THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 270, No. 45, Issue of November 10, pp. 26746 26749, 1995
1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.

Communication
A Novel Serum Protein Similar
to C1q, Produced Exclusively in
Adipocytes*
(Received for publication, July 25, 1995, and in revised form,
September 12, 1995)
Philipp E. Scherer, Suzanne Williams,
Michael Fogliano, Giulia Baldinii,
and Harvey F. Lodish**

We describe a novel 30-kDa secretory protein, Acrp30

(adipocyte complement-related protein of 30 kDa), that
is made exclusively in adipocytes and whose mRNA is
induced over 100-fold during adipocyte differentiation.
Acrp30 is structurally similar to complement factor C1q
and to a hibernation-specific protein isolated from the
plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced
by insulin, and Acrp30 is an abundant serum protein.
Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving
food intake and carbohydrate and lipid catabolism. Our
experiments also further corroborate the existence of an
insulin-regulated secretory pathway in adipocytes.

Insulin-induced glucose transport occurs in heart, striated

muscle, and fat tissue. In adipocytes, glucose uptake increases
20- to 30-fold in the presence of insulin. Glucose transport is
mediated by the sodium-independent facilitative glucose transporters GLUT1 and GLUT4, which, in response to insulin,
translocate from an intracellular compartment to the plasma
membrane (1, 2). GLUT4, which is expressed only in fat and
skeletal and cardiac muscle, is the primary transporter involved in this process and is the predominant transporter expressed in these tissues (3, 4). Insulin also causes translocation
of several receptor proteins from intracellular membranes to
the plasma membrane (57). Adipocytes are a principal storage
depot for triglycerides and express a specific transport protein
allowing them to import free fatty acids (8).
Adipocytes also secrete several proteins potentially important in homeostatic control of glucose and lipid metabolism.
Adipsin, equivalent to Factor D of the alternative complement
* This work was supported in part by National Institutes of Health
Grant DK 47618 and a grant from Pfizer Corp. (to H. F. L.). The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship from the Swiss National Science
Foundation.
i Supported by Fellowship 301033 from the Juvenile Diabetes Foundation. Present address: Dept. of Anatomy and Physiology, College of
Physicians and Surgeons 12-404, 630W 168th St., Columbia University,
New York, NY 10032.
To whom correspondence should be addressed. Tel.: 617-258-5216;
Fax: 617-258-9872; E-mail: lodish@wi.mit.edu.

EXPERIMENTAL PROCEDURES

Cloning of Acrp30 and DNA AnalysisA full-length cDNA library

templated by mRNA from 3T3-L1 adipocytes at day 8 of differentiation
(15) was screened with a digoxygenin-labeled cDNA fragment obtained
from the random sequencing screen. Labeling, hybridization, and detection were performed according to the manufacturers instructions
(Boehringer Mannheim). One of the positive clones obtained was subjected to automated sequencing on a Applied Biosystems 373-A Sequencer. The entire 1.3-kb insert was sequenced at least 2 independent
times on one and once on the complementary strand. Sequence analysis
was performed with the DNAstar package and showed an open reading
frame of 741 bp encoding a protein of 28 kDa. The sequence has been
submitted to GenBankTM and has the accession number U37222. Homology searches were performed at NCBI using the BLAST network
service, and alignments were performed with the Megalign program
from DNAstar using the Clustal algorithm. Only the globular domain
for the type X collagen was used for the alignment (residues 562 680).
Generation of Specific AntibodiesA peptide corresponding to the
sequence, EDDVTTTEELAPALV (residues 18 32), was used to generate specific anti-Acrp30 antibodies in rabbits (multiple antigen peptide
technology, Research Genetics).
mRNA Isolation and AnalysisIsolation of mRNA from tissues and
from 3T3-L1 cells at various stages of differentiation was as described
in Ref. 15, as was 32P labeling of DNA, agarose gel electrophoresis of
mRNA, and its transfer to nylon membranes.
Pulse-Chase Experiments and Immunoprecipitations3T3-L1 adipocytes were starved for 30 min in Dulbeccos modified Eagles medium
(ICN) lacking cysteine and methionine and then labeled for 10 min in
the same medium containing 0.5 mCi/ml Express Protein Labeling
Reagent (1000 Ci/mmol) (DuPont NEN). The cells were then washed
twice with Dulbeccos modified Eagles medium supplemented with
unlabeled cysteine and methionine, and then fresh growth medium
containing 300 mM cycloheximide was added. At the indicated time
points, the medium was collected. Insoluble material from the medium
was removed by centrifugation (15,000 3 g for 10 min); the supernatants were precleared with 50 ml of Protein A-Sepharose for 30 min at
4 C and then immunoprecipitated with 50 ml of affinity-purified antiAcrp30 antibody for 2 h at 4 C. Immunoprecipitates were washed 4
times in lysis buffer (1% Triton X-100, 60 mM octyl glucoside, 150 mM
NaCl, 20 mM Tris, pH 8.0, 2 mM EDTA, 1 mM phenylmethylsulfonyl
1
The abbreviations used are: Acrp30, adipocyte complement-related
protein of 30 kDa; Endo H, endoglycosidase H; BS3, bis(sulfosuccinimidyl)suberate; kb, kilobase(s); bp, base pair(s); PBS, phosphate-buffered
saline; PAGE, polyacrylamide gel electrophoresis.

26746

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From the Whitehead Institute for Biomedical Research,

Cambridge, Massachusetts 02142-1479, Pfizer Central
Research, Groton, Connecticut 06340, and the
**Department of Biology, Massachusetts Institute of
Technology, Cambridge Massachusetts 02139

pathway (9), is synthesized exclusively in adipocytes, and its

secretion is enhanced severalfold by insulin (10). The function
of adipsin in fat cell biology is not known, nor are the roles of
complement factors C3 and B that are also secreted by adipocytes (11). Tumor necrosis factor a, also secreted by adipocytes,
is a key mediator of insulin resistance in animal models of
non-insulin-dependent diabetes mellitus. Tumor necrosis factor a directly interferes with the signaling of insulin through
its receptor and consequently blocks biological actions of insulin including insulin-stimulated glucose uptake (reviewed in
Ref. 12). Adipocytes are the only cell type known to secrete the
ob gene product (13). In the absence of ob (ob2/ob2 mice) or its
presumed receptor, the db gene product (db2/db2 mice) the
mice overeat and become obese and diabetic (14). Here we
describe another novel protein, Acrp30,1 that is exclusively
synthesized in adipose tissue and secreted into serum. Like
adipsin, secretion of Acrp30 is enhanced severalfold by insulin.
While we do not know the function of this protein, its sequence
and structural resemblance to complement factor C1q is intriguing. Importantly, our experiments confirm the existence of
an insulin-regulated secretory pathway in adipocytes.

Acrp30, a Novel Serum Protein Similar to C1q

26747

fluoride, and 2 mg/ml leupeptin).

Cross-linking of Acrp30 A 10-cm plate of day 8 3T3-L1 adipocytes
was labeled overnight with [35S]methionine and cysteine as described
above. The medium was collected and, by means of several spins in a
Centricon 10 microconcentrator, the buffer was replaced with 150 mM
NaCl, 50 mM KPi, pH 8.5. A stock solution of 200 mg/ml bis(sulfosuccinimidyl)suberate (BS3, Pierce) in dimethyl sulfoxide was prepared
fresh and added to the final concentrations indicated in the figure
legends. Reactions were allowed to proceed for 30 min on ice, and excess
cross-linker was quenched by addition of 500 mM Tris buffer, pH 8.0.
Samples were diluted 1:1 with lysis buffer and subjected to immunoprecipitation with anti-Acrp30 antibodies.
Other MethodsSeparation of proteins by SDS-PAGE, fluorography,
immunoblotting, protein determinations, and densitometric scanning of
the gels were performed as described previously (16).
RESULTS

In order to identify novel adipocyte-specific proteins, we have

randomly sequenced portions of 1000 clones from a subtractive
cDNA library enriched in mRNAs induced during adipocyte
differentiation of 3T3-L1 fibroblasts (15). Northern blot analysis using one ;250-bp clone showed a marked induction during
adipocyte differentiation, and thus a full-length cDNA was
isolated and sequenced. The encoded protein, Acrp30, was
novel; it contained 247 amino acids with a predicted molecular
mass of 28 kDa. Acrp30 consists of a predicted amino-terminal
signal sequence, followed by a stretch of 27 amino acids that

does not show significant homology to any protein in the data

base and then by 22 perfect Gly-X-Pro or Gly-X-X repeats (Fig.
1, A and B). The carboxyl-terminal globular domain exhibits
striking homology to a number of proteins, such as the globular
domains of type VIII and type X collagens (17), the subunits of
complement factor C1q (18) and a protein found in the serum of
hibernating animals during the summer months (19). Structurally, albeit not at the primary sequence level, the protein
resembles the lung surfactant protein (20) and the hepatocyte
mannan-binding protein (21), both of which have collagen-like
domains and globular domains of similar size.
Northern blot analysis shows that Acrp30 is expressed exclusively in adipocytes. It is not expressed in 3T3-L1 fibroblasts and is
induced over 100-fold during adipocyte differentiation. Induction
occurs between days 2 and 4, at the same time as other adipocytespecific proteins such as GLUT4 (22) and Rab3D (15) (Fig. 1C).
These results were confirmed by Western blot analysis (data not
shown). The amount of Acrp30 mRNA may decline somewhat from
Day 6 to Day 8 (Fig. 1C), but this drop is not reproducible in other
experiments. In any case, we have not studied the accumulation or
stability of Acrp30 mRNA after Day 8.
An antibody raised against a peptide corresponding to the
unique amino-terminal domain of Acrp30 recognized a 3T3-L1
adipocyte protein of approximately 28 kDa (not shown). Acrp30

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FIG. 1. A, predicted structure of Acrp30. The protein consists of an amino-terminal signal sequence (SS) followed by a sequence of 27 amino acids
lacking significant homologies to any entries in the GenBankTM data base. This region is followed by a stretch of 22 collagen repeats with 7 perfect
Gly-X-Pro repeats (dark hatched boxes) clustered at the beginning and end of the domain interspersed with 15 imperfect Gly-X-Y repeats (light
hatched boxes). The C-terminal 137 amino acids probably form a globular domain. B, alignment of the amino acid sequences of Acrp30. Hib27, a
member of the hibernation-specific protein family; C1q-C, one of the subunits of complement C1q; Coll type X, the globular domain of the type X
collagen. Conserved residues are shaded. For simplicity, the other members of each family are not shown, but shaded conserved residues are in
most instances conserved within each protein family. Only the globular domain for the type X collagen was used for the alignment (residues
562 693). C, Northern blot analysis of Acrp30 expression. The left panel shows poly(A) RNA isolated from various tissues probed with the
full-length Acrp30 cDNA. The predominant Acrp30 mRNA is 1.4 kb and is expressed only in adipose tissue and cultured 3T3-L1 adipocytes.
Overexposure of the autoradiogram does not reveal expression in any other tissue. The right panel shows induction of the Acrp30 message during
differentiation of 3T3-L1 fibroblasts to adipocytes. Induction of Acrp30 occurs primarily between days 2 and 4 of differentiation, the same time as
induction of the insulin receptor and the insulin-responsive glucose transporter GLUT4. Numbers on the left indicate molecular mass standards
(in kb). Equal loading of RNA was documented by probing the stripped filter with a cDNA encoding the cytosolic hsp70 protein (lower panel).

26748

Acrp30, a Novel Serum Protein Similar to C1q

FIG. 2. Acrp30 is a secretory protein found in blood. Acrp30 can

be detected by Western blotting in serum from mice; the antibody does
not cross-react with calf, human, or rabbit serum. One microliter of fetal
calf, rabbit, mouse, and human serum was boiled for 5 min in 2 3
sample buffer and analyzed by SDS-PAGE and Western blotting with
the anti-Acrp30 antibody according to standard protocols. Antibody was
visualized with an anti-rabbit IgG antibody coupled to horseradish
peroxidase using a chemiluminesence kit from DuPont NEN.

FIG. 3. A, insulin stimulation of Acrp30 and adipsin secretion by

3T3-L1 adipocytes. Two 10-cm dishes of day 8 3T3-L1 adipocytes were
labeled for 10 min in medium containing [35S]methionine and cysteine
as described under Experimental Procedures. The cells were then
incubated in growth medium containing cycloheximide (to prevent further protein biosynthesis) and containing or lacking 100 nM insulin.
Every 30 min, the culture medium was removed and replaced with
fresh, prewarmed medium containing or lacking 100 nM insulin. The
media were subjected to sequential immunoprecipitations with antiAcrp30 and anti-adipsin antibodies as described under Experimental
Procedures and analyzed by electrophoresis through a 12% polyacrylamide gel containing SDS. As Acrp30 and adipsin contain a comparable
number of cysteine and methionine residues (7 and 9, respectively) and
equal exposures of the autoradiograms were used, one can determine
from the intensities of the bands that approximately equal amounts of
the two proteins are secreted. As judged by the amount of 35S-labeled
proteins remaining in the cells after the 2-h chase (not shown), all of the
35
S-labeled adipsin and about 40% of the 35S-labeled Acrp30 has been
secreted at this time. B, quantitation of Acrp30 and adipsin secretion by
3T3-L1 adipocytes in the presence (closed circles) and absence (open
circles) of insulin. The autoradiograms were scanned in a Molecular
Dynamics densitometer, and the cumulative amount secreted at each
time point was plotted. The amount of each protein secreted after 120
min in the presence of insulin was taken as 100%.

Fig. 4 show that Acrp30 has a similar oligomeric structure, but

is composed of a single type of polypeptide chain. When analyzed by velocity gradient sedimentation analysis, Acrp30 in
blood serum migrates as two species of apparent molecular
masses of 90 kDa and 300 kDa (Fig. 4C). Disregarding the
presumably nonglobular shape of the complex that could lead
to a slight distortion of the molecular weight determination,
the former is probably a trimer and the latter could be a
nonamer or dodecamer. Isoelectric focusing followed by SDSPAGE of 35S-Acrp30 secreted by 3T3-L1 adipocytes reveals
only a single polypeptide (not shown), suggesting that Acrp30
forms homo-oligomeric structures. Chemical cross-linking using low concentrations of BS3 of 35S medium from 3T3-L1
adipocytes, followed by specific immunoprecipitation and SDSPAGE under reducing conditions, shows mainly dimers and
trimers (lanes 1- 5, Fig. 4A). Larger concentrations of the BS3
cross-linking agent generated Acrp30 proteins that migrated as
hexamers as well as yet larger species. As extensively crosslinked proteins migrate aberrantly upon SDS-PAGE, it is difficult to determine the exact size of the high molecular mass
form indicated by an asterisk. It could represent either a
nonamer or a dodecameric structure. Together, panels A and C
of Fig. 4 show that Acrp30 forms homotrimers that interact
together to generate nonamers or dodecamers. Nonreducing
SDS-PAGE reveals that two of the subunits in a trimer are

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contains one potential N-glycosylation site, within the collagen

domain, but apparently is not glycosylated; Endo H treatment
did not cause a shift in molecular mass of Acrp30 at any time
during a metabolic pulse-chase experiment (not shown).
Acrp30 does become modified post-translationally, since, after
20 min of chase, there was a small but reproducible reduction
in gel mobility. This most likely represents hydroxylation of
collagen domain proline residues in the endoplasmic reticulum
or Golgi compartments, by analogy to a similar modification in
the structurally related mannan-binding protein (23). In
3T3-L1 adipocytes unstimulated by insulin, 50% of newly made
Acrp30 is secreted into the medium at 2.5 to 3 h of chase as
judged by densitometric scanning of the immunoprecipitates of
intracellular and extracellular 35S-labeled Acrp30. Indeed,
Acrp30 can be detected by Western blotting in normal mouse
serum. A protein of the identical molecular weight can be
detected by Western blot analysis of 3T3-L1 adipocytes (not
shown). The anti-peptide antibody is specific for the mouse
homologue, as it does not cross-react with bovine, human, or
rabbit serum (Fig. 2).
To examine effects of insulin on Acrp30 secretion, we monitored the discrete population of newly made protein generated
in a short pulse with labeled amino acids followed by inhibition
of further protein synthesis by cycloheximide. This offers increased sensitivity compared to examining secretion of the
entire cellular complement of Acrp30, particularly in light of
the very long t12 for secretion of Acrp30. Fig. 3 shows that,
during the first 60 min of chase, insulin causes a 4-fold increase
in secretion of newly made Acrp30. After 60 min, the rates of
Acrp30 secretion are the same in unstimulated and insulinstimulated cells. Similarly, insulin causes a 4-fold increase in
adipsin secretion during the first 30 min of chase, but, afterwards, the rate of adipsin secretion is the same in control and
insulin-treated cells (Fig. 3 and Ref. 10). The ability of insulin
to abolish the lag in adipsin secretion has been seen in several
separate experiments. We hypothesize that a fraction of newly
made adipsin and Acrp30 are sorted, probably in the transGolgi reticulum, into regulated secretory vesicles whose exocytosis is induced (in an unknown manner) by insulin, whereas
the balance is sorted into vesicles that are constitutively exocytosed. Partial sorting of protein hormones into regulated
secretory vesicles has been seen in other types of cultured cells
(24, 25). We do not know how insulin causes an increase in
protein secretion; insulin could cause a more efficient overall
processing of secretory proteins in 3T3-L1 adipocytes. We are
currently isolating other adipocyte-specific secretory proteins
to study this process in detail.
Complement factor C1q consists of three related polypeptides that form heterotrimeric subunits containing a threestranded collagen tail and three globular heads; six of these
subunits generate an 18-mer complex often referred to as a
bouquet of flowers (reviewed in Ref. 26). The experiments in

Acrp30, a Novel Serum Protein Similar to C1q

disulfide-bonded together (Fig. 4B), similar to other proteins

containing a collagen domain, including the macrophage scavenger receptor (27). Besides being a homo-oligomer, Acrp30
differs from C1q in containing an uninterrupted stretch of 22
perfect Gly-X-X repeats; this suggests that Acrp30 has a
straight collagen stalk as opposed to the characteristic kinked
collagen domain in C1q caused by imperfect Gly-X-X repeats in
two of the three subunits (26).
DISCUSSION

We do not yet know the function of Acrp30. However, its

expression exclusively in adipocytes, its enhanced secretion by
insulin, and its presence in normal serum, suggests that it is,
like the ob protein, involved in the control of the nutritional
status of the organism. Acrp30 is a relatively abundant serum
protein, accounting for up to 0.05% of total serum protein as

judged by quantitative Western blotting using recombinant

Acrp30 as a standard (data not shown). Even though we have
no evidence at this stage, we cannot exclude the possibility that
Acrp30, like C3 complement released by adipocytes (28), is
converted proteolytically to a bioactive molecule.
Our experiments also corroborate the existence of a regulated
secretory pathway in adipocytes. We do not yet know whether
adipsin and/or Acrp30 are in the same intracellular vesicles that
contain GLUT4 and that fuse with the plasma membrane in response to insulin, or whether they are in different types of vesicles.
Adipocytes express two members of the Rab3 family, Rab3A and
Rab3D (29); these are found in vesicles of different density. Rab3s
are small GTP-binding proteins involved in regulated exocytic
events. Except for adipocytes, Rab3A is found only in neuronal and
neuroendocrine cells; in neurons, Rab3A is localized to synaptic
vesicles and is important for their targeting to the plasma membrane (30). An attractive hypothesis under test is that, in adipocytes, Rab3A is localized to vesicles containing Acrp30 and/or adipsin, and that possibly Rab3D mediates insulin-triggered
exocytosis of vesicles containing GLUT4. In any case, the mechanism of signal transduction from the insulin receptor to regulated
exocytosis of intracellular vesicles remains an important unsolved
problem.
AcknowledgmentsWe thank Drs. Janice Chin, Andy Swick, Mike
Gibbs, and Walt Soeller for their help at several stages of this project,
Drs. Monty Krieger and Barbara Kahn for helpful discussions, Dr.
Bruce Spiegelman for anti-adipsin antibodies and Dr. Peter Murray for
the hsp70 cDNA.
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FIG. 4. A, incubation of 35S-labeled 3T3-L1 culture supernatant with

increasing amounts of the BS3 cross-linking reagent, followed by immunoprecipitation with Acrp30-specific antibodies, reveals a set of
cross-linked products whose molecular sizes are multiples of 30 kDa.
Predominant species are trimers, hexamers, and a high molecular mass
species (asterisk) that could correspond to a nonamer or a dodecamer. In
the lane Total, 1% of the amount of cell medium used for the crosslinking reactions was analyzed on the same gel; a comparison of the
Total lane and lane 1 demonstrates the specificity of the antibody used
for immunoprecipitation. Immunoprecipitates were analyzed by gradient SDS-PAGE (712.5% acrylamide) followed by fluorography. Rainbow markers (Amersham) together with a phosphorylase b ladder (Sigma) were used as molecular mass markers. B, reducing and
nonreducing SDS-PAGE of anti-Acrp30 immunocomplexes isolated
from 35S-labeled 3T3-L1 medium. Medium from day 8 3T3-L1 adipocytes labeled overnight with [35S]methionine and cysteine was immunoprecipitated with anti-Acrp30 antibodies as described under Experimental Procedures. The sample was subjected to SDS-PAGE (712.5%
acrylamide gradient) in the presence (reducing) or absence (nonreducing) of 50 mM dithiothreitol. Labeled proteins were detected by fluorography. C, velocity gradient centrifugation of mouse serum displays two
discrete Acrp30-immunoreactive species. The smallest corresponds to a
trimer of Acrp30 polypeptides and the larger a nonamer or dodecamer.
One microliter of mouse serum was diluted with 50 ml of PBS and
layered on top of a 4.5-ml linear 520% sucrose gradient in PBS and
centrifuged for 10 h at 60,000 rpm in a SW60 rotor of a Beckman
ultracentrifuge. Thirteen 340-ml fractions were collected from the top
and analyzed by SDS-PAGE and Western blotting using anti-Acrp30
antibodies. An identical gradient was run in parallel with a set of
molecular mass standards: cytochrome c (14 kDa), carbonic anhydrase
(29 kDa), bovine serum albumin (68 kDa), alcohol dehydrogenase (150
kDa), b-amylase (200 kDa), and apoferritin (443 kDa). The positions of
these markers are indicated below the panel with arrowheads.

26749

A Novel Serum Protein Similar to C1q, Produced Exclusively in Adipocytes

Philipp E. Scherer, Suzanne Williams, Michael Fogliano, Giulia Baldini and Harvey F.
Lodish
J. Biol. Chem. 1995, 270:26746-26749.
doi: 10.1074/jbc.270.45.26746

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