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J. Biol. Chem., Vol. 277, Issue 22, 19521-19529, May 31, 2002
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From the School of Biological Sciences, University of Auckland,
Private Bag 92019, Auckland, 1001 New Zealand
Received for publication, January 20, 2002, and in revised form, March 4, 2002
It has recently been shown that the fat-derived
hormone adiponectin has the ability to decrease hyperglycemia and to
reverse insulin resistance. However, bacterially produced full-length adiponectin is functionally inactive. Here, we show that endogenous adiponectin secreted by adipocytes is post-translationally modified into eight different isoforms, as shown by two-dimensional gel electrophoresis. Carbohydrate detection revealed that six of the adiponectin isoforms are glycosylated. The glycosylation sites were
mapped to several lysines (residues 68, 71, 80, and 104) located in the
collagenous domain of adiponectin, each having the surrounding motif of
GXKGE(D). These four lysines were found to be hydroxylated
and subsequently glycosylated. The glycosides attached to each of these
four hydroxylated lysines are possibly glucosylgalactosyl groups.
Functional analysis revealed that full-length adiponectin produced by
mammalian cells is much more potent than bacterially generated
adiponectin in enhancing the ability of subphysiological concentrations
of insulin to inhibit gluconeogenesis in primary rat hepatocytes,
whereas this insulin-sensitizing ability was significantly attenuated
when the four glycosylated lysines were substituted with arginines.
These results indicate that full-length adiponectin produced by
mammalian cells is functionally active as an insulin sensitizer and
that hydroxylation and glycosylation of the four lysines in the
collagenous domain might contribute to this activity.
In addition to serving as an energy storage depot for
triglycerides, adipose tissue is now recognized as an active endocrine organ that can secret a variety of biologically active molecules (adipocytokines) in response to extracellular signals (1-4). Some of
these adipocytokines, such as leptin, tumor necrosis factor- Adiponectin (also called ACRP30, adipoQ, and GBP28) is a protein
exclusively secreted by adipocytes and was originally cloned by four
research groups using different approaches (5-8). Several recent
studies suggest that adiponectin might be a critical, long sought after
hormone that links obesity, insulin resistance, and type II diabetes
(9-11). The adiponectin gene is located in chromosome 3q27, a
susceptibility locus for type II diabetes and related metabolic
syndromes (12-14). Circulating adiponectin is abundant in humans as
well as rodents, with plasma circulating levels in the microgram/ml
range, accounting for ~0.01% of the total plasma protein (15, 16).
mRNA expression and the secretion level of adiponectin are
dramatically decreased in a variety of animal models of insulin
resistance as well as in obese humans and type II diabetic patients
from different ethnic groups (15-20). Notably, treatment with the
insulin-sensitizing peroxisome proliferator-activated receptor- Evidence is accumulating that adiponectin or its synthetic analogs
might be useful in the treatment of type II diabetes and other
metabolic syndromes associated with insulin resistance. Lodish and
co-workers (10) have recently reported that acute in vivo
administration of truncated adiponectin decreases postprandial plasma
free fatty acid following a high fat meal and that chronic administration of this protein causes sustained weight loss in mice
without affecting food intake. Scherer and co-workers (9) demonstrated
that injection of recombinant adiponectin acutely abolishes
hyperglycemia in several diabetic animal models, including ob/ob,
non-obese diabetic, and streptozotocin-treated mice. Furthermore, Kadowaki and co-workers (11) showed that replenishment of adiponectin can restore insulin resistance in both high fat-fed and lipoatropic mice, perhaps by increasing Mouse adiponectin is composed of 247 amino acid residues with an
N-terminal collagenous domain and a C-terminal globular domain (7). The
N-terminal collagenous domain is required for the high order
multimerization of this protein (5). The three-dimensional structure of
the C-terminal globular region is strikingly similar to that of tumor
necrosis factor- Materials--
Dexamethasone, 3-isobutyl-1-methylxanthine,
Differentiation of 3T3-L1 Cells and Concentration of Proteins in
the Cell Culture Medium--
3T3-L1 cells were maintained as
subconfluent cultures in Dulbecco's modified Eagle's medium
(DMEM)1 supplemented with
10% fetal calf serum. For differentiation, post-confluent cells were
induced by incubation with 0.25 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and 10 µg/ml insulin for
2 days. This was followed by incubation with 10 µg/ml insulin for 2 days. The cells were then maintained in DMEM with 10% fetal calf serum
for another 4 days.
To harvest proteins secreted by adipocytes, the cells at day 8 following differentiation were washed three times with
phosphate-buffered saline and then incubated with serum-free medium for
another 4 h. The medium was collected, centrifuged at 3000 × g for 10 min, filtered through a 0.20-µm filter, and then
concentrated and desalted using a concentrator with a molecular mass
cutoff of 5000 Da (Vivascience Ltd.). The proteins were
quantitated using BCA reagent and stored at Two-dimensional Gel Electrophoresis, Immunoblotting, and
Carbohydrate Detection--
The proteins secreted by either adipocytes
or 3T3-L1 preadipocytes were separated by two-dimensional gel
electrophoresis as described previously (24). The separated proteins
were stained with either silver or Coomassie Brilliant Blue R-250. For
immunoblotting, proteins separated by two-dimensional gel
electrophoresis were transferred to nitrocellulose membranes using a
Multiphor II Novablot electrophoretic transfer unit (Amersham
Biosciences). The membranes were blocked and then incubated with rabbit
anti-adiponectin polyclonal antibody (1:1000) overnight at 4 °C.
After incubation with horseradish peroxidase-conjugated secondary
antibody for 1 h at room temperature, the bound antibodies were
detected using the ECL detection kit. Glycoproteins were detected using
the commercial Immu-Blot kit according to the manufacturer's instruction.
In-gel Trypsin Digestion and Reversed-phase High Performance
Liquid Chromatography (RP-HPLC)--
Protein spots of interest
separated by two-dimensional gel electrophoresis were excised and
subjected to in-gel trypsin digestion as described previously (25). The
extracted tryptic peptide mixtures were fractionated by RP-HPLC on a
Jupiter 5µ C18 column (250 × 2.00 mm;
Phenomenex Inc.). The prewarmed column (37 °C) was washed for 7 min
with 0.1% (v/v) trifluoroacetic acid, followed by elution using a
50-min linear gradient of 8-36% acetonitrile at a flow rate of 200 µl/min. Each fraction was collected manually and subjected to further
analysis as described below. 3H-Labeled glycopeptides were
detected by liquid scintillation counting.
Amino Acid Sequencing and Amino Acid Analysis--
Protein spots
separated by two-dimensional gel electrophoresis were transferred to
polyvinylidene difluoride membrane, stained with Coomassie Brilliant
Blue R-250, excised, and subjected to amino acid sequencing by Edman
degradation using a PerkinElmer Life Sciences Procise Model 492 protein
sequencer. Internal amino acid sequences were obtained by sequencing
the tryptic peptides following RP-HPLC fractionation.
For amino acid analysis, 5 µg of the tryptic peptides was
vacuum-dried and hydrolyzed in the gas phase with 6 M HCl
and 1% phenol for 24 h at 110 °C. This treatment destroyed
sugar residues, but still permitted detection and quantitation of
hydroxylysine and hydroxyproline (26). Free amino acid residues were
dissolved in 40 µl of 0.025% K3EDTA, derivatized with
phenyl isothiocyanate, separated on a Spheri-5 PTC 5µ column
(220 × 2.1 mm), and analyzed with an Applied Biosystems Model 421 amino acid analyzer.
Matrix-assisted Laser Desorption Ionization Time-of-Flight Mass
Spectrometry (MALDI-TOF-MS) Analysis--
0.5 µl of the tryptic
peptide mixtures or RP-HPLC-separated peptides was mixed with an equal
amount of Cloning, Recombinant Expression, and Purification of Adiponectin
and Its Variants--
Total RNA was obtained from 3T3-L1 adipocytes
using Trizol reagent according to the manufacturer's instructions.
Reverse transcription-PCR was performed based on the mouse
adiponectin nucleotide sequence (GenBankTM/EBI
accession number U37222). Full-length adiponectin cDNA was
then inserted into the pGEMT-easy vector, and its sequence was verified
by DNA sequencing. The protein sequence was counted starting
from the methionine residue.
For prokaryotic expression, the mouse adiponectin cDNA sequence was
amplified using 5'-ATCGGGATCCGAAGATGACGTTACTACAACT-3' as
the sense primer and 5'-TACGAATTCTCAGTTGGTATCATGGTAGAG-3' as the
antisense primer. The BamHI/SalI fragment of the
amplified DNA product was subcloned into the pPROEX-HTb plasmid,
resulting in expression vector pPRO-His-Ad, which encodes full-length
adiponectin with a His6 tag at its N terminus. A similar
strategy was used for the construction of prokaryotic expression vector
pPRO-His-gAd, which expresses the His6-tagged globular
region of adiponectin (amino acid residues 110-247), except
that the sense primer used was 5'-ATCGGGATCCGCCGCTTATATGTATCGCTC-3'.
The expression of His-tagged full-length adiponectin or its globular
region in BL21 cells was induced by the addition of 1 mM of
isopropyl-
The vector for mammalian expression was generated by amplification of
adiponectin cDNA using 5'-GCCCGCGGATCCATGCTACTGTTGCAAGCTCT-3' as
the sense primer and
5'-GGCCGCGAATTCTCACTTGTCATCGTCGTCCTTGTAGTCGTTGGTATCATGGTAGAG-3' as the
antisense primer. Following digestion with
BamHI/EcoRI, the fragment was inserted into
pcDNA3.1(+) to produce expression vector pcDNA-Ad-F, which
encodes full-length adiponectin with a FLAG epitope tag at its C
terminus. This expression vector was then used as a template to
construct the vectors encoding adiponectin variants in which the four
lysines (residues 68, 71, 80, and 104) were replaced with arginines
using the QuikChange site-directed mutagenesis kit. The mutagenic
oligonucleotide primers were designed according to the criteria
recommended by the manufacturer, with a codon change from AAG to CGG
(for residues 68 and 80) or from AAA to CGA (for residues 71 and 104).
A plasmid (pcDNA-Ad(K
These mammalian expression vectors were transfected into COS-7 cells
using FuGENE 6 transfection reagent, and the cells were allowed to
secrete adiponectin into serum-free medium for 48 h. The medium
was then harvested and precipitated by incubation with 40% ammonium
sulfate overnight at 4 °C. After subsequent centrifugation at
8000 × g for 1 h, the pellets were resuspended in
Tris-buffered saline and dialyzed against the same buffer using
SnakeSkin tubing with a molecular mass cutoff of 7000 Da (Pierce).
FLAG-tagged adiponectin was purified using anti-FLAG M2 affinity gel
and eluted with 150 µg/ml FLAG peptide.
Antibody Production--
His-tagged recombinant adiponectin
produced by Escherichia coli was mixed with Freund's
complete adjuvant and then intraperitoneally injected into female
Wistar rats (50 µg/rat) or subcutaneously injected into female New
Zealand rabbits (100 µg/rabbit). The animals were boosted twice with
the same amount of protein mixed with Freund's incomplete adjuvant,
and blood was collected 1 week after the last boost.
Isolation of Primary Rat Hepatocytes and Measurement of Hepatic
Glucose Production--
Primary hepatocytes were prepared from male
Wistar rats (200 g) as described previously (27) and plated on collagen
type I-coated 12-well plates in DMEM with 10% fetal bovine serum, 2 mM L-glutamine, 10 µM
dexamethasone, and 10 µg/ml insulin at a density of 5 × 105 cells/well. The cells were allowed to adhere to
the cell culture dishes for 24 h and then incubated overnight in
DMEM with 5.5 mM glucose and no insulin or dexamethasone.
Subsequently, the cells were stimulated with different concentrations
of insulin and/or adiponectin for another 24 h. The medium was
then replaced with 0.5 ml of glucose-free DMEM without phenol red and
supplemented with 5 mM each alanine, valine, glycine,
pyruvate, and lactate. After incubation for 6 h, the glucose level
in the medium was measured using the glucose Trinder assay kit.
Adiponectin Secreted by Adipocytes Exists as Multiple
Isoforms--
Two-dimensional gel electrophoresis analysis identified
eight protein spots that were preferentially expressed and secreted by
adipocytes, and not from undifferentiated 3T3-L1 preadipocytes (Fig.
1, A and B).
N-terminal amino acid sequencing revealed that all of these proteins
(spots 1-8) share an identical N-terminal sequence
(EDDVTTTE), which unequivocally matches amino acid residues 18-25 of
mouse adiponectin, a secretory protein expressed exclusively by
adipocytes (5, 7). This sequenced fragment (EDDVTTTE) is located
immediately after the hypothetical signal peptide cleavage site,
suggesting that the heterogeneous isoforms of adiponectin are not
caused by different protease cleavage during its secretion. The
identities of these proteins as adiponectin were further confirmed by
Western blot analysis, which showed that all eight proteins were
immunoreactive to an antibody against mouse adiponectin (Fig. 1C). Two-dimensional gel electrophoresis separation of
recombinant adiponectin produced by E. coli detected only a
single spot (data not shown), suggesting that the existence of multiple
isoforms of adiponectin produced by adipocytes is due to
post-translational modification occurring during its secretion.
Two-dimensional gel electrophoresis analysis of recombinant adiponectin
transiently expressed and secreted by COS-7 cells also demonstrated
multiple isoforms of this protein, a pattern similar to that in
adipocytes (data not shown).
Carbohydrate-based detection of proteins separated by two-dimensional
gel electrophoresis revealed that six isoforms of adiponectin (spots 3-8) derived from adipocytes are glycosylated (Fig.
1D), suggesting that glycosylation may at least partly
contribute to the heterogeneity of adiponectin. Although there are two
consensus N-linked glycosylation sites (Asn53
and Asn233), treatment with tunicamycin, an inhibitor of
N-linked glycosylation (28), did not affect the
glycosylation pattern (data not shown), thus excluding the possibility
of N-linked glycosylation of adiponectin. A previous study
using endoglycosidase H treatment also suggested that no
N-glycosylation occurs on adiponectin (5). Furthermore, there were no potential serine and threonine residues predicted to be
O-glycosylated using the NetOGlyc Version 2.0 prediction server (29), which produces network predictions of mucin-type O-glycosylation sites in mammalian proteins.
Glycosylation of Adiponectin Occurs at Several Conserved Lysine
Residues in the Collagenous Domain--
To further characterize the
nature of the glycosylation and to map the glycosylation sites of
adiponectin, tryptic peptide mixtures from each isoform of adiponectin
derived from adipocytes, transiently transfected COS-7 cells, or
E. coli were analyzed by MALDI-TOF-MS. Comparison of the
mass spectra for these samples detected three prominent peptide
fragments (with masses of 1679, 4260, and 4276 Da, respectively) that
existed only in the six glycosylated isoforms, but not in the two
unglycosylated isoforms or in adiponectin produced by E. coli (Fig. 2). Moreover, the masses
of these three tryptic peptide fragments did not match any of the
unmodified tryptic fragments of adiponectin, indicating that
glycosylation of adiponectin may occur in these three fragments.
To isolate these three peptide fragments, the tryptic peptide mixtures
from all of the glycosylated isoforms were pooled and separated by
RP-HPLC, and each fraction was analyzed by MALDI-TOF-MS (Fig.
3). This analysis found that fraction A,
which eluted at 16.4% acetonitrile, contains the peptide with a mass
of 1679 Da. The peptides with masses of 4276 and 4260 Da were detected
in fractions B and C, which eluted at 18 and 18.4% acetonitrile, respectively. Amino acid sequence analysis identified the peptide with
a mass of 1679 Da as KGEPGEAAYVYR, a fragment corresponding to amino acid residues 104-115 of mouse adiponectin. The peptides with
masses of 4276 and 4260 Da were derived from the same fragment (DGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPR),
which matches amino acid residues 62-95 of adiponectin. Notably, amino acid sequence analysis easily detected all of the amino acid residues in these three peptide fragments, except for the four lysine residues (lysine 104 in the peptide with a mass of 1679 Da and lysines 68, 71, and 80 in the peptides with masses of 4260 and 4276 Da). This
result indicates that these lysine residues might be modified by
hydrophilic groups such as carbohydrates so that the hydrophilic amino
acid derivatives could not be efficiently extracted in nonpolar solvent
by conventional liquid-phase sequencing. The conclusion that these four
lysine residues are modified was further supported by the observation
that they were resistant to digestion with trypsin, a proteinase that
specifically cleaves at the C terminus of either arginine or lysine
residues. Interestingly, these four lysines (residues 68, 71, 80, and
104) are located in the collagenous domain of adiponectin, with
surrounding motifs of GXKGE(D). Sequence alignment revealed
that these four lysines and their surrounding motifs are highly
conserved across all of the available adiponectin sequences (Fig.
4).
To verify that these four lysines were modified, the three peptides
purified above were further subjected to amino acid analysis following
hydrolysis with 6 M HCl for 24 h at 110 °C. The
results showed the absence of lysine residues at the predicted
positions in the elution gradient, although all of the other amino acid residues were detected with the expected molar ratios (Fig.
5). Further analysis of these spectra
revealed that all of the lysine residues in these three peptides are
hydroxylated. A hydroxylated proline residue was also detected in
peptide B. This hydroxyproline was subsequently assigned to
Pro94 (see below).
Because hydroxylation and subsequent glycosylation of hydroxylysine to
form
The assumption that each lysine in peptides B and C has an attached
glycoside group of 340 Da was further supported by digestion of these
two peptides with endoproteinase Asp-N, which specifically cleaves at
the N terminus of asparagine. MALDI-TOF-MS analysis showed that
the experimentally observed mass for the fragment containing
Lys80 is 340 Da larger than its theoretical mass, whereas
the actual mass for the fragment containing Lys68 and
Lys71 differs from its theoretical mass by 680 Da (Fig.
6). This result also indicated that an
extra hydroxylation in peptide B occurred at proline 94. Hydroxylation
of proline 94 was also verified by amino acid analysis and amino acid
sequencing.
To further verify that the glycosides attached to the four lysine
residues are modified by glucosylgalactosyl groups, COS-7 cells
transiently expressing FLAG-tagged adiponectin were radiolabeled with
[3H]galactose or [3H]glucose. The tryptic
mixtures of radiolabeled adiponectin purified from these cell culture
media were fractionated by RP-HPLC to isolate peptides A-C as
described for Fig. 3. Liquid scintillation counting revealed that both
[3H]galactose and [3H]glucose were
incorporated into these three peptides (Fig.
7).
Substitution of the Four Lysines (Residues 68, 71, 80, and 104) in
the Collagenous Domain of Adiponectin Attenuates Its
Insulin-sensitizing Activity--
To investigate whether the
glycosylation of the four hydroxylated lysines affects the biological
activities of adiponectin, we generated a construct
(pcDNA-Ad(K
Two recent studies have demonstrated that adiponectin can enhance the
action of insulin to inhibit hepatic glucose production (9, 32).
Consistent with these reports, our results showed that insulin at a
concentration of 50 pM did not significantly affect glucose
production in primary rat hepatocytes (Fig.
9, upper panel). Half-maximal
suppression by insulin was observed at a concentration of 200 pM. The ability of subphysiological concentrations of
insulin to suppress hepatic glucose production was significantly
enhanced by adiponectin produced by mammalian cells. In the presence of
20 µg/ml adiponectin, 50 pM insulin dramatically
decreased glucose production by 40 ± 3%. A concentration dependence study revealed that the EC50 of adiponectin is
at a level of ~4 µg/ml (Fig. 9, lower panel), a
concentration within the physiological range of adiponectin (15, 16).
Compared with wild-type adiponectin, the insulin-sensitizing ability of the Lys-to-Arg adiponectin variant on hepatic gluconeogenesis was
significantly attenuated. In the presence of the adiponectin variant at
4 µg/ml, 50 pM insulin showed no significant effect on
glucose production and caused only a 13 ± 1% decrease in the presence of this protein at 20 µg/ml. Bacterially generated
full-length adiponectin (Fig. 9) and the globular region (data not
shown) were biologically ineffective in enhancing the hepatic action of
insulin to suppress gluconeogenesis.
Although several recent reports independently demonstrated an
antidiabetic role of adiponectin, it is still controversial as to which
form of adiponectin is functionally active. Studies by Lodish and
co-workers (10) and Kadowaki and co-workers (11) found that a truncated
fragment corresponding to the globular domain of adiponectin is
effective in decreasing hyperglycemia and restoring insulin resistance,
whereas bacterially produced full-length adiponectin shows no activity.
Although these findings are certainly of pharmacological interest,
their physiological relevance remains uncertain. The preponderance of
plasma adiponectin exists as a full-length protein with an apparent
molecular mass of 30 kDa (5, 6). We were unable to detect any
proteolytic fragments of adiponectin in human and mouse sera by both
immunoprecipitation and Western blot analysis of the proteins separated
by two-dimensional gel electrophoresis (data not shown). Furthermore,
amino-terminal sequence analysis revealed that all of the major
isoforms of adiponectin secreted by adipocytes share the identical N
terminus (Fig. 1), suggesting that this protein is not cleaved
intracellularly during its secretion.
Scherer and co-workers (9) demonstrated that full-length adiponectin
produced by mammalian cells can acutely decrease hyperglycemia in
several diabetic animal models, whereas either full-length adiponectin
or its globular region derived from E. coli has no such
activities. This study also provided evidence to suggest that
hepatocytes are one of the potential physiological targets for
adiponectin. In vivo administration of adiponectin
significantly decreases glucose production by down-regulating the
hepatic expression of the key gluconeogenic enzymes phosphoenolpyruvate
carboxykinase and glucose-6-phosphatase (32). In line with these
reports, we showed that full-length adiponectin produced by mammalian
cells is much more potent than bacterially produced adiponectin in
enhancing the ability of a subphysiological concentration of insulin to suppress glucose production in primary rat hepatocytes (Fig. 9). These
observations indicate that post-translational modifications of
adiponectin are functionally important, at least regarding its role as
an insulin sensitizer involved in hepatic actions.
Our two-dimensional gel electrophoresis analysis revealed that
adiponectin secreted by adipocytes is extensively modified into
multiple isoforms with different pI values and molecular masses and
that this heterogeneity can be explained at least partly by
glycosylation (Fig. 1). Comparison of the mass spectra of the unglycosylated and glycosylated isoforms allowed us to identify the
four conserved lysines (residues 68, 71, 80, and 104) in the collagenous domain as potential glycosylation sites (Fig. 2). The
conclusion that these four lysines are glycosylated was further supported by the following evidence. First, these four lysines were not
able to be identified during sequencing and were also resistant to
trypsin cleavage, indicating that they may well be modified. Second,
amino acid analysis revealed that all four lysines were hydroxylated
(Fig. 5). Third, the glycosylation of adiponectin was substantially
decreased following substitution of these four lysines with arginines
(Fig. 8) or following treatment with Hydroxylation of lysine and subsequent glycosylation with galactose and
glucose to form Mutational analysis revealed that substitution of the four conserved
lysines with arginines in the collagenous domain significantly attenuated the ability of subphysiological concentrations of
adiponectin to enhance the hepatic action of insulin to suppress
glucose production (Fig. 9). This result indicates that these four
lysines in the collagenous domain are critically involved in the
insulin-sensitizing action of adiponectin and also suggests that
hydroxylation or the glycosides attached to these residues might be
functionally important. The mechanisms by which these four
hydroxylysine residues or their attached glycosides in the collagenous
domain enhance the insulin-sensitizing effect of adiponectin remain to
be defined. The insulin-sensitizing ability of the adiponectin variants
in which only one of the four lysine residues was replaced with
arginine was much lower than that of wild-type adiponectin, but
significantly higher than that of the variant with mutations at all
four sites (data not shown), suggesting that the hydroxylysines or the
attached glycosides might function in a cooperative manner.
Hydroxylation and glycosylation might be critical for the
three-dimensional structure required for the full biological activity
of the adiponectin molecule. The lack of hydroxylation and/or
glycosylation might destabilize the collagen-like stalk and thus
interfere with the formation of high order complexes. It is also
possible that hydroxyl or carbohydrate groups directly participate in
ligand-receptor interaction. These possibilities are currently under
investigation in our laboratory.
In summary, this study demonstrates that the four lysines (residues 68, 71, 80, and 104) in the collagenous domain of adiponectin are critical
for its insulin-sensitizing activity with respect to inhibition of
hepatic glucose production. These four lysine residues were found to be
hydroxylated and glycosylated, thus emphasizing the important role of
post-translational modifications in the biological activities of
adiponectin. The existence of multiple isoforms of adiponectin secreted
by adipocytes (Fig. 1) implies that there might be other
post-translational modifications that could also be functionally
relevant. Indeed, a recent study suggested the presence of disialic
acid residues as modifying groups in adiponectin (33). Further
characterization of other post-translational modifications occurring in
adiponectin will shed new light on the molecular mechanisms underlying
its biological activities.
We thank Drs. Christina Buchanan, Bernard
Choong, and David Crossman for technical assistance.
*
This work was supported by grants from the Health Research
Council of New Zealand, the New Zealand Lotteries Commission, the Endocore Research Trust, and the Foundation for Research Science and
Technology.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: School of Biological
Sciences, Level 4, Thomas Bldg., University of Auckland, Private Bag
92019, Auckland, 1001 New Zealand. Tel.: 64-9-3737599 (ext.
6265); Fax: 64-9-3737414; E-mail: a.xu@auckland.ac.nz.
Published, JBC Papers in Press, March 23, 2002, DOI 10.1074/jbc.M200601200
The abbreviations used are:
DMEM, Dulbecco's
modified Eagle's medium;
RP-HPLC, reversed-phase high performance
liquid chromatography;
MALDI-TOF-MS, matrix-assisted laser desorption
ionization time-of-flight mass spectrometry.
Hydroxylation and Glycosylation of the Four Conserved Lysine
Residues in the Collagenous Domain of Adiponectin
POTENTIAL ROLE IN THE MODULATION OF ITS INSULIN-SENSITIZING
ACTIVITY*
,§,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and
resistin, have been shown to play critical roles in the regulation of
systemic energy homeostasis, and altered expression and/or secretion of
these adipocytokines may contribute to the causation of insulin
resistance, type II diabetes, and its complications such as
cardiovascular diseases.
agonist thiazolidinedione in several insulin-resistant animal models
and human patients significantly increases the plasma concentration of
adiponectin (21, 22). Thus, adiponectin correlates well with the
insulin-sensitive state, and its absence is associated with insulin
resistance, dyslipidemia, and hyperglycemia.
-oxidation of fatty acid in muscle and
thus decreasing muscular triglyceride content.
, even though there is no homology at the primary
sequence level (23). The molecular mechanisms underlying the metabolic
functions of adiponectin are largely unknown. Bacterially produced
full-length recombinant adiponectin is inactive in restoring insulin
sensitivity as well as in decreasing hyperglycemia (9, 11), indicating
that post-translational modification might be critical for the
insulin-sensitizing actions of adiponectin. Here, we have shown that
adiponectin secreted by adipocytes exists in multiple glycosylated
isoforms. We also mapped the glycosylation sites to several
conserved lysines (residues 68, 71, 80, and 104) that are located in
the collagenous domain of adiponectin, with surrounding consensus
sequences of GXKGE(D). These lysine residues are
hydroxylated and subsequently glycosylated. Furthermore, the important
role of this hydroxylysyl glycosylation was confirmed by mutational
analysis, which showed that substitution of these lysine residues with
arginine significantly attenuated the ability of adiponectin to enhance
insulin's inhibitory actions on hepatic glucose production.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamic acid, collagenase, rat tail collagen type
I, amino acid standards, FLAG peptide, anti-FLAG M2 affinity gel, and
the glucose Trinder assay kit were purchased from Sigma. Human insulin
(Actrapid) was obtained from Novo Nordisk. The total cellular
RNA extraction reagent (Trizol), tobacco etch virus (TEV)
protease, mammalian expression vector pcDNA3.1(+), and prokaryotic
expression vector pPROEX-HTb were from Invitrogen. The QuikChange
site-directed mutagenesis kit was from Stratagene. BCA protein assay
reagent was from Pierce. The Immu-Blot kit for glycoprotein
detection was from Bio-Rad. FuGENE 6 transfection reagent, trypsin,
Asp-N endoproteinases, and the enhanced chemiluminescence detection system (ECL) were from Roche Molecular Biochemicals. The
Ni2+-nitrilotriacetic acid-agarose column was from QIAGEN
Inc. All consumables for two-dimensional gel electrophoresis,
[1-3H]galactose, and [1-3H]glucose were the
products of Amersham Biosciences. All amino acid analysis reagents and
Cal Mix 2 calibration standards for mass spectrometer were from
Applied Biosystems.
80 °C until used.
-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in 60%
acetonitrile and 0.3% trifluoroacetic acid), spotted onto the sample
plates, and air-dried. Reflectron mass spectrometric analyses
were performed on a Voyager DE PRO biospectrometry workstation (Applied
Biosystems) using a pulsed laser beam (nitrogen laser, 
= 337 nm). All ion spectra were recorded in the positive mode with an
accelerating voltage of 20.0 kV. The spectrometer was externally
calibrated using Cal Mix 2 standard mixture.
-D-thiogalactopyranoside to the growth
medium. Full-length adiponectin or its globular region was purified
from the bacterial lysates using the
Ni2+-nitrilotriacetic acid-agarose column according to
the manufacturer's instructions. Following purification, the
N-terminal tag was removed by cleavage with recombinant TEV protease.
The purity of the protein was confirmed by SDS-PAGE and HPLC.
R)-F) encoding a FLAG-tagged adiponectin
variant with all four lysine residues substituted with arginines
was obtained by sequential mutation of each site, and all mutations
were confirmed by DNA sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Endogenous adiponectin secreted by 3T3-L1
adipocytes exists as eight isoforms. The medium from
subconfluent 3T3-L1 preadipocytes (A) or adipocytes at day 8 after induction of differentiation (B) was collected and
concentrated as described under "Experimental Procedures." 50 µg
of proteins from each sample was separated by two-dimensional gel
electrophoresis and visualized by silver staining. The proteins
preferentially secreted by adipocytes are denoted by numbered
arrows. Secretory proteins from adipocytes separated by
two-dimensional gel electrophoresis as described above were detected
using rabbit anti-adiponectin antibody (C) or the Immu-Blot
carbohydrate detection kit (D). Note that all eight
indicated proteins are immunoreactive with anti-adiponectin antibody.
Six of the eight isoforms (spots 3-8) of adiponectin are
glycosylated.
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Fig. 2.
MALDI-TOF mass spectra of tryptic peptide
mixtures derived from different isoforms of adiponectin. Isoforms
1 (A) and 3 (B) of adiponectin secreted by
adipocytes were in-gel digested with trypsin, and the tryptic mixtures
were analyzed by MALDI-TOF-MS. Note that the three peptides (with
masses of 1679, 4260, and 4276 Da) denoted by arrows were
reproducibly observed in all of the glycosylated isoforms (isoforms
3-8) produced by both adipocytes and COS-7 cells, and not in the two
unglycosylated isoforms (isoforms 1 and 2) or the bacterially produced
adiponectin.
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Fig. 3.
Fractionation and characterization of the
tryptic peptides of adiponectin by RP-HPLC, MALDI-TOF-MS, and amino
acid sequencing. All of the glycosylated isoforms of adiponectin
separated by two-dimensional gel electrophoresis were excised from the
gels, pooled, and digested with trypsin. The tryptic peptide mixture
was separated by RP-HPLC. Each fraction was collected and analyzed by
MALDI-TOF-MS. The three fractions containing peptides with masses of
1679, 4276, and 4260 Da are denoted as A-C, respectively.
The table shows the amino acid sequences, the experimentally
observed masses, the theoretical masses, and the mass differences for
these three peptides.
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Fig. 4.
The four modified lysines (residues 68, 71, 80, and 104) in the collagenous domain of adiponectin are conserved
across all of the species investigated. The mouse, human, bovine,
monkey, and dog adiponectin sequences relate to
GenBankTM/EBI accession numbers BAB22597, NP_004788,
AAK58902, AAK92202, and AAL09702, respectively. The four modified
lysines and their surrounding motifs are shaded.
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Fig. 5.
Amino acid analysis of the three tryptic
peptides separated in Fig. 3. Peptide A (with a mass of
1679 Da), peptide B (with a mass of 4276 Da), and peptide C (with a
mass of 4260 Da) were digested with 6 M HCl at 110 °C
for 24 h and subjected to amino acid analysis as described under
"Experimental Procedures." Trace a, amino
acid standard; trace b, hydroxyproline standard; trace
c, hydroxylysine standard; trace d, hydrolysate of
peptide A; trace e, hydrolysate of peptide B; trace
f, hydrolysate of peptide C. The arrows and
star indicate the peaks of hydroxylysine (OH-K)
and hydroxyproline (OH-P), respectively.
-1,2-glucosylgalactosyl-O-hydroxylysine have previously been described in several secretory proteins with
collagen-like domains (30, 31), we speculate that the same type of
modification may occur on the four lysines (residues 68, 71, 80, and
104) in the three tryptic peptides isolated above. This speculation was supported by analysis of the MALDI-TOF-MS data for these three peptides (Fig. 3). For peptide A, the difference between the
experimentally observed mass (1679 Da) and the theoretical mass (1339 Da) is 340 Da, which is exactly the mass of a
glucosylgalactosylhydroxyl group. The experimentally observed mass for
peptide C differs from its predicted mass by 1020 Da, an expected mass
for three glucosylgalactosylhydroxyl groups that may attach to the
three lysines (residues 68, 71, and 80) in peptide C. The
experimentally observed mass of peptide B (4276 Da) differs from its
theoretical mass by 1036 Da, which is the expected size for three
glucosylgalactosylhydroxyl groups plus another hydroxyl group.
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Fig. 6.
MALDI-TOF mass spectra of peptide mixtures
from Asp-N-digested peptides B and C. Peptides B (panel
I) and C (panel II) separated in Fig. 3 were further
digested with Asp-N and then analyzed by MALDI-TOF-MS. The peptide
sequences and the potential modifications assigned to each mass are
indicated above each peak. Note that the assignment of
Pro94 as hydroxylated proline was also confirmed by amino
acid analysis. GG, glucosylgalactosyl.
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Fig. 7.
Evidence that glycosides attached to the four
hydroxylysines contain glucosyl and galactosyl groups. COS-7 cells
were transfected with pcDNA-Ad-F and then radiolabeled with 100 µCi/ml [1-3H]galactose in DMEM containing 2 mM glucose for 48 h or with 100 µCi/ml
[1-3H]glucose in DMEM containing 2 mM
galactose for 48 h. FLAG-tagged adiponectin was purified from the
cell culture medium, and tryptic peptide mixtures from unlabeled or
radiolabeled adiponectin were separated by RP-HPLC as described in the
legend to Fig. 3 to obtain peptides A-C. The fractions containing each
peptide were subjected to liquid scintillation counting.
R)-F) encoding an adiponectin variant in which the
four lysine residues have been replaced with arginines. SDS-PAGE
analysis revealed that wild-type adiponectin secreted by COS-7 cells
migrated as three bands with slightly different molecular masses (Fig.
8). The upper two bands, which accounted
for ~85% of the total adiponectin, were glycosylated. In contrast,
the Lys-to-Arg adiponectin variant consisted mainly of a single
unglycosylated band that migrated with a lower apparent molecular mass
than the two major glycosylated bands of wide-type adiponectin. This
result further confirms that glycosylation of adiponectin mainly occurs
at the four lysine residues in the collagenous domain.
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Fig. 8.
Expression and carbohydrate detection of the
FLAG-tagged Lys-to-Arg adiponectin variant. COS-7 cells were
transfected with pcDNA-Ad-F or pcDNA-Ad(K R)-F. 48 h
later, FLAG-tagged adiponectin or the Lys-to-Arg adiponectin variant
was purified from the cell culture medium. 1 µg of protein from each
sample was separated by 15% SDS-PAGE and stained with Coomassie
Brilliant Blue R-250 (A) or detected with the Immu-Blot
glycoprotein detection kit (B). Note that the majority of
glycosylation was abolished in the Lys-to-Arg adiponectin
variant.
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Fig. 9.
Effect of adiponectin and adiponectin
variants on insulin-induced inhibition of glucose production in primary
rat hepatocytes. Upper panel, inhibition of hepatic
glucose production following treatment with increasing amounts of
insulin in the absence or presence of 20 µg/ml adiponectin
(Ad) or Lys-to-Arg adiponectin variant (Ad
variant) generated from COS-7 cells or 20 µg/ml bacterially
produced adiponectin (pAd); lower panel,
inhibition of hepatic glucose production following treatment with 50 pM insulin plus increasing amounts of adiponectin or the
Lys-to-Arg adiponectin variant generated from COS-7 cells or
bacterially produced adiponectin. The results are represented as
decreased percentage of glucose production relative to the untreated
cells and as means ± S.D. (n = 4).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,'-dipyridyl, a hydroxylase
inhibitor (data not shown). Notably, hydroxylysyl glycosylation of
these four sites was detected in all six major glycosylated isoforms,
which account for >85% of the total adiponectin secreted by
adipocytes, suggesting that glycosylation is one of the major
post-translational modifications occurring in adiponectin.
-1,2-glucosylgalactosyl-O-hydroxylysine have been previously observed in several secretory proteins with collagen-like domains, including complement component C1q and pulmonary
surfactant proteins (30). Although the exact carbohydrate composition
remains to be further defined, we have obtained evidence suggesting
that the glycosides attached to the four lysines are possibly
glucosylgalactosyl groups. Mass spectrometric analysis indicated that
the mass of the glycoside group at lysines 80 and 104 is 340 Da, an
expected size for a glucosylgalactosylhydroxyl residue (Fig. 6).
Radiolabeling experiments also revealed that the glycosides contain
both [3H]galactose and [3H]glucose (Fig.
7).
ACKNOWLEDGEMENTS
FOOTNOTES
Both authors contributed equally to this work.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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