From the Department of Biostructure and Function, School of Dental
Medicine, University of Connecticut Health Center, Farmington,
Connecticut 06030-3705
Each globular domain of exported multiglobular
proteins putatively undergoes chaperone surveillance in the endoplasmic
reticulum lumen. It is difficult to visualize how surveillance of
multiple globular domains might be orchestrated and regulated. Aggrecan core protein has been used as a prototype for this problem by examining
transfection of informative constructs into Chinese hamster ovary
cells. The salient results are as follows: 1) aggrecan's N-terminal G1
domain is minimally secreted, and its flanking Golgi reporter sites are
not decorated with glycsoaminoglycan chains; in contrast, its
C-terminal G3 domain is readily secreted with flanking GAG chains, and
G3 also facilitates G1 secretion; 2) G3 but not G1 can be
intracellularly cross-linked to chaperone Hsp25; 3) G3 and Hsp25 remain
noncovalently bound and are secreted together when G3 is situated
N-terminal to its normal location; 4) exon 15, which encodes the center
of G3's C-lectin subdomain, is necessary and sufficient for G3
secretion. A model is proposed in which Hsp25 piggybacks onto nascent
G3 in the cytosol during a translocational pause and enters the ER
lumen with G3, and once G3 properly folds, Hsp25 releases G3 and
recycles to the nucleus while G3 continues to the Golgi stacks,
providing passage for the entire core protein.
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INTRODUCTION |
Globular proteins that are destined for export or for transit to
the cell surface putatively undergo surveillance by molecular chaperones in the lumen of the endoplasmic reticulum (1, 2). This
quality control mechanism ensures that globular proteins have achieved
proper conformation prior to their progression in the routing process
from the endoplasmic reticulum to the Golgi stacks. In the case of
proteins with two or more globular domains, each individual domain is
potentially subject to chaperone surveillance. It is difficult to
visualize, for an individual molecule, how surveillance of multiple
globular domains might be orchestrated and regulated.
Insight into this problem of chaperone surveillance of multiglobular
proteins may be achieved using aggrecan as a model system. The aggrecan
family of secreted proteoglycans has various members with similar
molecular features (3). The N-terminal globular G1 domains of the
family members are highly conserved, are virtually identical, and are
lectins that bind hyaluronic acid, producing aggregates in
situ (4). The C-terminal globular G3 domains are also highly
conserved, are virtually identical, and contain C-lectin domains that
bind monosaccharides (5) and also bind tenascin-R by calcium-mediated,
non-lectin interactions (6). Thus, the C-lectin domain has at least one
site for protein recognition and binding.
The importance of the G3 domain for aggrecan secretion is exemplified
by the nanomelic mutation in which mutant aggrecan core protein,
lacking G3, fails to leave the cell and accumulates in the lumen of the
ER (7). The mutant protein is truncated due to a premature stop codon
and lacks both G3 and a portion of the adjacent chondroitin sulfate
consensus region (8). Transfection with various constructs has shown
that it is G3, and not G1 or the chondroitin sulfate consensus domain,
that is critical for aggrecan core protein intracellular trafficking
and aggrecan secretion (9). The present study was designed to further
examine this role of G3 in modulating aggrecan core protein
intracellular routing.
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EXPERIMENTAL PROCEDURES |
Construct Assembly (Figs. 1 and 2)--
The G1-glycosaminoglycan
(GAG)1-G3 miniaggrecan
construct contains the endogenous avian aggrecan signal peptide (SP),
endogenous avian N-terminal G1 domain, endogenous glycosaminoglycan
consensus sites, endogenous avian C-terminal G3 domain, and a
six-histidine (His6) tag and is abbreviated
SP-G1-GAG-G3-His6. SP-G1 was amplified by polymerase chain
reaction (PCR) using oligonucleotide 203 (Table I) plus oligonucleotide 201. The GAG-G3
sequence was amplified by PCR using oligonucleotide 202 plus
oligonucleotide 104, which contains His6 and a stop codon.
PCR products were reamplified by overlapping PCR using oligonucleotides
203 and 104 to obtain full-length SP-G1-GAG-G3-His6, which
was digested with SrfI and ligated to predigested pCR-Script
SK(+) vector (Stratagene), which was then used to transform
Escherichia coli XL1-blue MRF' Kan supercompetent cells.
E. coli colonies were screened, and suitable inserts were
released by BamHI and XhoI and then ligated into corresponding sites of the pcDNA3 vector (Invitrogen).
A contralateral G1 construct contains SP, endogenous GAG, N-terminal G1
domain, and His6 and is abbreviated
SP-GAG-G1-His6. SP-GAG was amplified by PCR using
oligonucleotide 203 plus oligonucleotide 302. G1 was amplified by PCR
using oligonucleotide 303 plus oligonucleotide 304, which contains
His6 and a stop codon. The PCR products were digested with
XhoI and then ligated into pCR-Script SK(+) and redigested
with BamHI and XbaI and ligated into the
pcDNA3 vector.
A contralateral G3 construct contains SP, endogenous C-terminal G3
domain, GAG, and His6 and is abbreviated
SP-G3-GAG-His6. SP was amplified by PCR using
oligonucleotide 101 plus oligonucleotide 117. G3 was amplified by PCR
using oligonucleotide 116 plus 119. SP-G3 was then reamplified by
overlapping PCR using oligonucleotides 101 and 119. GAG was amplified
by PCR using oligonucleotide 118 plus oligonucleotide 111, which
contains His6 and a stop codon. SP-G3-GAG-His6
was then reamplified by overlapping PCR using oligonucleotides 101 and
111. The final PCR products were digested with HindIII and
XhoI and ligated into the pcDNA3 vector.
G3 and G1 constructs were produced as described by Luo et
al. (9).
Exon combination constructs were made with relevant oligonucleotides
(Table II) as follows. The P1 construct
was amplified by PCR with the SP-G1-GAG-G3 construct as a template
using oligonucleotides 203 and 401, which contains His6 and
a stop codon. The P2 construct was amplified by PCR with the
SP-G1-GAG-G3 construct as a template using oligonucleotides 203 and
402, which contains His6 and a stop codon. The P3 construct
was amplified by PCR with the SP-G1-GAG-G3 construct as a template
using oligonucleotides 203 and 403, which contains His6 and
a stop codon. The P1, P2, and P3 constructs were individually digested
with BamHI and XhoI and then ligated into
pcDNA3 vector.
For the P4 construct, SP-G1 sequence was amplified by PCR using
oligonucleotides 203 and 409. These PCR products were digested with
BamHI and EcoRI and then ligated into a
pcDNA3 vector. The GAG consensus sequence was amplified by PCR
using oligonucleotides 408 and 404. The exon 15 and 16 sequence was
amplified by PCR using oligonucleotides 405 and 401. The two PCR
products were purified and then reamplified by overlapping PCR using
oligonucleotides 408 and 401. The final products were digested with
EcoRI and XhoI and then ligated into the
pcDNA3-SP-G1 vector.
For the P5 construct, the GAG consensus sequence was amplified as for
P4. Exon 15 was amplified by PCR using oligonucleotides 405 and 402. The GAG PCR and exon 15 PCR products were purified and then reamplified
by overlapping PCR using oligonucleotides 408 and 402. The final
products were digested with EcoRI and XhoI and
then ligated into the pcDNA3-SP-G1 vector.
For the P6 construct, the GAG consensus sequence was amplified by PCR
using oligonucleotides 408 and 406. Exon 16 sequence was amplified by
PCR using oligonucleotides 407 and 401. The two PCR products were
purified and then reamplified by overlapping PCR using oligonucleotides
408 and 401. The final PCR products were digested with EcoRI
and XhoI and ligated into the pcDNA3-SP-G1 vector.
All PCR products were directly cloned into pCR-Script vector
(Stratagene) following the vendor's instructions and then digested with suitable enzymes prior to ligating them into a pcDNA3 vector. All constructs were separately transformed into E. coli
strain TOP10F' (Invitrogen) and amplified; the plasmid DNA was purified by Qiafilter Maxiprep (Qiagen) and sequenced by a university facility on a fee for service basis. Only constructs showing sequence identity to the desired gene sequences were used for transfection; any mutant
constructs were discarded.
Cell Culture and Transfection--
Chinese hamster ovary (CHO)
cells were grown in minimal essential medium culture medium (Life
Technologies, Inc.) supplemented with 5% fetal bovine serum and
antibiotics (Life Technologies, Inc.; 100 units/ml penicillin, 100 µg/ml streptomycin, 250 ng/ml amphotericin) at 37 °C in humidified
air with 5% CO2. 1.8 million cells were seeded per 100-mm
culture dish and cultured for 36 h prior to transfection. 15 µg
of DNA, 52.5 µl of LipofectAMINE reagent (Life Technologies, Inc.)
were each added to 500 µl of Opti-MEM (Life Technologies, Inc.) and
mixed for 30 min. Concurrently, a dish of cells was twice washed with
Opti-MEM and then cultured with 5 ml of Opti-MEM. The DNA/LipofectAMINE
mixture was then added to the cells, which were incubated for 6 h.
In Situ Cross-linking--
Labeled cells were washed twice with
ice-cold 130 mM NaCl, 20 mM Bicine, pH 8.0;
lysed in ice-cold detergent plus cross-linker (50 mM
Bicine, pH 8.0, 150 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.2% digitonin with 1.5 mM DTSSP, Pierce); and incubated on ice for 30 min. Excess
cross-linker was inactivated with 10 mM glycine for 10 min
on ice. The lysates were processed by a chelation chromatography method, described below. The cross-linked samples were electrophoresed in SDS-polyacrylamide gel electrophoresis in the presence of
dithiothreitol, which cleaves an internal disulfide bond in DTSSP,
releasing cross-linked proteins from each other.
Radioactive Labeling and Chelation Chromatography--
After
transfection, the medium was aspirated. For
35SO4 labeling, 5 ml of fresh Opti-MEM and 250 µl of [35S]Na2SO4 (NEN Life
Science Products) were added to the cells, which were incubated for
16 h. For [35S]methionine labeling, 5 ml of
methionine- and cysteine-free Opti-MEM medium and 125 µl of
35S-protein labeling mix (Amersham Pharmacia Biotech) were
added to the cells, which were incubated for 16 h. The medium was
collected, and protease inhibitors were added to final concentrations
of 1 mM PMSF, 2 mg/ml leupeptin, 0.4 mg/ml antipain, 2 mg/ml benzamidine, 2 mg/ml aprotinin, 1 mg/ml chymostatin, 1 mg/ml
pepstatin. Debris was removed by centrifuging at 1000 rpm for 1 min.
900 µl of medium was adjusted to 0.5% Triton X-100 concentration and
added to 50 µl of nickel resin (Qiagen) for 1 h. Resin had been
prepared by equilibration with 0.1 M NaCl, 44 mM NaHCO3, 1 mM imidazole, 0.5% Triton X-100. Sample-containing resin was spun at 14,000 × g for 15 s, and the liquid was decanted. Resin was
washed three times with buffer A (10 mM HEPES, 2 M NaCl, 10% glycerol, 0.1 mM PMSF, 2 mM imidazole, 0.5% Triton X-100, 6 M urea, 250 mM dithiothreitol, pH 8.0) for 30 min and then buffer B (10 mM HEPES, 750 mM NaCl, 10% glycerol, 0.1 mM PMSF, 10 mM imidazole, 0.5% Triton X-100, 1 mg/ml bovine serum albumin, pH 8.0) for 30 min, and buffer C (10 mM HEPES, 750 mM NaCl, 10% glycerol, 0.1 mM PMSF, 10 mM imidazole, pH 8.0) for 15 min
and eluted with 10 mM HEPES, 750 mM NaCl, 10% glycerol, 100 mM EDTA, 0.1 mM PMSF, 250 mM imidazole, pH 8.0, for 1 h. Cells were washed twice
with Hanks' medium (Life Technologies, Inc.) and lysed with 1 ml
of 0.5% Nonidet P-40 at 0 °C for 10 min. The lysate was spun at
14,000 × g for 5 min. The clear lysate was transferred
to a new tube. 200 µl of cell extract plus 700 µl of Opti-MEM were
adjusted to 0.5% Triton X-100 concentration and added to 50 µl of
nickel resin prepared as above. The resin was washed three times with
buffer A for 30 min, buffer B for 30 min, and buffer C for 15 min and
then eluted as described above.
Immunocapture--
Cell lysates or spent media were incubated
with antibody to Hsp25 (Stressgen) and protein A beads (Pierce) on a
rotating table at ice temperature for 5 h, following which the
beads were harvested by centrifugation and serially washed with 50 mM Tris-HCl, pH 7.5, containing 1 M NaCl and
1% Triton X-100 to elute nonspecific proteins. Then antibody-bound
proteins were released by boiling the beads in sample buffer prior to
SDS-polyacrylamide gel electrophoresis.
SDS-Polyacrylamide Gel Electrophoresis and
Autoradiography--
The eluates or digested products were
electrophoresed in a 5-15% gradient SDS-polyacrylamide gel
electrophoresis gel, which was passed through an Instant Imager
(Packard) and then dried and exposed to x-ray film.
Radioactivity Quantitation--
Samples were collected at
various times after adding radioactive isotope and electrophoresed.
Radioactive protein bands were measured using an Instant Imager
(Packard); G1 and G3 signals were corrected by subtracting background
from cell lysates obtained after simultaneous transfection with control
(no insert) vectors. Samples loaded onto gels represent aliqout
portions of total cell lysate or medium. Total amounts of G1 and G3 in
each sample were calculated by normalizing to the initial volume of the
harvested samples. The secretion of G1 and G3 proteins is expressed as
the total amount of G1 or G3 in the medium divided by the sum of the total amount of G1 or G3 in the medium plus cell lysate.
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RESULTS |
This study was designed to further analyze how domain G3 governs
the progression of aggrecan core protein through the intracellular routing pathway from the site of core protein synthesis in the ER to
aggrecan's release at the cell surface. Various constructs were
produced (Figs. 1 and
2) and individually transfected into CHO
cells. Fig. 3 shows that the construct
synthesized as a "miniaggrecan" core protein containing G1, GAG
consensus sites and G3 was secreted at a rate intermediate between
analogous proteins containing G1 or G3 alone. As described previously
(9), the small amount of secreted G1 protein did not contain detectable
GAG chains (data not shown), whereas secreted G3 protein or just GAG
consensus sequences alone become neoproteoglycans (Fig.
3d).

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Fig. 1.
Diagram of aggrecan organization compared
with G1 and G3 domain organization in various constructs. Signal
peptide, G1, GAG consensus sites, and G3 are the chicken endogenous
sequences. His6 is a histidine hexapeptide. Five tandem
consensus sequences for GAG chain addition are present in the
constructs, as described previously (9). The "reverse" G1 and G3
amino acid sequences are identical to G1 and G3, respectively, except
that they have been placed opposite their natural loci relative to the
GAG consensus sites. All constructs were sequenced to determine
fidelity prior to insertion into a eukaryotic expression vector.
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Fig. 2.
Diagram of constructs designed to test the
putative role of aggrecan's C-lectin domain in neoproteoglycan
formation and secretion. Various sequences are identified in Fig.
1. The complement regulatory protein-like domain is synonymous with the
short consensus repeat and sushi domains. The exons coding for their
respective domains are depicted. All constructs were sequenced to
determine fidelity prior to insertion into a eukaryotic expression
vector.
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Fig. 3.
G1, G3, and G1 + G3 (miniaggrecan) protein
secretion, labeled by [35S]methionine and secretion of
neoproteoglycans, labeled by
[35S]Na2SO4. a,
[35S]methionine label; spent media; autoradiography of
empty vector proteins (lane 1), G1 protein (lane
2), G3 protein (lane 3), and G1 + G3 protein
(lane 4). The arrows depict the locations of the
secreted proteins. b, cell lysates (same samples as in
panel a). c, percentage of each protein secreted
by cells. Cells were labeled for 16 h in these experiments.
d, [35S]Na2SO4 label;
spent media; autoradiography of empty vector proteins (lane
1), SP-GAG5-G3-HIS6 protein (lane 2), and
SP-GAG5-His6 protein (lane 3).
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Globular proteins destined for secretion or targeted to the plasma
membrane generally interact as nascent chains with ER intraluminal molecular chaperones; G1 and G3 domains would be expected to behave in
a similar fashion. Pilot experiments showed that the molecular chaperone, Hsp25 (10, 11), appeared to interact with miniaggrecan core
protein, and these observations were expanded. Fig.
4 shows that Hsp25 is present in CHO
cells transfected with an empty vector or with a vector containing G1
or G3 constructs. Fig. 5a
demonstrates that intracellular Hsp25 can be cross-linked to G3 protein
but not to G1 protein; Fig. 5b shows that Hsp25 does not
appear in the spent media in these experiments. As noted under
"Experimental Procedures," electrophoresis of the cross-linked
proteins in the presence of dithiothreitol cleaves an internal
disulfide bond in the DTSSP cross-link, liberating cross-linked
proteins, in this case Hsp25 and G3.

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Fig. 4.
The presence of Hsp25 in cell lysates of CHO
cells, labeled by [35S]methionine. Cell lysates were
immunoprecipitated with nonspecific antibody (Ab, ) or
anti-Hsp25 antibody (Ab, +). Empty vector transfection
(lanes 1 and 2), G1 construct transfection
(lanes 3 and 4), and G3 construct transfection
(lanes 5 and 6) are shown. Cells were labeled for
6 h in these experiments.
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Fig. 5.
Cross-linking of Hsp25 to intracellular G3
but not G1. a, cell lysates labeled by
[35S]methionine for 6 h and cross-linked with DTSSP;
autoradiography of empty vector proteins (lane 1), G1
protein (lane 2), and G3 protein (lane 3). The
arrows depict the locations of the intracellular proteins.
b, spent media (same samples as in panel a).
Samples were electrophoresed in the presence of dithiothreitol, which
cleaves an internal disulfide bond in the DTSSP cross-link, releasing
the cross-linked proteins.
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The molecular positions of the G1 domain and the G3 domain are
invariant throughout the members of the aggrecan family; G1 is always
N-terminal to G3, implying that the locations of these globular domains
are significant. Suitable constructs were transfected to test this
hypothesis. Fig. 6 shows that locating G1
on the opposite flank of GAG consensus sites, in a construct termed
RG1, reduces secretion compared with G1 construct; the small amounts of
G1 and RG1 proteins in the media are not decorated with GAG chains
(data not shown). Fig. 7 shows, in
contrast, that locating G3 on the opposite flank of GAG consensus
sites, in a construct termed RG3, does not affect the secretion rate
and that G3 and RG3 proteins each become neoproteoglycans. It is
noteworthy that, without chemical cross-linking, secreted RG3 protein
is bound to Hsp25 (Fig. 7a) and that these two proteins
remain firmly bound during chelation chromatography plus stringent
washes followed by immunoprecipitation of the chromatographic eluate
plus stringent washes (Fig. 8).

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Fig. 6.
G1 and RG1 protein secretion, labeled by
[35S]methionine. a, spent media;
autoradiography of empty vector proteins (lane 1), G1
protein (lane 2), and RG1 protein (lane 3). The
arrow depicts the location of the secreted proteins.
b, cell lysates (same samples as in panel a).
c, percentage of each protein secreted by cells. Cells were
labeled for 16 h in these experiments.
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Fig. 7.
G3 and RG3 protein secretion, labeled by
[35S]methionine, and secretion of neoproteoglycans,
labeled by [35S]Na2SO4.
a, spent media, methionine-labeled proteins; autoradiography
of empty vector proteins (lane 1), G3 protein (lane
2), and RG3 protein (lane 3). The arrows
depict the location of the secreted proteins. Note the 25-kDa protein
in the media of the RG3 sample. b, cell lysates (same
samples as panel a). c, percentage of each
protein secreted by cells. d, spent media, sulfate-labeled
proteoglycans; autoradiography of empty vector proteins (lane
1), G3 protein (lane 2), and RG3 protein (lane
3).
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Fig. 8.
Identification of secreted Hsp25 complexed to
RG3 protein. Spent media were passed through chelation
chromatography to retain RG3, followed by stringent washes and elution
and then immunocapture of the eluate with antibody versus
Hsp25, plus protein A beads, and then by stringent washes.
Autoradiography of empty vector proteins or G3 protein (lane
1) and RG3 protein (lane 2) is shown. Positions of
Hsp25 and RG3 are depicted.
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Exon-deleted constructs were used to determine which part(s) of domain
G3 might underlie its ability to enhance intracellular routing and
secretion of aggrecan-related proteins. Miniaggrecan core proteins
lacking G3's epidermal growth factor domain (exon 13) or its sushi
domain (exons 17-18) were fully functional (data not shown); only the
C-lectin domain (exons 14-16) is needed for effective GAG chain
O-glycosylation (Fig. 9) and
miniaggrecan secretion (Fig. 10).
Within the C-lectin domain, exon 15 accounts for virtually all of G3's
properties (Figs. 9 and 10); exons 14 and 16 contribute little to exon
15's properties, but each, by itself, suppresses those properties.
Interestingly, two separate proteins originate from the exon 15 construct (Fig. 10a, lane 7), and two different
populations of neoproteoglycans seem to arise from those proteins (Fig.
9, lane 7).

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Fig. 9.
Secretion of neoproteoglycans, labeled by
[35S]Na2SO4. Secretion of
neoproteoglycans containing exon-based deletions; autoradiography of
empty vector proteins (lane 1),
SP-G1-GAG-G3-His6 or miniaggrecan protein (lane
2) and proteins P1 (lane 3), P2 (lane 4), P3
(lane 5), P4 (lane 6), P5 (lane 7),
and P6 (lane 8). One bracket depicts the
neoproteoglycan based on miniaggrecan protein while the other
bracket depicts the neoproteoglycan based on protein P1 or
P5.
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Fig. 10.
Miniaggrecan protein and exon-deleted
miniaggrecan secretion, labeled by [35S]methionine.
a, spent media; autoradiography of empty vector proteins
(lane 1), miniaggrecan protein (lane 2), P1
protein (lane 3), P2 protein (lane 4), P3 protein
(lane 5), P4 protein (lane 6), P5 protein
(lane 7), and P6 protein (lane 8). b,
cell lysates (same samples as in panel a). c,
percentage of each protein secreted by cells. Cells were labeled for
6 h in these experiments.
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DISCUSSION |
Secreted proteins with multiple globular domains, e.g.
members of the aggrecan family, would be expected to undergo
chaperone-mediated surveillance in the ER lumen. It is difficult to
visualize, however, how multidomain surveillance might be orchestrated
and regulated for an individual molecule. Various possibilities include
separate chaperones for each globular domain (1) and/or
"intramolecular" chaperones (12).
The experimental results in this study confirm earlier work that
proteins bearing domain G3 are secreted more rapidly than those
containing domain G1 (9). Interestingly, a G1 plus G3-containing protein is secreted at a rate intermediate between a protein bearing G1
alone and one bearing G3 alone. These observations suggest that G3
facilitates secretion of G1 and, consequently, of aggrecan core protein
itself.
Aggrecan Globular Domains--
Members of the aggrecan family
contain G1 at the N-terminal flank of the GAG consensus sites and G3 at
their C-terminal flank. Reversing these orientations decreases the
secretory rate of G1 protein but does not affect the G3 protein
secretory rate, suggesting that both individual globular conformation
and globular location are important determinants contributing to
intracellular passage of G1/G3-containing proteins.
The three-dimensional structure of rat mannose-binding protein has been
deciphered from its crystalline form (13); the highly conserved
secondary structure of its C-lectin domain is prevalent in a wide
variety of otherwise unrelated proteins (14). Using the atomic
coordinates of rat mannose-binding protein, molecular modeling has
yielded homologous secondary structure (Figs.
11 and 12) of the C-lectin domain of mouse IgE
cell surface receptor (15); this model appears prototypical of the
secondary structure of all the related C-lectin domains (16) and is
reflected in the primary structure as well. For example, comparison of
the amino acid sequences of mouse IGE receptor C-lectin domain and
chicken aggrecan C-lectin domain shows 38% conservation (Fig.
13); the other proteins in this
superfamily generally show 20-40% conservation of secondary
structure, compared with the mouse IGE receptor C-lectin domain (14).
Recent expansion and further refinement of these analyses shows that a
superfamily of 131 secretory or cell surface proteins has a consensus
sequence of 136 residues, with 32% of them being conserved (17); one
of the two calcium binding sites is partially encoded by exon 15, and
the remainder is encoded by exon 16 in the avian domain.

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Fig. 11.
Ribbon representation of the C-lectin domain
of mouse IgE receptor, a prototypical member of a superfamily of 131 different proteins, including chicken aggrecan core protein. This
conceptual model (15) was based on the known structure of rat
mannose-binding protein, a member of the superfamily. The
color-coded sections represent the equivalent regions
encoded by chicken aggrecan exons. The graphics programs
Swiss-PdbViewer (available on the World Wide Web at
expasy.hcuge.ch/spdbv/mainpage.html) and POV-Ray (available at
www.povray.org/) were used to create this figure.
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Fig. 12.
Space-filling representation of the C-lectin
domain of mouse IgE receptor. This figure was created
in the same way as Fig. 11 except that the space-filling mode of
representation was substituted for the ribbon mode.
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Fig. 13.
Sequence comparison of the C-lectin domains
of mouse IgE receptor and chicken aggrecan. These two domains are
members of a superfamily of 131 different proteins that are either on
the cell surface or are secreted (17). Residues with greater than 50%
identity and similarity are shaded in black and
gray, respectively. Alignment was performed by the Genetics
Computer Group program CLUSTALW, and the results are expressed in
PileUp format (available at www.ibc.wustl.edu/msa/clustal_old.cgi). The
segmented horizontal line below the sequences
indicates the tandem order of chicken exons 14-16.
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The C-lectin domain of G3 has recently been shown to bind tenascin R
via calcium-mediated protein-protein interaction (6). Assuming that it
is exon 15 protein that binds tenascin R, it may also be the site that
binds Hsp25. The environment of the ER lumen provides ideal
circumstances for such interactions because it has high concentrations
of calcium, molecular chaperones, and nascent proteins (18, 19). The
protein encoded by exon 15 has two prominent lobules, which we
speculate may, in their nascent state, be the actual binding site for
Hsp25. Once the lobules are formed, Hsp25 may dissociate, enabling the
lobules to interact elsewhere. As proposed below, once Hsp25 is
released, the G3 lobules may bind to the nascent G1 region of core
proteins, acting as an "intramolecular" chaperone. The failure of
Hsp25 to dissociate from RG3 may potentially provide insight into the
recognition mechanisms between that molecular chaperone and nascent
aggrecan core proteins.
Conservation of the C-lectin domain structure and its widespread
occurrence as a common unit of many exported and cell surface proteins
imply that it is an important recognition signal for ER quality control
mechanisms. In some cases, the C-lectin domain structure may be
expendable once the parent protein reaches its final destination. This
may, in fact, be the case for extracellular aggrecan, which
progressively loses its entire G3 domain (20).
Molecular Chaperone Hsp25--
The small heat shock proteins,
including Hsp25 (11, 21), are well conserved in prokaryotes, animals,
and plants, sharing internal domains of about 35 amino acids within the
carboxyl-terminal half of the proteins (22, 23). To date, plant cells
show the most diverse intracellular distribution of small heat shock
proteins, which are distributed in cytoplasmic classes I and II, in
chloroplast class III, and in the ER lumen as class IV (24). This
latter class was found most recently, and the authors suggest that it may have gone unobserved in other eukaryotes. Although three
alternatively spliced Hsp25 isoforms are seen in cultured murine
osteoblasts and their relative expression is controlled by two separate
promoter elements, none of these isoforms has an N-terminal signal
sequence (25).
One possibility is that cytosolic Hsp25 may bind to nascent G3 during a
translocational pause; such pauses have been linked to potential
nascent chain interactions with cytosolic components (26, 27). Pause
sites have been mapped in nascent apolipoprotein B-100 but do not show
linear consensus sequences (27); the authors invoke topogenic consensus
sequences, conceptually analogous to chaperone topogenic recognition
sequences.
G3, but not G1, interacts with molecular chaperone Hsp25; such
specificity is consistent with chaperone quality control mechanisms in
the ER lumen and trafficking of properly folded proteins. This point is
emphasized by the unexpected observation that Hsp25 and RG3 protein
remain associated in a complex that is released from the cell,
suggesting that intracellular Hsp25 fails to dissociate once RG3
has achieved proper conformation. It is also noteworthy that 1)
persistent binding of Hsp25 does not prevent GAG chain addition to
consensus sites in RG3 protein and 2) the precise molecular location of
the G3 domain in a protein has a profound effect on Hsp25
interactions.
Scheme for Hsp25-G3 Interactions--
Fig.
14 illustrates one possible scheme that
is consistent with current results. Nascent domain G3 initially binds
cytosolic Hsp25 during a translocational pause, bringing Hsp25 into the ER lumen and preventing G3 aggregation, thereby enabling intraluminal G3 to adopt its correct conformation. Intraluminal Hsp25 then dissociates from properly folded G3, which in turn serves as an intramolecular chaperone and binds to G1, enabling G1 to progressively fold into proper conformation. G3 could, of course, interact with G1 in
the same nascent molecule and/or with G1 in adjacent nascent molecules,
similar to schemes proposed for other intramolecular chaperones (28).
Meanwhile, released Hsp25 is probably recycled to the nucleus,
consistent with its intranuclear localization in stressed cells (29);
it is generally recognized that stress reactions simply emphasize
preexisting physiological pathways.

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Fig. 14.
Schematic depicting a postulated chaperone
pathway for intracellular aggrecan core protein. Aggrecan core
protein nascent chains in the ER lumen can either coalesce into
nonproductive complexes or can interact with the Hsp25 molecular
chaperone, which recognizes the G3 domain. The interaction leads to
perfection of G3 structure, dissociation of Hsp25, and G3's subsequent
interaction with G1 domain, either in the same nascent aggrecan chain
("intramolecular") or with nearby nascent aggrecan chains
("intermolecular"). G3/G1 interaction leads to an improved G1
structure, followed by aggrecan core protein entry into the Golgi
stacks where GAG chains are added and finished aggrecan is released
into the extracellular compartment. As described in the text, cytosolic
Hsp25 may initially bind translated G3 during a translocational pause
and then enter the ER lumen piggybacked to G3, release properly folded
G3, and recycle via the nucleus.
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Although G3 enhances G1 routing through the cell, G1 appears to be
immature when it leaves the cell, denoted by its initial weak avidity
for hyaluronic acid. That avidity progressively increases once aggrecan
is in the extracellular matrix (30), apparently due to G1 achieving
correct disulfide bond arrangements (31). Thus, while G3 enables G1 to
traverse the intracellular quality control pathway, such passage is
insufficient for G1 to perfect its final conformation. Extracellular G1
gradually matures, eventually fulfilling its major role in
participating in an extended network with hyaluronic acid and link
proteins. Simultaneously, extracellular aggrecan progressively loses
its G3 domain (20), consistent with a predominantly intracellular
role.
Ramifications of Hsp25 Expression and Regulation--
Recent
research has implicated Hsp25 as being protective against oxidative
stress and subsequent apoptosis (32). Constitutive expression of human
Hsp25 in murine cells blocks receptor-initiated cell death (33); Hsp25
levels are developmentally regulated and have been linked to cell
growth and differentiation (34). Hsp25 transient expression is critical
for preventing differentiating ES cells from undergoing apoptosis, by a
switch that may be redox-regulated (35). Redox levels in the ER lumen
are maintained by inflow of cysteine and glutathione (36); enhanced
synthesis of secretory and membrane proteins stimulates such thiol
influx to counteract the vectorial import of reduced cysteine residues
that enter the ER lumen as part of nascent proteins. The precise
relationships among Hsp25 regulation, cellular redox levels, and
apoptosis have not been elucidated.
Hsp25 is constitutively expressed in a wide variety of vertebrate
tissues (37). Of particular interest is the localization of this
molecular chaperone to the upper part of the hypertrophic zone of the
rat tibial growth plate (38). This region is a site of active synthesis
of cartilage matrix components including aggrecan. Growth plate
hypertrophic chondrocytes progressively move toward bone, successively
becoming apoptotic (39-41) Thus, down-regulation of Hsp25 in
hypertrophic chondrocytes correlates with diminished synthesis of
aggrecan and switching to an apoptotic pathway. Chondrocytes in
postnatal cartilage, a relatively anoxic environment, may be especially
sensitive to such a redox-regulated switch. This point is reflected by
chondrocytes in culture, since thiol levels in the culture medium are
found to be critical in modulating events linked to chondrocyte
maturation and cartilage matrix synthesis (42).
In summary, molecular chaperone Hsp25 has been implicated in the
intracellular trafficking of aggrecan core protein, and, most likely,
Hsp25 participates in a quality control system that determines whether
the core protein will leave the ER lumen to enter the Golgi stacks.
Hsp25 interacts with the C-lectin motif of domain G3; strikingly, Hsp25
occurs throughout eukaryotes, and, concomitantly, the C-lectin motif is
seen in an extensive variety of cell surface and secreted eukaryotic
proteins, implying that an evolutionary partnership between the
molecular chaperone and the protein motif has been very well
conserved.
We thank Dr. Barbara Vertel for stimulating
discussions about these studies.