From the Diabetes Research and Training Center and the Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, November 26, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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In single-chain insulins (SCIs), the C
terminus of the insulin B-chain is contiguous with the N terminus of
the A-chain, connected by a short bioengineered linker sequence. SCIs
have been proposed to offer potential benefit for gene therapy of
diabetes (Lee, H. C., Kim, S. J., Kim, K. S., Shin,
H. C., and Yoon, J. W. (2000) Nature 408, 483-488) yet relatively little is known about their folding,
intracellular transport, or secretion from mammalian cells. Because
SCIs can be considered as mutant proinsulin (with selective shortening
of the 35-amino acid connecting peptide that normally includes two sets
of flanking dibasic residues), they offer insights into understanding
the role of the connecting peptide in insulin biosynthesis. Herein
we have explored the relationship of the linker sequence to SCI
biosynthesis, folding, and intracellular transport in transiently
transfected HEK293 or Chinese hamster ovary cells or in stably
transfected AtT20 cells. Despite previous reports that direct linkage
of B- and A-chains produces a structure isomorphous with authentic
two-chain insulin, we find that constructs with short linkers tend to
be synthesized at lower levels, with a significant fraction of
molecules exhibiting improper disulfide bonding. Nevertheless,
disulfide-mispaired isoforms from a number of different SCI constructs
are secreted. While this suggests that a novel folded state goes
unrecognized by secretory pathway quality control, we find that
misfolded SCIs are detected at higher levels in Chinese hamster ovary
cells with artificially activated unfolded protein response mediated by
inducible overexpression of active ATF-6. Such a maneuver allows
analysis of more seriously misfolded mutants with further
foreshortening of the linker sequence or loss (by mutation) of the
insulin interchain disulfide bonds.
Insulin, a peptide hormone involved in carbohydrate and lipid
metabolism, consists of a 30-residue B-chain and 21-residue A-chain.
Although information contained in the sequence of these chains is
sufficient for formation of the native molecule (1, 2), insulin is
normally synthesized in pancreatic Single-chain insulins (SCIs) are insulin analogs in which the C-peptide
of proinsulin has been replaced by an artificial linker peptide whose
length and sequence is controlled by bioengineering. Unlike the
direct linkage construct noted above, some SCI constructs clearly have
potent biological activity on insulin receptors (9), making these
constructs of potential interest for gene therapy of type 1 diabetes
where a replacement insulin gene might encode a product that does not
require endoproteolytic activation for expression in non- In the course of recent studies on the role of B-chain point mutations
in insulin trafficking in the secretory pathway (12), we were surprised
to discover that H10D, a point mutation that is known to improve the
thermodynamic stability of insulin, nevertheless causes insulin
disulfide bond mispairing in the ER of mammalian (and yeast) cells,
underscoring differences in disulfide bond formation in
vitro and in vivo (13, 14). In the current study, we
have expanded our analysis to re-examine the role of the linker peptide
on insulin biosynthesis. The evidence to be presented suggests that,
contrary to previous beliefs, there is a requirement for a linker
peptide in order for insulin to form all three native disulfide bonds
in mammalian cells in vivo. As the presence of these
disulfide bonds is required for full insulin potency (15, 16), such an
investigation provides new guidance for the design of SCIs for gene
therapy of diabetes and also sheds light on the role of the C-peptide
in insulin biosynthesis.
Antibodies and Other Materials--
Polyclonal
anti-insulin was made in guinea pigs (Linco Research, St. Charles, MO).
Secondary antibodies and peroxidase conjugates were from Jackson
ImmunoLaboratories (West Grove, PA); Zysorbin was from Zymed
Laboratories Inc., and protein A-agarose was from Sigma.
[35S]cysteine and [35S]methionine/cysteine
mixture (Expre35S35S) were purchased from New
England Nuclear (New Bedford, MA). Methionine/cysteine-deficient mammalian cell culture media, brefeldin A, and stock chemicals were from Sigma.
Linker Sequence Mutagenesis--
The pRSV-InsDC plasmid
(encoding a preproinsulin mutant in which the B-chain is directly
contiguous with the A-chain, lacking any C-peptide) was obtained from
Dr. H. P. Moore (University of California, Berkeley, CA), and this
was subcloned to pcDNA3 and was used as double-stranded DNA
template. All of the artificial linker sequences or other point
mutations were prepared by PCR mutagenesis using the four-primer
method. In brief one primer pair encodes a restriction site at one end
and the desired mutation sequence at the other end. The second primer
pair contains the desired mutation sequence encoded on the reverse
orientation primer (21-24-base overlap in sequence with the first
pair) and encodes a second restriction site at the opposite end. The
PCR products of the first two rounds of PCR were then used as template
for a final PCR reaction using the external flanking primers encoding the restriction sites, and this product was subcloned initially into
the pGEM T-vector, sequenced for confirmation of each mutation, and
finally subcloned into the pcDNA3 expression vector.
Mammalian Cell Culture and Transfection--
CHO-Lac cells
(originally called CHO-3.6lac, which stably express the
Lac-transactivator under control of the CMV immediate-early promoter)
were obtained from Dr. M. G. Roth (University of Texas Southwestern Medical Center, Dallas, TX) (17) and maintained in the
same medium plus 200 µg/ml hygromycin. Two plasmids, called pCGN.ATF61-373 (N-terminal HA tag) and
pCGN-ATF61-670 were obtained from Dr. R. Prywes (Columbia
University, New York, NY). The first of these plasmids encodes the
peptide MASSYPYDVPDYASLGGPSR containing a single HA tag
(underlined) immediately upstream of ATF6 residue 1 with a
stop codon following residue 373 and a 3'-BamHI site
beginning nine nucleotides downstream. However, upon sequencing of this
construct we identified a point mutation encoding a M313I substitution. To correct the point mutation, we used
pCGN-ATF61-670 as template and PCR-amplified a fragment
from an internal KpnI site and a newly engineered TAG stop
codon after amino acid 373 followed by BamHI, and we used
this amplified product to replace the KpnI-BamHI
fragment of ATF61-373 (N-terminal HA tag). Another PCR was
performed to add 5'-NotI and 3'-XhoI restriction
sites flanking the insert. Finally, a NotI-XhoI
digest allowed subcloning the corrected ATF61-373
(N-terminal HA tag) into pCMV3RLuc in which the luciferase insert was
excised by NotI-XhoI digestion. This plasmid
contains 450 bases of repeated Lac repressor binding sites immediately
downstream of the CMV promoter and upstream of the inserted cDNA of
interest. The final plasmid called pCMV3R.HA-ATF6cyt was
transfected into CHO-Lac cells, and stable clones were selected with
G418 at 800 µg/ml. The clone used in these experiments, CLA14, is
characterized more fully in a forthcoming
study.2
HEK293 cells and CHO cells were cultured in high-glucose Dulbecco's
modified Eagle's medium plus 10% fetal bovine serum and 0.1%
penicillin-streptomycin (Invitrogen) at 37 °C with 5%
CO2. AtT20 cells were grown in the same medium plus 10%
NuSerum (BD Biosciences). CLA14-CHO cells were maintained in
Dulbecco's modified Eagle's medium (5.5 mM glucose) plus
400 µg/ml G418 and 200 µg/ml hygromycin. Cells were transfected
with plasmid DNA 1 day after seeding (at ~70% confluence) using
LipofectAMINE (Invitrogen) in OptiMEM for 5 h at 37 °C and then
recovered overnight in medium containing 10% fetal bovine serum, with
a change to normal growth medium at 20-24 h and assays of protein
expression at 40-48 h.
Metabolic Labeling, Immunoprecipitation, and
Tricine-Urea-SDS-PAGE--
Cells were metabolically radiolabeled with
[35S]methionine/cysteine mixture in
methionine/cysteine-deficient medium and chased in complete medium for
the times indicated. Where indicated, brefeldin A (BFA) was included
during labeling and chase at a concentration of 10 µg/ml. At the end
of the chase, medium was collected and the cells were lysed in 100 mM NaCl, 1% Triton X-100, 0.2% sodium deoxycholate, 0.1%
SDS, 10 mM EDTA, and 25 mM Tris, pH 7.4. Cell lysates and chase media were treated with a proteinase inhibitor mixture (Roche Applied Science, Indianapolis, IN), precleared with
Zysorbin (Zymed Laboratories Inc.) for 30 min and then
subjected to immunoprecipitation with guinea pig anti-insulin plus
additional Zysorbin. Immunocomplexes were sedimented at 12,000 × g for 1 min, and pellets were washed twice with cell lysis
buffer and once in high salt buffer (0.5 M NaCl, 1% Triton
X-100, 10 mM EDTA, and 25 mM Tris, pH 7.4).
The samples were then analyzed by Tricine-urea-SDS-PAGE system (18)
with previously noted modifications (12); samples were allowed to enter
the gel at 50 V for 1 h and then run at 100 V overnight without
cooling. (As described previously, the gel solutions were not routinely
de-gassed before use (12); however, after the experiments for this
paper were completed we discovered that a small but variable degree of
thiol reoxidation can occur in vitro for our samples during
the running of the gel itself, and this can be effectively decreased by
de-gassing the gel solutions.) Insulin gels were fixed initially in
20% trichloroacetic acid without alcohol, then in 12.5%
trichloroacetic acid plus 50% methanol, then incubated briefly with
water, and finally either phosphorimaged or incubated with 1 M sodium salicylate for 20 min and exposed to XAR
film at Abnormal Nonreducing SDS-PAGE Mobility for Single-chain Insulin
Bearing a Short Linker Sequence--
It has been reported that the
insulin B-chain guides folding and disulfide bond formation within the
A-chain (19-21). Nevertheless, we found surprising the recent result
that certain B-chain point mutations, including ones known to provide
thermodynamic stability to insulin that has been folded in
vitro (such a substitution of insulin HisB10 by Asp), induce
insulin disulfide mispairing within the secretory pathway in
vivo (12). In this study, we have primarily used analysis by
nonreducing SDS-PAGE to assess insulin disulfide maturation, which is
generally recognized to occur within the endoplasmic reticulum (22),
focusing primarily on the length of the linker peptide contained within
SCIs. In an initial series of constructs, we created "artificial
C-peptides" from five to nine residues in which the linker sequence
begins and ends with a Met residue, flanking a stretch of glycine
residues. When these SCI constructs were expressed in transiently
transfected 293 cells, they exhibited nearly ideal behavior under
nonreducing and reducing conditions, although the migration was slower
(higher) under reducing conditions, reflecting a more open rod-like
conformation expected in SDS-PAGE upon dissolution of the disulfide
bonds (right half of each gel shown in Fig.
1).
We prepared additional constructs in which the linker sequence was
foreshortened either to four, three, or two Gly residues or in which
the B-chain had direct linkage to A-chain with no linker whatsoever.
The SCI constructs with short Gly-containing linkers showed diminished
incorporation of [35S]Met/Cys label at least in part
because of absence of methionines from the linker sequence; however, it
was clear that these constructs were also synthesized at low levels, as
indicated by the fact that even direct linkage of B- and A-chains with
no C-peptide showed a reproducibly stronger signal upon transient
expression (left half of each gel shown in Fig. 1).
Moreover, while each of the foreshortened linker constructs showed
nearly ideal behavior under reducing conditions, they showed a far more
complex pattern, with more than one band of anomalous mobility
(indicating non-ideal behavior) under nonreducing conditions (Fig. 1).
These data are highly reminiscent of disulfide mispairing observed upon
introduction of B-chain point mutations in SCIs or proinsulin (12).
Although not exhaustively explored in this study, when the linker was
short, small sequence changes did affect the nonreducing SDS-PAGE
mobility of the predominant isoform of insulin. As shown in Fig.
2, an MGGM tetrapeptide linker resulted
in most SCI molecules migrating with a mobility that was only slightly
slower (higher) than authentic insulin (either biosynthetically labeled
from
On the one hand, some of the slower nonreducing SDS-PAGE mobility of
SCIs could be directly attributed to the presence of the linker
peptide, as demonstrated by an experiment in which an
immunoprecipitated SCI bearing a nine-residue linker sequence was
either partially digested or not digested with CNBr (Fig. 3). Without CNBr digestion, the reduced
SCI shifts up (to a slower mobility), precisely as in Fig. 1. After
partial CNBr digestion, two bands are recovered upon nonreducing
SDS-PAGE: the upper band precisely co-migrates with the undigested
species, while the lower band precisely co-migrates with the two-chain
insulin standard (lane marked I). Upon reduction with DTT,
the upper band co-migrating with undigested SCI shifts up whereas the
lower band co-migrating with two-chain insulin shifts down (in Fig. 3,
the identical gel exposure is shown twice; on the left are
the original data, while the right shows superimposed
arrows clarifying this interpretation). The upward shift of
the upper band is consistent with disulfide reduction allowing the
uncleaved SCI protein to assume a rod-like open conformation, whereas
the downward shift of the lower band is consistent with dissociation of
the two chains from the cleaved SCI (as is also shown in Fig. 3 for
authentic insulin standard). Thus, for SCIs shown with linkers longer
than four or five residues (such as in Fig. 3), the difference in
mobility on nonreducing SDS-PAGE from that of authentic two-chain
insulin can be attributed entirely to the presence of the linker
itself, without invoking any insulin structural perturbation. On the
other hand, for direct linkage of the B- and A-chains (Fig. 2), it
appeared that the slow (high) mobility observed for the major species
could only be explained by disulfide malformation.
Single Chain Insulins Missing Individual Disulfide
Pairs--
We considered the possibility that upon shortening the
C-peptide, one or more insulin disulfide bonds might fail to form
(resulting in free thiols) versus actual disulfide
mispairing. Previously we found that introduction of selected B-chain
point mutations caused similar abnormal upward mobility shifting upon
nonreducing SDS-PAGE but without reactivity to alkylating agents that
modify free thiols (12). In this report, using an SCI with a
seven-residue linker as template, we elected to individually mutate
each of the three conserved cysteine pairs in the hopes of identifying their relative contributions to protein biosynthesis, secretion, and
especially to nonreducing SDS-PAGE mobility. Loss of the insulin disulfide bond between the sixth and eleventh positions of the A-chain
by mutation of these Cys residues to Ser (C43S, C48S) caused no loss of
recombinant protein production, no loss of recombinant protein
secretion and no detectable mobility shift of the predominant insulin
species identified by nonreducing SDS-PAGE (Fig.
4A). By contrast, deletion of
the disulfide normally existing between B-chain residue 19 and A-chain
residue 20 (Fig. 4B, C19S, C56S) caused decreased production of the recombinant protein, being retained
intracellularly rather than being secreted, as well as a slower
(higher) SCI band mobility by nonreducing SDS-PAGE. The foregoing data
demonstrate that presence of the intra-A-chain disulfide bond is not
required for formation of the fast-migrating compact form detected
electrophoretically nor in secretion of the SCI; however, it must be
emphasized that this does not exclude that A6 or A11 might be involved
in disulfide mispairing for SCIs bearing a foreshortened C-peptide
(23). By contrast, the B19-A20 disulfide bond is important for protein
biosynthesis, formation of the compact form detected
electrophoretically, and secretion. (As described further (below), the
B7-A7 disulfide bond is also important for protein biosynthesis and
secretion.)
Misfolded SCIs Traverse the Secretory Pathway of Mammalian Cells
That Have No Known Defects in Secretory Pathway Quality
Control--
We examined secretory kinetics both for an SCI bearing a
seven-residue linker and apparently normal disulfide bonding and for an
SCI bearing direct linkage of B- and A-chains with apparent disulfide
mispairing (see Figs. 1 and 2). In pulse-chase experiments with
35S-labeled amino acids, both newly synthesized constructs
showed an apparent secretory half-time of 30 min (Fig.
5A). At 3 h of chase,
~90% of both constructs had been secreted, whereas no secretion of
either construct was detected at this time in cells treated with
brefeldin A (Fig. 5B). The data indicate that both the
properly and improperly disulfide bonded forms of SCI traverse the
secretory pathway of 293 cells. Identical results were also obtained in CHO cells (and AtT20 cells, see below).
Proof That an Insufficient Linker between B- and A-chains Results
in a Predominant Species with Mispaired Disulfide Bonds, Corrected by
Re-folding in Vitro--
Several SCI constructs bearing different
linkers were exposed to in vitro disulfide reshuffling
conditions using a mixture of reduced and oxidized glutathione at a
10:1 ratio. In Fig. 6A, an SCI
with a nine-residue linker (that is potently bioactive on insulin
receptors)3 was compared with
direct linkage of B- and A-chains. After exposure to the glutathione
mixture, the SCI with a nine-residue linker showed relatively modest
changes in mobility upon nonreducing SDS-PAGE, whereas for the direct
linkage, the major species refolded to a form with a substantially
faster gel mobility. This downward mobility shift of the directly
linked chains does not reflect a proteolytic cleavage between B- and
A-chains because upon re-reduction of the product with 100 mM DTT the predominant species once again shifted back to a
slower migrating form, similar to the behavior of the control SCI with
a nine-residue linker (Fig. 6A, last two lanes).
A modification of the direct linkage construct was also tested in which
Thr-30 of the B-chain was replaced with methionine. (Cleavage with CNBr
to yield a two-chain form of the B30Met direct linkage construct (not
shown) yields a nonreduced band with persistently slower mobility from
that of authentic two-chain insulin despite virtual amino acid identity
providing further confirmation that the anomalous gel mobility stems
from disulfide mispairing.) The T30M mutation caused essentially no
change in the dramatic ability of the direct linkage construct to
reshuffle its disulfide bonds to the more compact form or to reduce
them subsequently with DTT (Fig. 6B). In addition, as shown
in Fig. 6B, disulfide reshuffling conditions did not show
such dramatic mobility changes for the SCI bearing a seven-residue
linker with deletion of the A6-A11 disulfide bond (a construct first
introduced in Fig. 4A), further supporting the idea that the
two interchain disulfide bonds formed properly despite absence of the
intra-A-chain disulfide.
One might expect that unlike the disulfide reshuffling conditions
provided by a mixture of reduced and oxidized glutathione, creation of
free sulfhydryl groups in SCIs by reduction with DTT, followed by
addition of iodoacetamide (IAA) to irreversibly modify those
sulfhydryls, would result exclusively in reduced and carboxymethylated SCIs with no possibility of disulfide re-formation. However, for some
cysteine-containing polypeptides it has been shown that upon addition
of alkylating agent, thiol-disulfide rearrangement occurs on the same
time scale as alkylation; thus, a kinetic competition exists between
the two reactions (24, 25). Indeed, we recently reported that in
vitro reshuffling of insulin disulfide bonds in some SCIs occurs
for a fraction of the molecules upon adding IAA immediately after DTT
treatment (12). In the main gel of Fig.
7, cells bearing either the direct
linkage construct or the presence of four- or seven-residue linkers
underwent pulse-chase (1 h label, 1 h chase) with both cell lysate
and chase media collected. While the production of the SCI bearing a
four-residue (-GGGG-) linker was poor as before, the protein encoded by
each construct was secreted. Each sample was then split in half and
boiled in SDS gel sample buffer containing or not containing 20 mM DTT, and this was followed by incubation with 50 mM IAA for 30 min at 37 °C. The SCI bearing a
seven-residue linker appeared as if it were only partially reduced,
trifurcating into three major species, the lowest (fastest) of which
co-migrated with the original samples that had been treated with IAA
only. However, despite the appearance of partial reduction, this more
likely represents complete reduction followed by re-formation of
disulfide bonds in a fraction of the molecules as both the four-residue
linker and direct linkage constructs also trifurcated after DTT/IAA
treatments, in this case clearly reoxidizing in part to species more
compact (faster migrating) than those contained in the original
samples. In a separate experiment shown in the two lanes on the
right of Fig. 7, we found that after the trifurcation of the
species from the direct linkage construct following reduction in DTT
and partial reoxidation in IAA (lane marked D/I,
with a result identical to that shown in the main gel for
DTT/IAA treatments), the faster migrating forms
shifted back yet again to a slower migrating form upon re-reduction
with 100 mM DTT. Taken together, the foregoing data (Figs.
6 and 7) provide definite proof that the anomalous mobility of SCI
constructs with very short linker peptides is due to formation of
mispaired disulfide isomers in vivo in which the mispairing
can be at least partially corrected upon disulfide reshuffling in
vitro.
Aspects of the ER Environment in the
Biosynthesis/Secretion of SCIs--
Recently we showed that
there is a poor correlation between thermodynamic stability of the
mature SCI protein and the formation of mispaired disulfide insulin
isomers during SCI folding in the ER. There are many possible
explanations for this, including the incomplete availability of the
entire polypeptide for the folding that occurs co-translationally (26),
the unknown kinetic relationship(s) between initial disulfide bond
formation and insulin signal peptide cleavage (which can affect
subsequent folding of mature polypeptides (27)), and the possible role
of ER-specific thiol-interactor proteins that can influence disulfide
bond formation (28). In addition one might postulate that because
mispaired disulfide isomers are relatively rapidly secreted (Fig. 7),
there might be insufficient time in the ER for proper disulfide
isomerization of SCIs. We considered this unlikely because constructs
with a suitable linker length show proper SCI disulfide isomerization despite nearly identical secretory kinetics (Fig. 5A);
nevertheless, we chose to examine the effect of extending the residence
of SCIs in the ER with BFA. For both a seven-residue linker bearing a substitution of serine B9 with Asp (12), as well as for direct linkage
of B- and A-chains, retention in the ER for up to 6 h of chase did
not detectably convert the major disulfide-mispaired isoform into a
more compact disulfide-bonded isoform (Fig.
8). Thus the fact that such isoforms are
misfolded as judged by disulfide mispairing does not preclude their
stability (21), which is consistent with their lack of detection by ER
retention machinery or other secretory pathway quality control (Figs. 5
and 7). Nevertheless, it seemed of interest to know if manipulation of
the unfolded protein response pathway might influence the
biosynthesis or secretion of SCIs with (or without) disulfide
mispairing.
To begin to explore this question, we created a stable CHO cell line in
which to induce expression of the active ATF6 transcription factor that
drives ER chaperone expression in the absence of any ER stress.
Normally during ER stress, the ~90-kDa integral membrane form of ATF6
migrates to the Golgi complex (29, 30) and is proteolysed (31) to
release an ~50-kDa cytosolic domain containing the leucine zipper
motif, which translocates to the nucleus and activates transcription of
ER chaperones and folding enzymes directly (32, 33) as well as
indirectly via XBP-1 (34, 35). However, cDNA-mediated expression of
recombinant cytosolic ATF6 domain constitutively activates
transcription of these (and other) genes without ER stress (36). We
therefore engineered an HA-tagged ATF6cyt into the pCMV3R
plasmid for IPTG-inducible expression in CLA14 cells (see
"Experimental Procedures"). After a 2-day exposure with 15 mM IPTG, the cells express a specific Western-blottable ~50-kDa HA-ATF6cyt band, and without any added ER stress,
this leads to expression of a representative ER molecule chaperone (GRP94) at a level exceeding that obtained from cells treated overnight
with 15 µg/ml tunicamycin (Fig.
9A).
Cells exposed to inducer for 1 d were then transfected with an
expression plasmid encoding either SCI bearing direct linkage of B- and
A-chains or containing the same seven-residue linker plus an S9D point
mutation shown in Fig. 8. Although these mutant constructs are
ordinarily well expressed (Fig. 8), 1 d after transfection (and
2 d after IPTG treatment) there was an obvious further enhancement in the biosynthesis of both constructs (Fig. 9B). The CLA14
cells will be more extensively characterized
elsewhere,2 but as SCI production is not affected in
other cell lines treated with IPTG (not shown), the foregoing results
suggest that induced expression of active ATF6cyt either
increases the efficiency of expression after stress associated with
transfection (in effect, increasing transfection efficiency) or does
not change transfection efficiency but increases recombinant SCI
protein biosynthesis in transfected cells. Regardless of the mechanism
(which is still under investigation), this tool allowed us not only to
increase the levels of constructs that are already well expressed under ordinary conditions but also permitted detection of SCI constructs that
ordinarily were nearly undetectable. Not surprisingly in Fig.
10, an SCI bearing wild-type insulin
chains and a seven-residue linker is well synthesized/secreted, and
this is increased further in CLA14 cells after induced expression of
active ATF6cyt. More remarkably, an SCI with the same
linker but in which the B7-A7 interchain disulfide bond has been
deleted (by double point mutation of these Cys residues to serine)
resulted in a protein produced at extremely low levels and not
detectably secreted; while induced expression of active
ATF6cyt did not rescue secretion of the construct it
nevertheless dramatically increased protein production from transient
transfection (Fig. 10, last lane). The low biosynthetic level for this construct was reminiscent of that seen upon deletion of
the B19-A20 interchain disulfide bond (indeed, induced expression of
active ATF6cyt was employed for experiments with this
mutant shown in Fig. 4B); thus it is apparent that the
presence of both interchain disulfide bonds are essential to escape
secretory pathway quality control. However, only loss of the B19-A20
(and not the B7-A7) disulfide caused significantly slower
mobility of the SCI band by nonreducing SDS-PAGE.
Further Foreshortening of the SCI Linker--
The foregoing
results challenge the assumption that no linker peptide is required for
proper folding of insulin in vivo, thereby raising questions
about the in vivo validity of in vitro
observations suggesting that further foreshortening of the linkage
between B- and A-chains, such as deletion of B30Thr or deletion of this residue in addition to B29Lys, would nevertheless permit insulin to be
synthesized into a structure essentially isomorphous with authentic
two-chain insulin (e.g. Ref. 5). We decided to test this
question in the context of SCI folding in the mammalian ER. As these
constructs were synthesized at low levels under ordinary conditions, we
elected to use CLA14 cells in which ATF6 was induced with IPTG. As
shown in Fig. 11, both a B29-A1 direct
linkage and a B28-A1 direct linkage construct were produced and
secreted from these cells. However, the B29-A1 construct, like the
B30-A1 direct linkage construct, showed only a minor fraction of
molecules with normal or near-normal mobility by nonreducing SDS-PAGE;
instead, the predominant form migrated with abnormally slow gel
mobility indicative of a mispaired disulfide-bonded monomeric isomer
(which nevertheless was secreted). Even more remarkable was the B28-A1 direct linkage, in which virtually no monomeric isoform could be
detected. Instead, almost all of the SCI protein was recovered as a
disulfide-linked homodimer (Fig. 11, nonreduced gel), which ran ideally
as a monomer upon SDS-PAGE under conditions reduced with 100 mM DTT (Fig. 11, reduced gel). These data strongly indicate that direct linkage of the B- and A-chains synthesized in
vivo tends to result in structures that are not
isomorphous with two-chain insulin (due to disulfide mispairing), and
further foreshortening of the linkage between the chains makes matters
worse, decreasing protein biosynthesis and more seriously impairing the
nascent monomer folding pathway.
SCI Folding in AtT20 Cells--
Because direct B- and A-chain
linkage does not preclude normal disulfide bond formation in
vitro but does impair this process within the ER of HEK293 cells
and CHO cells, we did consider the possibility that the profile of ER
luminal chaperone activities (37) in cells with a classical regulated
secretory pathway might be different, and this might alter the folding
outcome. Indeed it has been shown that upon expression in AtT20 cells,
an SCI with direct linkage of B- and A-chains is transported
efficiently through the secretory pathway and also is stored in
secretory granules with an efficiency that is as high or higher than
that of authentic insulin, raising the question of whether the
construct does or does not fold properly in such cells (8). We
therefore introduced into AtT20 cells by transient transfection SCI
constructs bearing a seven-residue linker that has normal nonreducing
SDS-PAGE mobility (Fig. 1), the complete C-peptide and cleavage sites
(i.e. proinsulin), or direct linkage of the chains. With a
60-min pulse and no chase (Fig. 12),
each of the constructs was synthesized, and little had yet been
secreted to the medium (although all constructs were eventually
secreted, not shown). Importantly, absence of a C-peptide caused the
same formation of mispaired disulfide isomers in these cells, with a
mobility upon nonreducing SDS-PAGE mid-way between that of the
seven-residue linker and authentic proinsulin (Fig. 12). These data
support the previous presumption that direct linkage of B- and A-chains
are misfolded in vivo, but despite this, the construct forms
a sufficiently intact three-dimensional structure to escape secretory
pathway quality control (8).
Although information contained solely within the B- and A-chains
of insulin is sufficient for insulin protein structure (1, 2), a
previous report examining the apparently normal intracellular trafficking of a construct called InsDC (i.e. B- and
A-chains directly linked with no C-peptide) was not clear about whether the insulin moiety is properly or improperly folded within the secretory pathway of mammalian cells (8). This matter was seemingly irrelevant to insulin action because it has long been clear that such a
construct, even if the disulfide bonds are correctly assembled, is
almost completely unable to bind insulin receptors (5, 6, 38). However,
in light of recent studies indicating that SCI constructs may have
utility in gene therapy of type 1 diabetes (9), it now seems surprising
that there is a paucity of information about issues relating to
biosynthesis, folding, and secretion of SCIs from mammalian cells. In
the present study, we have endeavored to determine to explore just one
aspect of this question, namely, the role of a connecting peptide in
insulin folding and intracellular transport in the secretory pathway.
Our data lead to the conclusion that a C-peptide is required for proper
insulin folding in the ER. Specifically, although the possible
number and combination of bioengineered amino acids comprising
such a linker is endless and conclusions cannot be drawn about
sequences that have never been tested, from our previous and current
studies using a simple set of constructs we suggest that insulin
disulfide bonds form early during protein folding in the ER (22) with
subpopulations of disulfide isomers that are not readily interconverted
thereafter (Fig. 8 and Ref. 12). A minimum linker length of five
residues is needed to facilitate formation of the proper disulfide
isomer (Figs. 1 and 2). While only a small slowing of electrophoretic
mobility can be directly attributed to the presence of the linker
itself (Fig. 3), loss of the proper disulfide pairings, and this is
proved for the B19-A20 pairing (Fig. 4B), causes loss of
compactness as measured by additional slowing of mobility upon SDS-PAGE
selectively under nonreducing conditions. The fact that loss of the
A6-A11 or B7-A7 disulfide pair causes no discernable slowing of
nonreducing gel mobility (Figs. 4 and 10), whereas direct linkage of B-
and A-chains clearly does (Figs. 1 and 2), provides strong
circumstantial evidence that the gel mobility assay reflects more about
the gain of improper insulin disulfide bonds than the loss of normal
insulin disulfide bonds. Nevertheless, consistent with previous reports
(16), it is likely that the severity of misfolding cannot be correlated solely with the nonreducing SDS-PAGE mobility because direct linkage of
B- and A-chains is secreted from cells with near-normal kinetics and
efficiency (Fig. 5), whereas loss of either of the disulfide linkages
between B- and A-chains causes a profound inhibition of insulin
secretion (Figs. 4 and 10). (However, loss of the intra-A-chain disulfide bond is compatible with good protein biosynthesis and secretion, suggesting the least severe folding defect (Fig. 4).) Thus,
insulin with mispaired disulfide bonds forms a novel structure or
structures capable of escaping secretory pathway quality control, whereas insulin lacking interchain disulfide bonds apparently exposes
structural information to the secretory pathway quality control
machinery (most likely, ER chaperones (39)) that is recognized by the
cell as misfolded. Similar conclusions have been reached about the
importance of the interchain disulfide bonds for insulin expressed in
the secretory pathway of the yeast, Saccharomyces cerevisiae
(40).
Our strongest evidence that the disulfide isomerization occurs within
the secretory pathway and does not reflect some artifact of our method
of sample analysis comes from the fact that identical sample analysis
after exposure of misfolded constructs to in vitro disulfide
reshuffling conditions allows for recovery of a faster migrating
species upon nonreducing SDS-PAGE, and this faster migrating species
can once again be converted to a slower migrating species upon
reduction of disulfide bonds (Figs. 6 and 7). That fact that an SCI
with a -GGGG- linker migrates more slowly upon full reduction than for
an SCI with direct linkage of B- and A-chains and yet both constructs
migrate anomalously upon nonreducing SDS-PAGE with the direct linker
construct exhibiting an even slower gel mobility strongly implies that
more than one possible disulfide mispairing is achieved upon
foreshortening of the C-peptide, with different isomers preferentially
enriched depending upon the precise length and sequence of the linker
(Figs. 1, 2, and 7) as a consequence of structural stress imposed by a
tight turn between B- and A-chains (41).
In our pursuit of the question of whether disulfide-mispaired SCI
constructs are recognized by mammalian cells as misfolded, we have
begun analysis of cells in which the unfolded protein response is
artificially activated upon IPTG-inducible expression of active
ATF6cyt (Fig. 9A). Indeed, such a maneuver
increases biosynthesis of most of the constructs studied in this
report. Because of the protocol we used, we do not presently know
whether this increase in expression reflects primarily improved
transfection efficiency of CLA14 cells, improved translation of SCI
proteins, or decreased ER-associated degradation of SCI proteins. These possibilities will be pursued elsewhere.2 However,
we have noted that active ATF6cyt expression does not lead
to major changes in SCI disulfide mispairing or in the fraction of SCI
protein that gets secreted. Thus at present, the primary utility of the
CLA14 cells is the detection of secretory proteins that are otherwise
difficult to produce. With this in mind we have examined the
biosynthesis of SCI constructs with further foreshortening of the
linker, involving deletion of residue B30 and B29. From this
analysis we find that while both constructs are synthesized at
relatively low levels, upon direct linkage of B28-A1 monomeric insulin
is essentially no longer detected, with essentially all of this SCI
recovered as a disulfide-linked dimer. Such a result begins to sound
suggestive of the autosomal-dominant phenotype observed in the
Mody/Akita diabetic mouse (42) in which the B7Cys of proinsulin is
replaced by Tyr, providing an unpaired cysteine that may have the
potential to cross-link to other proinsulin molecules, a possibility
that needs to be pursued.
The present findings help to amplify our understanding of results from
certain previous studies. In particular, we find that direct linkage of
B- and A-chains causes the same misfolding in AtT20 cells (Fig. 12) as
is observed in 293 and CHO cells, strongly suggesting that previous
reports indicating entry of this SCI into secretory granules of AtT20
cells does not even require a native protein structure (8). Given this
fact, the present data raise a potential note of caution when using
insulin mutagenesis to study protein targeting in the secretory
pathway, as it is possible that certain mutants could form entirely
novel, non-physiological structures (12). Finally, while the present
data indicate that a C-peptide of five residues is required for proper
insulin folding, they do not shed further insight into the fact that
the authentic C-peptide of proinsulin is actually ~30 residues,
i.e. much longer than the minimum required for proper
nascent insulin disulfide bond formation. It therefore must be argued
that since uncleaved proinsulin has much less insulin receptor
bioactivity than that previously reported for certain bioactive SCIs
(9), evolution of the authentic C-peptide of proinsulin is indeed
likely to be associated with the presentation of cleavage sites for
endoproteolysis by prohormone convertases (4, 7), which is necessary
for hormone activation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells as a longer, single-chain
prohormone (proinsulin) in which the B- and A-chains are linked via the
"connecting peptide"
(C-peptide),1 a 31-amino acid
sequence flanked by dibasic amino acids (3). Thus by definition within
proinsulin, the C-peptide brings the B- and A-chains together such that
initial folding and disulfide bond formation in the endoplasmic
reticulum (ER) can occur intramolecularly (4). However it has been
shown that, at least in vitro, simply linking the B- and
A-chains together sequentially, without any C-peptide, also will result
in a normally folded "miniproinsulin" (5, 6). It can be argued
alternatively that the primary importance of the C-peptide spacer
sequence is that it provides suitable flexibility for presenting the
dibasic cleavage sites (4) for endoproteolysis by prohormone
convertases (7). Such cleavage is of enormous biological significance,
since an insulin-like molecule in which the N terminus of the A-chain
is tethered directly to the C terminus of the B-chain is devoid of
activity on insulin receptors (because of inability of the B-chain C
terminus to be displaced, thereby disturbing proper contact of other
key residues with the receptor). Nevertheless, the fact that such a
bioengineered "direct linkage" construct can traverse the secretory
pathway of AtT20 cells, become stored in granules, and be released to the medium upon secretagogue stimulation (8), in conjunction with the
aforementioned findings (5, 6), would seem to argue that a linker
peptide is dispensable at least for achieving a native insulin protein structure.
cells.
(Such genetic manipulation seems reasonable given that C-peptide of
proinsulin is not particularly well conserved during evolution (10) and
is shorter in guinea pig by two residues, in duck by five residues, and
in dog by eight residues (11)). However, to our knowledge, there is
only very limited information regarding the folding of SCIs in the
context of the ER leading to intracellular transport and secretion from
mammalian cells.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Anomalous nonreducing SDS-PAGE mobility of
SCI constructs bearing a highly foreshortened connecting peptide.
Identical wells of HEK293 cells were transiently transfected with 2 µg pcDNA3 containing either no cDNA insert (Empty
Vector) or cDNAs encoding SCIs with the linker sequences
shown. An SCI in which no connecting peptide links B- and A-chains is
termed Direct Linkage. Each well was pulse-labeled with
35S-labeled amino acids and chased for 1 h. The cells
were lysed, and both lysates and chase media were immunoprecipitated
with anti-insulin. Each immunoprecipitate was divided in two, and each
half was analyzed by SDS-PAGE either under nonreducing conditions
(left gel) or after reduction with 20 mM
dithiothreitol (right gel) followed by fluorography. In this
figure, only immunoprecipitates from the chase media are shown;
however, the pattern of bands was identical from the cell lysates. For
comparison, a nonreduced radioiodinated insulin standard is shown in
the first lane.
TC-3 cells or iodinated standard), whereas an SCI with the GGGG
tetrapeptide linker (in addition to being synthesized weakly) was
higher still, despite a virtually identical molecular mass.
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Fig. 2.
Distinct anomalous nonreducing SDS-PAGE band
mobilities for two constructs bearing a linker sequence of identical
(four-residue) length but differing sequence. HEK293 cells were
transfected with cDNAs encoding SCIs bearing the linker sequences
shown or with the empty expression vector. An SCI in which no
connecting peptide links B- and A-chains is termed Direct
Link. The transfected cells were then analyzed exactly as in Fig.
1. Only nonreducing SDS-PAGE (followed by fluorography) is shown. A
radioiodinated insulin standard and an anti-insulin immunoprecipitate
from 35S-labeled amino acid labeled TC-3 cells are shown
at left to identify the SDS-PAGE mobility of proinsulin and
insulin.
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Fig. 3.
Changes in mobility of uncleaved and cyanogen
bromide-cleaved SCI as a consequence of disulfide bond reduction.
A seven-lane gel bearing six samples (and one blank lane) is shown
twice in exact repeat. The lanes marked I contain
radiodinated insulin standard. An immunoprecipitated, metabolically
labeled SCI (secreted from transfected HEK293 cells; linker sequence
shown at bottom) is analyzed by SDS-PAGE under nonreducing conditions
or reduced with 20 mM DTT in the third and
sixth lanes, respectively. For CNBr-mediated
cleavage of approximately half of the molecules, the immunoprecipitate
was treated with 50 mg/ml CNBr in 6 M guanidine plus 20 mM HCl at room temperature overnight, and this was then was
spun through a Microcon YM-3 ultrafilter (Millipore, New Bedford, MA)
at 10,000 × g for 50 min to de-salt the sample before
mixing with SDS-gel sample buffer. After CNBr treatment, the SCI band
can be seen to be split in two under nonreducing conditions: half
co-migrating with the original uncleaved SCI and the other half
co-migrating with the insulin standard. Upon reduction of the
CNBr-digested sample, approximately half the molecules shift up in the
gel, co-migrating with the reduced SCI exhibiting diminished SDS-PAGE
mobility that indicates lack of peptide chain cleavage, while the other
half shifts down in the gel, co-migrating with the insulin standard in
which the interchain disulfide bonds have been reduced to release two
chains, indicating that this faster-migrating SCI band has been
proteolytically cleaved by CNBr.
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Fig. 4.
Nonreducing SDS-PAGE mobility of SCIs bearing
a seven-residue linker either lacking the A6-A11 intrachain- or B19-A20
interchain-disulfide bond. A, CHO cells were
pulse-labeled for 1 h with 35S-labeled amino acids and
chased for 1.5 h; the cells were lysed, and both lysates and chase
media were immunoprecipitated with anti-insulin. The numbering of
residues in the construct begins with residue 1 of the B-chain; the
mutant C43S/C48S indicates loss of the A6-A11 disulfide).
B, CLA14 cells (see "Experimental Procedures") were
pulse-labeled with 35S-labeled amino acids for 30 min and
chased for 40 min. The cells were lysed, and both lysates
(C) and chase media (M) were immunoprecipitated
with anti-insulin (note that the order in which the cell and media
lanes were loaded on the gel differ from that shown in panel
A). An arrowhead at right marks the position of the
anomalously migrating SCI consequent to loss of the B19-A20 bond (C19S,
C56S). At later chase times also (not shown), this construct remains
defective for secretion to the medium.
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Fig. 5.
SCI release to the medium via the classical
secretory pathway. HEK293 cells were transfected with cDNAs
encoding SCIs either bearing no connecting peptide (Direct
Linkage) or the seven-reside linker shown. A, the cells
were pulse-labeled with 35S-labeled amino acids for 30 min
and chased for the times indicated; anti-insulin immunoprecipitates
from cells (C) and chase media (M) are shown.
Note that despite the obvious anomalous nonreducing SDS-PAGE mobility
(suggesting abnormal disulfide bond formation) of the direct linkage
construct, the kinetics of release of this SCI is not dramatically
altered. B, cells transfected with the same constructs as in
A were labeled and chased for 3 h in the continuous
absence or presence of BFA.
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Fig. 6.
Glutathione-mediated SCI disulfide
interchange to a state of improved disulfide pairing from that observed
in vivo as measured by nonreducing SDS-PAGE
mobility. 35S-labeled SCIs bearing the linker
sequences shown were secreted and immunoprecipitated with anti-insulin
(an aliquot taken, first two lanes) and were then exposed to
a reduced/oxidized glutathione mixture in a 10:1 ratio (an aliquot
taken, middle two lanes) and finally reduced with 100 mM DTT (last two lanes). A, upon
direct linkage of B- and A-chains, despite a markedly slower initial
SDS-PAGE mobility of the major isoform, this construct actually
exhibits a faster gel mobility than the control (nine-residue linker)
construct after exposure to in vitro disulfide reshuffling
conditions. B, a similarly anomalous band mobility and
dramatic disulfide isomerization is observed when B30Thr is replaced by
methionine in the direct linkage construct. A second construct contains
the seven-residue linker sequence shown plus absence of the A6-A11
intrachain disulfide bond (C43S, C48S); such a construct shows more
modest band mobility shift upon glutathione-mediated disulfide
reshuffling.
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Fig. 7.
SCI disulfide reshuffling during a standard
reduction-alkylation protocol. HEK293 cells were transfected with
cDNAs encoding SCIs bearing the linker sequences shown and were
then pulse-labeled with 35S-labled amino acids and chased
as described in Fig. 1. Immunoprecipitates with anti-insulin from cells
(C) and media (M) were then exposed to 50 mM IAA either without prior reduction or after initial
reduction by boiling in SDS-gel sample buffer containing 20 mM DTT. In a separate experiment shown in lanes
15 and 16, an SCI bearing no connecting peptide
(Direct Link) that had undergone the reduction/alkylation
protocol (D/I, identical to lane 4)
was then further reduced ( D) with 100 mM DTT
prior to SDS-PAGE and fluorography.
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Fig. 8.
Nonreducing SDS-PAGE mobility of SCIs with
mispaired disulfide bonds upon prolonged entrapment in the ER as a
consequence of BFA treatment. Cells were transiently transfected
with cDNAs encoding SCIs bearing either the seven-residue linker
shown, no linker (Direct Link), or the same seven-residue
linker but including point mutation in which B9Ser is replaced by Asp
(S9D), which is known to cause disulfide mispairing (12).
Cells were exposed to 10 µg/ml BFA during pulse labeling with
35S-labeled amino acids, and this treatment was continued
during the chase periods shown.
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Fig. 9.
Expression of active ATF6cyt
induced without ER stress improves the biosynthesis of SCIs with
mispaired disulfide bonds. A, Western blot of CLA14
cell lysates with anti-HA and anti-GRP94 under control conditions or
after overnight treatment with tunicamycin (TUN
O/N) or for 2 d with IPTG. B, CLA14
cells were either treated or untreated with IPTG beginning on day 0, transfected with the cDNAs encoding SCIs bearing the linker
sequences shown on day 1, and pulse-labeled with
35S-labeled amino acids for 50 min and chased 6 h on
day 2. Cell lysates and media were immunoprecipitated with anti-insulin
and analyzed by nonreducing SDS-PAGE and fluorography.
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Fig. 10.
Elimination of the B7-A7 interchain
disulfide bond inhibits protein production but does not change SDS-PAGE
mobility under nonreducing conditions. CLA14 cells were either
induced or not induced to express ATF6 exactly as in Fig. 9 and were
transfected to express SCIs bearing the linker sequences shown. Cells
were pulse-labeled with 35S-labeled amino acids for 30 min
and chased for 40 min. Both chase media (M) and cell lysates
(C) were immunoprecipitated with anti-insulin.
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Fig. 11.
Further tightening of the tether between
insulin B- and A-chains by deletion of residues
B30 ± B29 inhibits protein
production and shows worsening of disulfide bond pairing in
vivo. CLA14 cells induced to express
ATF6cyt with IPTG were transfected with cDNAs encoding
the five distinct SCI constructs shown and the next day were
pulse-labeled with 35S-labeled amino acids for 1 h and
chased for 1 h. SCIs immunoprecipitated from both cell lysates
(C) and chase media (M) with anti-insulin were
analyzed by SDS-PAGE under nonreducing conditions or after reduction
with 100 mM DTT followed by fluorography. Note that while
all the constructs exhibit ideal behavior under reducing conditions,
both constructs linking B30-A1 showed similarly anomalous nonreducing
SDS-PAGE mobility to that of B29 linked to A1, while the B28-A1 linkage
results in near-exclusive formation of disulfide-linked SCI
dimers.
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Fig. 12.
Expression of SCI constructs in AtT20
cells. The cells were transiently transfected with cDNAs
encoding the linker sequences shown and were pulse-labeled for 1 h
without chase. Cell lysates (C) and pulse media
(M) were immunoprecipitated with anti-insulin and analyzed
by nonreducing SDS-PAGE and fluorography.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank members of the Arvan laboratory for suggestions during the course of these studies.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK48280 (to P. A.).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.
Holder of an academic license from INSP, Mexico.
§ To whom correspondence should be addressed: Div. of Endocrinology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8685; Fax: 718-430-8557; E-mail: arvan@aecom.yu.edu.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M212070200
2 J. Ramos-Castañeda, M. Liu, P. Arvan, manuscript in preparation.
3 M. Liu and P. Arvan, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
C-peptide, connecting peptide;
ER, endoplasmic reticulum;
SCIs, single-chain
insulins;
CMV, cytomegalovirus;
HA, hemagglutinin;
BFA, brefeldin A;
DTT, dithiothreitol;
IAA, iodoacetamide;
IPTG, isopropyl-1-thio--D-galactopyranoside.
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---|
1. | Tang, J. G., and Tsou, C. L. (1990) Biochem. J. 268, 429-435[Medline] [Order article via Infotrieve] |
2. |
Hua, Q. X.,
Chu, Y. C.,
Jia, W.,
Phillips, N. F.,
Wang, R. Y.,
Katsoyannis, P. G.,
and Weiss, M. A.
(2002)
J. Biol. Chem.
277,
43443-43453 |
3. | Steiner, D. F., Kemmler, W., Clark, J. L., Oyer, P. E., and Rubenstein, A. H. (1972) in Handbook of Physiology (Steiner, D. F. , and Freinkel, N., eds) , pp. 175-198, Williams and Wilkins, Baltimore, MD |
4. |
Steiner, D. F.,
and Rubenstein, A. H.
(1997)
Science
277,
531-532 |
5. | Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Bing, X., and Markussen, J. (1991) J. Mol. Biol. 220, 425-433[Medline] [Order article via Infotrieve] |
6. | Hua, Q. X., Hu, S. Q., Jia, W., Chu, Y. C., Burke, G. T., Wang, S. H., Wang, R. Y., Katsoyannis, P. G., and Weiss, M. A. (1998) J. Mol. Biol. 277, 103-118[CrossRef][Medline] [Order article via Infotrieve] |
7. | Steiner, D. F. (1998) Curr. Opin. Chem. Biol. 2, 31-39[CrossRef][Medline] [Order article via Infotrieve] |
8. | Powell, S. K., Orci, L., Craik, C. S., and Moore, H. P. H. (1988) J. Cell Biol. 106, 1843-1851[Abstract] |
9. | Lee, H. C., Kim, S. J., Kim, K. S., Shin, H. C., and Yoon, J. W. (2000) Nature 408, 483-488[CrossRef][Medline] [Order article via Infotrieve] |
10. | Steiner, D. F., Chan, S. J., Welsh, J. M., and Kwok, S. C. (1985) Annu. Rev. Genet. 19, 463-484[CrossRef][Medline] [Order article via Infotrieve] |
11. | Permutt, M. A. (1981) in The Islets of Langerhans (Cooperstein, S. J. , and Watkins, D., eds) , pp. 75-95, Academic Press, New York, NY |
12. | Zhang, B.-y., Liu, M., and Arvan, P. (2002) J. Biol. Chem. 278, 3687-3693 |
13. | Fassio, A., and Sitia, R. (2002) Histochem. Cell Biol. 117, 151-157[CrossRef][Medline] [Order article via Infotrieve] |
14. | Sevier, C. S., and Kaiser, C. A. (2002) Nat. Rev. Mol. Cell. Biol. 3, 836-847[CrossRef][Medline] [Order article via Infotrieve] |
15. | Sieber, P., Eisler, K., Kamber, B., Riniker, B., Rittel, W., Marki, F., and de Gasparo, M. (1978) Hoppe Seyler's Z. Physiol. Chem. 359, 113-123[Medline] [Order article via Infotrieve] |
16. | Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138[Medline] [Order article via Infotrieve] |
17. | Lucocq, J., Manifava, M., Bi, K., Roth, M. G., and Ktistakis, N. T. (2001) Eur. J. Cell Biol. 80, 508-520[Medline] [Order article via Infotrieve] |
18. | Schagger, H., and vonJagow, G. (1987) Anal. Biochem. 166, 368-379[Medline] [Order article via Infotrieve] |
19. | Hua, Q. X., Nakagawa, S. H., Jia, W., Hu, S. Q., Chu, Y. C., Katsoyannis, P. G., and Weiss, M. A. (2001) Biochemistry 40, 12299-12311[CrossRef][Medline] [Order article via Infotrieve] |
20. | Guo, Z. Y., Shen, L., and Feng, Y. M. (2002) Biochemistry 41, 10585-10592[CrossRef][Medline] [Order article via Infotrieve] |
21. | Guo, Z. Y., Shen, L., and Feng, Y. M. (2002) Biochemistry 41, 1556-1567[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Huang, X. F.,
and Arvan, P.
(1995)
J. Biol. Chem.
270,
20417-20423 |
23. | Qiao, Z. S., Guo, Z. Y., and Feng, Y. M. (2001) Biochemistry 40, 2662-2668[CrossRef][Medline] [Order article via Infotrieve] |
24. | Weissman, J. S., and Kim, P. S. (1991) Science 253, 1386-1392[Medline] [Order article via Infotrieve] |
25. | Chang, J. Y. (1996) Biochemistry 35, 11702-11709[CrossRef][Medline] [Order article via Infotrieve] |
26. | Kowarik, M., Küng, S., Martoglio, B., and Helenius, A. (2002) Mol. Cell 10, 769-778[Medline] [Order article via Infotrieve] |
27. |
Scheele, G.,
and Jacoby, R.
(1983)
J. Biol. Chem.
258,
2005-2009 |
28. |
Anelli, T.,
Alessio, M.,
Mezghrani, A.,
Simmen, T.,
Talamo, F.,
Bachi, A.,
and Sitia, R.
(2002)
EMBO J.
21,
835-844 |
29. |
Chen, X.,
Shen, J.,
and Prywes, R.
(2002)
J. Biol. Chem.
277,
13045-13052 |
30. | Shen, J., Chen, X., Hendershot, L., and Prywes, R. (2002) Dev. Cell 3, 99-111[Medline] [Order article via Infotrieve] |
31. | Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355-1364[Medline] [Order article via Infotrieve] |
32. |
Haze, K.,
Yoshida, H.,
Yanagi, H.,
Yura, T.,
and Mori, K.
(1999)
Mol. Biol. Cell
10,
3787-3799 |
33. | Okada, T., Yoshida, H., Akazawa, R., Negishi, M., and Mori, K. (2002) Biochem. J. 366, 585-594[CrossRef][Medline] [Order article via Infotrieve] |
34. | Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001) Cell 107, 881-891[Medline] [Order article via Infotrieve] |
35. |
Lee, K.,
Tirasophon, W.,
Shen, X.,
Michalak, M.,
Prywes, R.,
Okada, T.,
Yoshida, H.,
Mori, K.,
and Kaufman, R. J.
(2002)
Genes Dev.
16,
452-466 |
36. |
Yoshida, H.,
Okada, T.,
Haze, K.,
Yanagi, H.,
Yura, T.,
Negishi, M.,
and Mori, K.
(2000)
Mol. Cell. Biol.
20,
6755-6767 |
37. |
Winter, J.,
Klappa, P.,
Freedman, R. B.,
Lilie, H.,
and Rudolph, R.
(2002)
J. Biol. Chem.
277,
310-317 |
38. |
Nakagawa, S. H.,
and Tager, H. S.
(1989)
J. Biol. Chem.
264,
272-279 |
39. | Arvan, P., Zhao, X., Ramos-Castañeda, J., and Chang, A. (2002) Traffic 3, 771-780[CrossRef][Medline] [Order article via Infotrieve] |
40. | Guo, Z. Y., and Feng, Y. M. (2001) Biol. Chem. 382, 443-448[Medline] [Order article via Infotrieve] |
41. | Chou, K. C. (2000) Anal. Biochem. 286, 1-16[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Wang, J.,
Takeuchi, T.,
Tanaka, S.,
Kubo, S. K.,
Kayo, T.,
Lu, D.,
Takata, K.,
Koizumi, A.,
and Izumi, T.
(1999)
J. Clin. Invest.
103,
27-37 |