19-Gly107 of human adiponectin). In VC-Hyp4 and VC-Hyp4/Ghl4, Pro44, 47, 53,
and 91, which are commonly reported to be hydroxylated in the literature, are substituted by 4(R)-hydroxyproline (Hyp). Furthermore, in VC-Hyp4/Ghl4, Lys65,
68, 77, and 101 are substituted by glucosyl-galactosylhydroxylysine (Ghl), in which an a-D-glucopyranosyl(1?2)-b-D-galactopyranosyl moiety is attached to
(2S,5R)-5-hydroxylysine (Hyl) via a b-O-glycoside linkage (Fig. 1). VC-Hyp4 and VC-Hyp4/Ghl4 were synthesized using a solid-phase synthesis method and a native
chemical ligation method as previously reported [16].
In addition, VC without any modifications was prepared using an E. coli expression system as a control.
To evaluate whether these three VC peptides formed
a triple helix, CD measurements were performed. It is
established that collagen-like triple helices show a signature pattern with a positive peak around 225 nm
originating from a polyproline type II helix. CD spectra of VC peptides at 4 C are shown in Fig. S1.
Because all spectra had a maximum at 222 nm, it was
considered that all VC peptides formed collagen-like
triple helices. The thermal stability of the triple helix
was estimated from the temperature dependency of
ellipticity at 222 nm (Fig. 2). All profiles show a single
cooperative transition supporting triple helix formation. As shown in Fig. 2, the transition temperature
(Tm) of VC-Hyp4 was 18.0 C which is 5.9 C higher
than that of VC. From this result, it was elucidated
that hydroxylation of Pro in the adiponectin collagenous domain has a role in stabilizing the triple helices.
It was reported that 4(R)-Hyp at the Yaa position stabilized the triple helix in model peptides with the

Fig. 2. Thermal unfolding of the triple helices of adiponectin VC

series peptides, VC (red), VC-Hyp4 (blue) and VC-Hyp4/Ghl4
(yellow). Dependence of ellipticity [h] at 222 nm as a function of
temperature is shown. Transition temperature of VC, VC-Hyp4, and
VC-Hyp4/Ghl4 are calculated as 12.1, 18.0 and 33.4  0.4 C
respectively. All experiments were performed in 10 mM Tris-HCl,
150 mM NaCl, pH 7.5.

characteristic repeat of Gly-Xaa-Yaa of collagen [19].

Of the four Pro residues hydroxylated in VC-Hyp4,
three Pro are at the Yaa position, whereas the other
Pro is at the Xaa position. In this study, all Pro were
modified as 4(R)-Hyp. Although it has been reported
that 4(R)-Hyp at the Xaa position may destabilize triple helices [20], the other three 4(R)-Hyp at the Yaa
position sufficiently stabilized the triple helical structure in VC-Hyp4. Interestingly, the transition temperature of the triple helix in VC-Hyp4/Ghl4 was 15.4 C
and 21.3 C higher than those of VC-Hyp4 and VC
respectively; thus, revealing that glycosylation at Lys
causes a significant increase in Tm. Such a stabilizing
effect of the collagen triple helix by glycosylation has
only been reported for glycosylation at Thr found in
collagen of annelida [21]. Our findings indicate that
glucosyl-galactosyl hydroxylation of Lys residue,
which is frequently found in collagen and collagen-like
proteins of vertebrata [22], contributes to stabilizing
the collagen-like triple helical structure. We confirmed
the stabilizing effect of post-translational modifications
in full-length adiponectin. The CD melting profiles of
adiponectin with and without the modifications as
obtained from the human serum and using bacterial
expression system, respectively, indicate that the modifications stabilize the collagen-like triple helical structure in endogenous adiponectin (Fig. S2).
We next examined the role of various modifications
for higher order oligomerization using sedimentation
velocity analytical ultracentrifugation (SV-AUC) [17].

FEBS Letters 590 (2016) 195201 2015 Federation of European Biochemical Societies


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Assembly of adiponectin collagenous domain

Fig. 3. Higher order oligomerization of the VC series peptides.

(A) VC and VC-Hyp4, and (B) VC-Hyp4/Ghl4 were analysed
by sedimentation velocity c(s) distribution. In panel B, the
sedimentation profile of VC-Hyp4/Ghl4 under reducing condition is
also shown. All experiments were performed in 10 mM Tris-HCl,
150 mM NaCl, pH 7.5, at 4 C (A) or 20 C (B).

In this method, higher order oligomerization could be

estimated from the distribution of the sedimentation
coefficient (s value). Each distribution plots of s value
corrected for water at 20 C is shown in Fig. 3. The
result indicated that both VC and VC-Hyp4 consisted
of two components whose s values were 1.2 S and 1.8
S and the calculated molecular weights of these species
were approximately 15 and 27 kDa respectively. These
results are consistent with the observations that adiponectin without glycosylation at lysines do not form
HMW [15]. In contrast, the sedimentation profile of
VC-Hyp4/Ghl4 showed four major components and
one minor component. Sedimentation coefficients and

calculated molecular weights of the major components

were 1.1 S (calculated molecular weight was 11 kDa),
2.0 S (30 kDa), 3.3 S (59 kDa), and 6.7 S (167 kDa)
and they coincide with the molecular weight of a
monomer, trimer, hexamer and octadecamer, because
the monomer of VC-Hyp4/Ghl4 was 10083 Da. The
minor component of 4.7 S with a molecular weight of
98 kDa could represent a nonamer. Sedimentation
equilibrium analysis, which is a more quantitative
method than SV-AUC, confirmed that the heaviest
component was an octadecamer with a weight-average
molecular weight of 172  8 kDa (Fig. S3). To examine whether the formation of observed multimers is
mediated by an intertriple-helix disulphide linkage in
the variable region, SV-AUC analysis of VC-Hyp4/
Ghl4 under reducing conditions with tris-(2-carboxyethyl)-phosphine (TCEP) was performed. In the sedimentation profile of reduced VC-Hyp4/Ghl4, the peak
corresponding to the hexamer was disappeared,
whereas the octadecamer peak remained as the main
component (Fig. 3). In plasma, HMW adiponectin has
been found as a mixture consisting of nonamer, dodecamer and octadecamer forms with the latter representing the most abundant of these components [23].
Moreover, it has been reported that the hexamer of
adiponectin is broken into trimers by treatment with a
reducing agent, but the octadecamer of adiponectin is
resistant to this [24,25]. Taken together, it can be concluded that VC-Hyp4/Ghl4 essentially reproduces the
self-assembly profile of adiponectin. Our results clearly
demonstrate that noncovalent interactions mediated by
glucosyl-galactosyl hydroxylation of Lys afford the
magic stability for sextuple trimer formation by the
collagenous domain of adiponectin.
In a fibre-forming collagen such as type I collagen, it
has been considered that five staggered triple helices are
arranged to form a quasi-hexagonal lattice, in which a
triple helix is surrounded by six triple helices to form a
close-packed arrangement, resulting in a nanofibrous
structure [26]. However, this packing corresponds to a
septuple trimer and may not be applicable to VC-Hyp4/
Ghl4, and thus, to the collagenous domain of adiponectin. Indeed, electron micrographs of adiponectin HMW
showed a bouquet-like shape of six trimers, suggesting
that bundled collagenous domains cause a hexagonal
ring arrangement of the globular domain [27,28]. To
rationalize a sextuple trimer bundle structure of the collagenous domain, we attempted to construct a model of
this structure. The three chains in the collagen triple
helix are not equivalent because they are coiled around
each other with a 1-residue stagger resulting in a
quasi-3-fold screw axis. Therefore, assuming a six-fold
rotational symmetry, each trimer should be rotated

FEBS Letters 590 (2016) 195201 2015 Federation of European Biochemical Societies

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Assembly of adiponectin collagenous domain

value observed for VC-Hyp4/Ghl4 (Fig. 2). It is noteworthy that these interactions are closed within the six
triple helices, which face the same surfaces outside, and
another triple helix cannot interact in the same manner.
In this study, we revealed that glucosyl-galactosylhydroxylysine residues play a critical role in the selfassembly and the stability of collagenous domain of
adiponectin. Currently, the function of chaperones and
the redox system in the endoplasmic reticulum for the
assembly of adiponectin HMW via disulphide linkages
has been emphasized [12,24,25,2931]. Even if these
factors may be important for the kinetics of association of the trimer, our findings indicate that interactions between glycosylated lysine moieties are essential
for the thermodynamic stability of HMW, and in particular, the octadecamer of adiponectin. Given that the
oligomeric distribution of adiponectin correlates with
the pathophysiology of adiponectin-related diseases,
such as type 2 diabetes, more detailed studies on the
glycosylation state of adiponectin will lead to novel
insights into such diseases. Furthermore, collagenous
peptides are attracting interests in biomimetic design.
To date, engineered collagenous peptides, which have
complementary charged residues, disulphide linkages
or multivalent terminal capping groups, are found to
self-assemble and form fibres, sheets and gel structures
[3234]. Our study implies that glucosyl-galactosylhydroxylysine provides a unique option, which is probably useful to make a hexagonal core via noncovalent
interactions, to biomimetic designers.
Fig. 4. A proposed model for the octadecamer of VC-Hyp4/Ghl4.
(A, B) The region corresponding to the collagenous domain is
shown (A: side view, B: top view). Glucosyl-galactosylhydroxylysine residues are illustrated as spheres in panel A. Six
triple helices are packed in a hexagonal arrangement. (C, D, E)
Interactions among glucosyl-galactosyl-hydroxylysine residues.
Possible hydrogen bonds are shown as dashed orange lines.

approximately 60 degrees to the adjacent one with

respect to the triple helix axis. Such arrangement may
be favourable to minimize the steric clash between
globular domains in full-length adiponectin. Figure 4
shows a possible model for the hexagonal arrangement
of triple helices with a diameter of about 40 A,
is consistent with the electron micrographs of adiponectin HMW [28]. At the interface of two adjacent triple
helices (chains A, B, C and A0 , B0 , C0 ), pairs of glucosyl-galactosyl-hydroxylysine residues, C65-C0 65, B65A0 68, C65-A0 68 and B77-B0 77 form a network of
hydrogen bonds including intratriple-helix interactions
at B65-C65 (Fig. 4 and Supplementary data). These
interactions can stabilize the octadecamer and are attributable to a cooperative melting curve with high Tm

Author contributions
A.T. performed the physico-chemical analyses. M.M.
and Y.N. prepared the peptide materials. T.M. provided technical assistance with AUC. A.T. and T.Y.
wrote the manuscript and performed the modelling
study. K.K, Y.K. and T.O. edited the manuscript.
T.Y. and T.O. conceived and designed the study.

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Supporting information
Additional supporting information may be found in the
online version of this article at the publishers web site:
Fig. S1. Triple helix formation of adiponectin VC
series peptides.
Fig. S2. Thermal unfolding profiles of full-length
Fig. S3. Sedimentation equilibrium analysis of VC-Hyp4/
Ghl4 under reducing conditions.

FEBS Letters 590 (2016) 195201 2015 Federation of European Biochemical Societies