From the Department of Developmental and Molecular
Biology and the § Division of Endocrinology, Albert
Einstein College of Medicine, Bronx, New York 10461
Received for publication, September 16, 2002, and in revised form, November 5, 2002
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ABSTRACT |
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In the secretory pathway, endoproteolytic
cleavage of the insulin precursor protein promotes a change in the
biophysical properties of the processed insulin product, and this may
be relevant for its intracellular trafficking. We have now studied
several independent point mutants contained within the insulin B-chain,
S9D, H10D, V12E (called B9D, B10D, and B12E), as well as the double
point mutant P28K,K29P (B28K,B29P), that have been reported to inhibit insulin oligomerization. In yeast cells, the unprocessed precursor of
each of these mutants is secreted, whereas >90% of the
endoproteolytically released single-chain insulin moiety is retained
intracellularly; a large portion of the B9D, B10D, and B12E
single-chain insulins exhibit abnormally slow mobility upon nonreducing
SDS-PAGE, despite normal mobility upon reducing SDS-PAGE. Although no
free thiols can be detected, each of these mutants exhibits increased
disulfide accessibility to dithiothreitol. After dithiothreitol
treatment, a portion of the molecules can reoxidize to a form more
compact than the original single-chain insulin mutants formed in
vivo (indicating initial disulfide mispairing). Disulfide
mispairing of a fraction of B9D, B10D, and B12E mutants also occurs in
the context of single-chain insulin and even in authentic proinsulin expressed within the secretory pathway of mammalian cells. We conclude that analyses of the intracellular trafficking of certain oligomerization-defective insulin mutants is complicated by the formation of disulfide isomers in the secretory pathway.
Clinically relevant production of insulin in the eukaryotic
secretory pathway occurs as a consequence of endogenous expression of
proinsulin in pancreatic beta cells (1), as a consequence of artificial
expression in heterologous mammalian cells (2), or from expression of
an insulin-containing fusion protein
(ICFP1; encoding the yeast
preproalpha factor leader peptide fused via a KR cleavage site to a
single-chain insulin) in Saccharomyces cerevisiae (3). In
the latter case, upon ICFP cleavage in the distal secretory pathway by
Kex2p endoprotease to liberate the alpha leader peptide, a
crystallizable single-chain insulin is produced with a structure
essentially isomorphous with that of authentic two-chain insulin (3,
4).
We have shown previously that upon expression of ICFP at modest levels,
the fraction of molecules remaining unprocessed is secreted rapidly,
whereas the other fraction undergoing Kex2p-mediated endoproteolysis
results in a single-chain insulin that primarily is not secreted (5).
Moreover, this sorting, determined by Kex2p-mediated processing, is
saturable. Failure to secrete the single-chain insulin moiety cannot be
explained by ICFP endoproteolysis within a "dead-end" compartment
from which molecules can no longer be secreted, because simple deletion
of the yeast KEX2 gene not only prevents ICFP
processing but also increases the quantity of ICFP secreted. It is
known that by liberating insulin from its precursor (in either
mammalian cells or yeast), cleavage promotes a change in the
biophysical properties of the processed product (5-11), and this might
play a role in protein sorting in the lumen of the distal secretory
pathway (12, 13).
The structure of insulin has been examined extensively (14) and has
been the subject of detailed mutagenesis studies. From the crystal
structure (9), residues B9S, B12V, and B28P (B referring to the insulin
B-chain), among others (15), participate in the dimer-forming surface
of the insulin monomer; mutations such as B9D, B12E, and B28K,B29P have
been described as having diminished tendency to dimerization (16-18),
whereas a B10D mutant is thought to be impaired primarily in hexamer
assembly from dimers (19). Given that some assembly-deficient B-chain
point mutants are thought to exhibit folding stability comparable with
that of native insulin (20), we have now examined the expression of
these B-chain point mutants in the secretory pathway of yeast and in
transiently transfected mammalian cells. Although the B28K,B29P double
mutant was expressed generally at the lowest levels, each of the B9D,
B10D, and B12E mutants produced more than one isomer differing in
disulfide bonding, suggesting that these mutants have varying degrees
of predisposition for disulfide mispairing during initial folding in
the secretory pathway.
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 ImmunoResearch
Laboratories (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 PerkinElmer
Life Sciences. Methionine/cysteine-deficient mammalian cell culture
medium, brefeldin A, and stock chemicals were from Sigma.
Yeast Strains and Plasmids--
Unless otherwise indicated,
strains employed in this study are isogenic to PA13 (MATa,
kss1, ura3-52,
leu2-3,112, his3,
trp1^63, ade2, GAL+), which
has a complete deletion of the PEP4 gene, marked with URA3
(5). The VPS10 gene was then disrupted by transformation with XhoI and BamHI fragment of pEM10-103 (21)
to produce strain PA11D. pMIGLC, a TRP1-marked centromeric plasmid (22)
bearing the wild-type ICFP sequence is as described previously (with
the clarification that the strong promoter/terminator previously
reported as belonging to the glycerol-3-phosphate dehydrogenase gene,
GPD1, in fact belongs to the promoter of
glyceraldehyde-3-phosphate dehydrogenase, known as TDH3, and
the terminator of 3-phosphoglycerate kinase, known as
PGK1) (5). In one set of experiments examining the
effects of vps10, each of the insulin B-chain point
mutations was prepared by PCR mutagenesis (of an ~0.4-kb
EcoRI-HindIII PCR-amplified fragment containing
the relevant mutation that was used to replace the matching
EcoRI-HindIII fragment of ICFP, confirmed by DNA sequencing) in the context of the pMIGLC vector, and these were transformed into PA11D and the PA13 parental strain. In a second set of
experiments, strain AHY63 was employed (the kind gift of Dr. S. Nothwehr, University of Missouri, Columbia, MO) which is isogenic with
W303-1A (MATa; ura3-1;
leu2-3,112; his3-11,15;
trp1-1, can1-100; ade2-1) but
contains pho8
For experiments examining the nonreducing SDS-PAGE mobility of insulin
bearing different B-chain point mutations, the URA3 marker
of PA13 was disrupted by insertion of HIS3
(pUC119-ura3::HIS3 cut with XmaI) to
produce strain YB118. Then, each of the pGMU series of vectors bearing
insulin B-chain point mutations was transformed into YB118.
Metabolic Labeling and Immunoprecipitation in Yeast
Cells--
Cells were grown to exponential phase in minimal medium
supplemented with required amino acids but without methionine and cysteine, using 2% glucose as the carbon source, and then sedimented at 3000 × g for 5 min and resuspended at 4 A600/ml in the same medium plus 1 mg/ml
bovine serum albumin. For pulse labeling, [35S]cysteine was added at 50 µCi/A600 for 5 min (unless otherwise indicated). The cells were chased in synthetic complete (SC) medium plus 10 mM methionine and cysteine. At 45 min of chase
(unless otherwise indicated), 1-ml aliquots were transferred to ice,
and 10 mM sodium azide and a protease inhibitor mixture
(aprotinin, 0.25 µg/ml; leupeptin, 0.1 mM; pepstatin, 10 µM; EDTA, 5 mM; E64, 10 µM;
diisopropyl fluorophosphate, 1 mM, bovine serum
albumin 0.02%) were added immediately. Cells were then sedimented at
3000 × g for 5 min, and the supernatant containing
secreted proteins was collected for further analysis. The cells were
resuspended in 100 µl of breaking buffer containing 6 M
urea, 1% SDS, 1 mM EDTA, and 50 mM Tris, pH
7.5. The cells were lysed by vortexing with glass beads and then
boiling for 4 min. Samples were diluted 10-fold to a final
concentration of 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5, precleared with 20 µl of zysorbin for 30 min (Zymed Laboratories Inc.,
San Francisco, CA), and then immunoprecipitated with 5 µl of guinea
pig anti-insulin plus 20 µl of zysorbin. Immunoprecipitates were
digested with PNGase F (New England Biolabs, Beverly, MA) and analyzed
by SDS-PAGE in the absence or presence of reducing conditions as
described in the text.
For the sample preparation in the experiment shown in Fig. 2, yeast
cells were lysed in the absence of detergent, Triton X-100 was added to
0.9%, and the samples were immunoprecipitated with anti-insulin as
described above. The immunoprecipitates were then exposed
sequentially to 18 mM dithiothreitol (DTT) for 30 min at
37 °C, to iodoacetamide at a final concentration of 36 mM (37 °C, 5 min), and finally to SDS gel sample buffer
before analysis by SDS-PAGE.
Tricine-Urea-SDS-PAGE--
We modified the Tricine-urea-SDS-PAGE
system (24) as follows. In addition to TEMED and ammonium persulfate as
described, the resolving gel contained 11.2% acrylamide, 0.35%
bisacrylamide, 10% Prosieve-50 (BioWhittaker), and a final
concentration of 6.0 M urea in 0.1% SDS and 1.0 M Tris, pH 8.45. Before this was allowed to polymerize, it
was overlaid with a small quantity (sufficient for adding 0.2 cm of
overlay) of "spacer gel" containing, in addition to TEMED and
persulfate, 9.3% acrylamide and 0.6% bisacrylamide without Prosieve
or urea in 0.1% SDS and 1.0 M Tris, pH 8.45. 30 min after
polymerization of the resolving and spacer gel, a stacking gel was
poured containing, in addition to TEMED and persulfate, 3.7%
acrylamide and 0.24% bisacrylamide in 0.075% SDS and 0.75 M Tris, pH 8.45. None of the gel solutions were degassed
before use. The sample buffer we used resulted in a final concentration of 2% SDS, 12% glycerol, and 0.0025% Serva blue in 50 mM
Tris, pH 6.8. Typically, insulin samples were boiled for 5 min before gel loading, although we found that omitting the boiling step had
essentially no effect on the outcome of these experiments. The tank
buffers were the same as those described previously: a running buffer
of 0.1% SDS in 0.1 M Tris, 0.1 M Tricine, pH 8.25, and an anode buffer of 0.2 M Tris, pH 8.9 (24). This
gel system was essential to the detection of mobility differences between the insulin species described.
Mammalian Cell Culture, Transfection, and Labeling--
HEK293
cells were cultured in Dulbecco's modified Eagle's medium plus 10%
fetal bovine serum and 0.1% penicillin-streptomycin (Invitrogen) at
37 °C with 5% CO2. A cDNA encoding wild-type human proinsulin was cloned into the pcDNA3 (CMV promoter-driven)
expression vector (Invitrogen). Each of the insulin B-chain point
mutations was prepared by PCR mutagenesis within an ~0.4-kb
PCR-amplified fragment within the pGEM-T vector and then subcloned to
replace the appropriate wild-type proinsulin sequence in pcDNA3
with successful completion of the mutagenesis confirmed by DNA
sequencing. The pRSV-InsDC plasmid (encoding a preproinsulin in which
the C-peptide and endoproteolytic cleavage sites are completely
lacking), obtained from Dr. H. P. Moore (University of California,
Berkeley), was subcloned to pcDNA3 and used as the template for
further PCR mutagenesis to encode an artificial 7-amino acid C-peptide
sequence comprising MGGGGGM (Met-Gly-Gly-Gly-Gly-Gly-Met). The
resulting construct is referred to in this manuscript as SCI
(single-chain insulin). The SCI construct was then submitted to
additional rounds of PCR mutagenesis to produce the same specific
B-chain point mutations as those produced in the yeast ICFP and in proinsulin.
Plasmid DNA was transfected into HEK293 cells using
LipofectAMINE (Invitrogen). Cells were metabolically radiolabeled with a [35S]methionine/cysteine mixture for 1 h in
methionine/cysteine-deficient medium and chased for 1 h in
complete medium. Where indicated, brefeldin A was included during
labeling and chase at a concentration of 10 µg/ml. At the end of the
chase, the medium was collected, and the cells were lysed in 100 mM NaCl, 1% Triton X-100, 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 Molecular
Biochemicals), precleared with zysorbin (Zymed Laboratories Inc.), and then subjected to immunoprecipitation with guinea pig anti-insulin as described above. Zysorbin-bound 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) before analysis by Tricine-urea-SDS-PAGE
(24) in the absence or presence of reducing conditions as described above.
Reactivity with 4-Acetamido-4'-maleimidylstilbene-2,2'-disulfonic
Acid (AMS)--
AMS was purchased from Molecular Probes (catalog No.
A-485). Immunoprecipitates with anti-insulin, derived either from yeast or mammalian cells, were divided in 50 µl of either 80 mM
Tris-HCl, pH 6.8, 1% SDS, 1 mM phenylmethylsulfonyl
fluoride, and 20 mM AMS or an identical buffer without AMS.
The samples were then resolved by nonreducing SDS-PAGE. In some cases,
identical aliquots of radiolabeled yeast or mammalian cell lysate were
incubated with AMS prior to lysis and insulin immunoprecipitation, but
the results were identical to those obtained when the AMS reaction was
performed after immunoprecipitation.
Secretion from Yeast of Unprocessed Insulin-containing Fusion
Proteins with Lack of Secretion of Processed Single-chain Insulins
Containing B-chain Point Mutations--
We previously found that in
yeast, after Kex2p-mediated endoproteolytic removal of the alpha leader
peptide from ICFP (bearing the wild-type B-chain residues 1-29 and
A-chain residues 1-21 made contiguous by an AAK tripeptide linker
sequence), a single-chain insulin was produced that was not efficiently
secreted and instead accumulated intracellularly in a manner dependent
upon PEP4, encoding the major yeast vacuolar proteinase (5).
Intriguingly, this intracellular targeting was entirely dependent upon
the catalytic activity encoded by KEX2, and the
sorting function provided by this gene could be replaced in
trans by expression of a truncated, membraneless, secretory Kex2p.
In contrast, intracellular insulin targeting was not perturbed by
deletion of the VPS10 gene encoding a vacuolar sorting receptor.
The present study was initiated to examine the effect of B-chain point
mutations on the expression and trafficking of insulin. Because
substitution of proximal B-chain residues can provoke increased
conformational changes of the insulin moiety including increased
B-chain flexibility (25, 26), and the B-subdomain may provide a
template to guide folding of the A-chain (27, 28), it seemed possible
that such B-chain point mutants might be recognized as misfolded by
Vps10p (29-31). We therefore employed a pulse-chase protocol to
examine the behavior of ICFP constructs bearing B9D, B10D, B12E, or
B28K,B29P point mutations, driven by the strong TDH3
promoter, in strains that were (or were not) deleted for
VPS10. At this level of substrate expression the
processing activity of Kex2p is limiting (5). As shown in Fig.
1 (bottom left), for the
wild-type insulin sequence the unprocessed ICFP was well secreted, and
the processed form was stored (in the vacuole) of pep4 null
cells. In agreement with our previous study, introduction of the B5T
mutation greatly augmented ICFP expression, but the small amount of
cleaved B5T-insulin was not retained efficiently intracellularly (Fig.
1). Each of the other point mutants was expressed (although the
B28K,B29P samples had to be overloaded in order to achieve a comparable
radioactive protein signal), and in each case, the fusion protein was
secreted, whereas the processed insulin mutant was retained efficiently
(Fig. 1). For the vps10 mutant strains, there was at most a
modest effect observed for B10D, B12E, and B28K,B29P mutants, as the
final ratio of unprocessed ICFP in the medium to processed insulin in
the cells was not greatly affected. As shown in the enlarged image of
the gel analyzing the B10D mutant (Fig. 1, right), only a
small decrease in intracellular insulin, coupled with a small increase
in secreted ICFP, was detectable.
Single-chain Insulins Containing B-chain Point Mutations Exhibit
Increased Sensitivity to Reduction with Dithiothreitol--
Upon
endoproteolytic processing in yeast, the resulting single-chain insulin
containing the wild-type sequence has a structure that tends to resist
disulfide reduction with DTT under nondenaturing conditions (5). To
further explore the stability of single-chain insulins bearing B-chain
point mutations, immunoprecipitates from cells expressing various
mutant constructs were exposed to 20 mM DTT under
nondenaturing conditions (see "Experimental Procedures"). After
such treatment, single-chain insulin without additional B-chain point
mutations was largely recovered as a band with an identical mobility to
that of a parallel sample not treated with DTT (Fig.
2, open arrow). However, each
of the B-chain point mutants exhibited enhanced sensitivity to DTT as
demonstrated by diminished recovery of the single-chain insulin band
upon SDS-PAGE. These data suggest differences in the conformational
stability of the single-chain insulins bearing wild-type and mutant
sequences.
In Yeast, Some Single-chain Insulins Containing B-chain Point
Mutations Exhibit Abnormal Mobility upon Nonreducing
SDS-PAGE--
Although many structural analyses of insulin B-chain
point mutants have begun with chemically or bacterially synthesized
peptides or otherwise purified material that has been folded or
refolded in vitro or purified for structural homogeneity by
selection of a specific HPLC peak (32), relatively few studies have
examined the folding of such mutants as they are produced in
vivo within the eukaryotic secretory pathway. It is known that
substitution of proximal B-chain residues, such as the B9S
substitution with a Glu residue, can provoke conformational changes of
the B-chain (25, 26) that may influence formation of one or more
interchain disulfide bonds (33). (Disulfide mispairing is also known to occur during production of recombinant IGF-1; see "Discussion".) To
study this question, we examined the mobility of intracellular single-chain insulins bearing various B-chain point mutations upon
reducing versus nonreducing SDS-PAGE. In short, upon
reducing SDS-PAGE, all single-chain insulin constructs exhibited an
identical mobility (Fig. 3A,
left). However, upon nonreducing SDS-PAGE, single-chain insulins
bearing B9D, B10D, or B12E point mutations consistently exhibited a
slower SDS-PAGE mobility (arrows in Fig. 3A,
right). The B28K,B29P double mutant was always more poorly expressed than the other insulin constructs; however, its mobility by
nonreducing SDS-PAGE appeared normal (Fig. 3A). The
isoelectric points of the constructs were unlikely to explain the
anomalous mobility of the B-chain point mutants, as these parameters
did not strictly correlate (Fig. 3A, and data not shown). To
determine whether any of the mutant forms contained unpaired cysteine
residues bearing free thiols, each construct was incubated (or not) in the presence of AMS (34). This compound adds >500 daltons to the
molecular mass of the polypeptide upon thiol reaction with an AMS
residue, and such a molecular mass increase should be readily detected
as loss of the 6-kDa single-chain insulin band (with shift to a higher
molecular weight). However, as shown in Fig. 3B, none of the
B-chain point mutants showed detectable reactivity with AMS (unless
disulfide bonds were first broken by treatment with dithiothreitol; not
shown). Thus, no positive evidence for unpaired cysteine residues could
be found for any of the B-chain point mutants studied.
Distinct Isoforms of Single-chain Insulin Bearing B-chain Point
Mutations--
Pulse-labeled yeast cells expressing wild-type control
ICFP or ICFP bearing B9D, B10D, B12E, or B28K,B29P mutations were
chased for 45 min in complete medium, and both the lysed cells and
chase media were analyzed by insulin immunoprecipitation and
nonreducing SDS-PAGE. As shown in Fig. 4
(upper panels), in which equal proportions of cell lysate
and medium were analyzed, a nearly undetectable quantity of processed
insulin was released to the medium (amounting to no more than a few
percent) for any of the constructs examined. However, when
markedly unequal exposures of these gels were obtained, significantly
underexposing the cell lysate insulin while overexposing that recovered
in the medium (Fig 4, lower panels), two interesting features became apparent. First, although the bulk of intracellular B9D, B10D, and B12E mutants exhibited abnormal mobility on nonreducing gels, a much smaller quantity of these mutant insulins exhibited a
mobility that was indistinguishable from that of the single-chain insulin bearing the wild-type control sequence (Fig. 4,
left). Second, for these mutants, the very little
processed insulin that was secreted was enriched in a species for which
mobility was indistinguishable from that of the single-chain insulin
bearing the wild-type control sequence (Fig. 4, right).
Together, the data in Figs. 3 and 4 demonstrate that for single-chain
insulin bearing certain B-chain point mutations, distinct isoforms are detectable upon nonreducing SDS-PAGE, which can no longer be
distinguished upon disulfide reduction. Assuming that the faster
species co-migrating with single-chain insulin bearing the wild-type
control sequence represents a form containing three correct (A6-A11,
B7-A7, B19-A20) disulfide pairs, then a substantial portion of these
B-chain point mutants is likely to represent an isomeric by-product of
equal molecular mass but with one or more disulfide bonds interchanged (33, 35-38). Moreover, although the processed insulin moiety is very
poorly secreted, in the case of these selected B-chain point mutants,
the secretion tends to be enriched in the better folded isomer, whereas
what is retained intracellularly tends to be enriched in the misfolded
isomer.
In Vitro Disulfide Isomerizaton of Single-chain Insulin Bearing
B-chain Point Mutations--
In the course of these studies it was
observed that unlike in authentic two-chain insulin, which, after SDS
denaturation, is very sensitive to in vitro disulfide
reduction because of chain dissociation, it was more difficult to
obtain completely reduced and alkylated single-chain insulin in
vitro. An example of this for the single-chain insulin bearing the
wild-type control sequence is shown in Fig.
5 (right) in which the protein
becomes divided into three bands: a fast migrating species identical to
the initial mobility under nonreduced conditions and two slower
migrating species, the slowest of which migrates in the approximate
position of fully reduced single-chain insulin (see Fig.
3A, left panel). Although this complex pattern of
bands could be attributed to incomplete initial insulin reduction with
20 mM DTT (Fig. 5), a remarkable finding was obtained when
examining the behavior of processed single-chain insulin recovered from
yeast cells bearing ICFP with various B-chain point mutations.
Specifically, for the single-chain insulin mutants B9D, B10D, and B12E
(which exhibit abnormal mobility upon nonreducing SDS-PAGE), after
initial reduction with 20 mM DTT followed by treatment with
40 mM iodoacetamide, intended to alkylate all free
sulfhydryls, the same three-species banding pattern was obtained in
proportions similar to that observed for the wild-type control protein
(Fig. 5). Indeed, the faster migrating isoform (similar to the mobility
of the nonreduced wild-type control protein) was actually faster after
DTT/iodoacetamide treatments than before. Although these
findings do not establish whether initial reduction was complete, the
data do establish that (for at least a portion of the molecules) the
disulfide reduction is followed by in vitro reoxidation of
single-chain insulins. For certain B-chain point mutants, this
reoxidation clearly matches a better folded species than that which
accumulated in living cells in the first place. Similar reoxidation
results were obtained by combining reduced single-chain insulin with
glutathione containing mixtures of reduced (GSH) and oxidized (GS-SG)
species (not shown). These results are consistent with previously
published studies of single-chain insulins in vitro
(33).
Folding and Transport of Single-chain Insulin Bearing B-chain Point
Mutations in the Secretory Pathway of Mammalian Cells--
The
foregoing data are consistent with the possibility that B9D, B10D, and
B12E point mutations may have placed conformational stress on the
single-chain insulin structure (25), which might lead to disulfide
mispairing in the oxidizing environment of the endoplasmic reticulum
(ER). As the overall oxidoreductase capability of the yeast and
mammalian ER may differ (39), it seemed of interest to examine the
folding of single-chain insulin constructs in the context of the
mammalian secretory pathway. A cDNA encoding a single-chain insulin
(SCI) bearing the wild-type preproinsulin signal peptide, B-chain and
A-chain, in which the chains are linked by the 7-amino acid
noncleavable sequence MGGGGGM, was constructed. When driven by a CMV
promoter and introduced by transient transfection in HEK293 cells, an
insulin-immunoprecipitable band was produced that migrated upon
nonreducing SDS-PAGE slightly slower than an authentic iodoinsulin
standard (Fig. 6A). This
slightly slower mobility was attributable solely to the presence of the
linker peptide, as cyanogen bromide excision of the linker caused the SCI to co-migrate precisely with the two-chain insulin
standard.2 The SCI was then
mutagenized to include B9D, B10D, or B12E point mutations. As shown in
the nonreducing gel of Fig. 6A, immunoprecipitation of B9D
or B12E point mutants produced two monomeric isoforms: a faster species
that co-migrated with the wild-type control SCI sequence and a second,
slower form (in addition small quantities of dimeric B9D mutant insulin
were observed). As in yeast (compare with Fig. 4), less than half of
the B9D or B12E mutant monomers migrated with the normal mobility. The
B10D point mutant exhibited even greater perturbation of mobility upon
nonreducing SDS-PAGE. Additionally, as has been reported for a known
disulfide-mispaired isomer of insulin (38), none of the bands was
changed by incubation with alkylating agent (+AMS,
Fig. 6B). Once again, these data suggest disulfide
mispairing of the B9D, B10D, and B12E mutant SCIs.
Remarkably, despite large-scale generation of a single-chain insulin
by-product with disulfide interchange, which must constitute significant misfolding, the mutant SCI constructs were secreted with an
efficiency comparable with that of SCI bearing the wild-type insulin
sequence. Moreover, unlike what was observed in yeast, secretion
efficiency was very high and was comparable for both disulfide isomers
of mutant SCI. An example of this is shown for the B9D mutant in Fig.
7 (but this was also observed for B10D and B12E mutants; not shown). Following a 1-h pulse labeling plus an
additional 1.5-h chase, the majority of the wild-type SCI and both
monomeric disulfide isomers of the B9D mutant were released to the
medium. This release represents genuine secretion rather than
nonspecific release (such as due to impaired cell viability) because it
was abolished by treatment of the cells with brefeldin A, which blocks
intracellular protein transport through the secretory pathway (Fig. 7).
Thus, at least in HEK293 cells, misfolded disulfide isomers of
single-chain insulin are apparently invisible both to ER
retention/quality control machinery (40) and to Golgi quality control
machinery (13).
Introduction of B-chain Point Mutations within the Coding Sequence
of Authentic Proinsulin--
The ICFP expressed in yeast employed a
short (3-amino acid) linker peptide between B- and A-chain sequences,
and the SCI expressed in mammalian cells employed a short (7-amino
acid) linker. By contrast, authentic human proinsulin employs a
35-amino acid linker that includes the 31-amino acid C-peptide plus two
sets of flanking dibasic residues. B10D is a naturally occurring point
mutation within proinsulin, associated with autosomal dominant
hyperproinsulinemia and glucose intolerance (41). Steiner and
colleagues (42) have raised the suggestion that some B10D proinsulin
molecules might undergo misfolding, including incorrect sulfhydryl
oxidation. Moreover, the proinsulin cDNA bearing the B9D point
mutation has been expressed in AtT-20 cells, where it was found to be
secreted, and this was taken as evidence suggesting that B9D proinsulin is not misfolded in the secretory pathway (43). It was therefore of
interest to re-examine the folding of proinsulin bearing B-chain point
mutations. As shown in the nonreducing gel of Fig.
8, each of the B9D, B10D, and B12E
proinsulin point mutants showed some slower mobility isoforms
consistent with disulfide mispairing. Moreover, all of the mutant
proinsulin constructs ran as a single fully reduced species under
reducing conditions. However, unlike for the ICFP and SCI constructs,
the majority of the B9D, B10D, or B12E mutant proinsulins migrated upon
nonreducing SDS-PAGE with the normal gel mobility (as exhibited by
wild-type proinsulin). Thus, these B-chain point mutations are
evidently better tolerated within the context of the full 35-amino acid
linker of proinsulin than within single-chain insulins expressed in
yeast or mammalian cells (32).
Many previous structural analyses of proinsulin, insulin, and
insulin mutants have combined to provide an extremely valuable contribution to the biotechnology of insulin production (3). However,
relatively few studies have examined the folding of such proteins
within the context of the eukaryotic secretory pathway. Our laboratory
has long been interested in the relationship between insulin
self-assembly and insulin storage in the secretory granule compartment
of mammalian cells (10, 44-46). Recently, we have expanded our
interest to include the possible relationship between the assembly of
insulin and its trafficking in the secretory pathway of yeast (5),
using a single-chain insulin like that which forms the basis for
pharmaceutical insulin production (47). For both of these systems, it
seemed of interest to study point mutants with reported defects in
insulin assembly, although it would need to be excluded that such point
mutations might interfere with proper disulfide bond maturation. Such a
concern might seem unlikely in the case of a mutant such as B10D, which
in vitro is known to stabilize insulin structure and
in vivo generally improves insulin expression. However, it
is already known that thermodynamic stability does not necessarily
correlate with biological activity (48, 49), and it is equally
important to consider that thermodynamic stability may have incomplete
predictive value with regards to the co-translational folding pathway
that occurs during translocation into the specialized environment of
the ER. In the cell, the polypeptide may fold as it enters the ER from N terminus to C terminus (50), in the immediate proximity of the
protein-conducting channel (51), timed in relation to signal peptide
cleavage (52), and in the presence of ER-specific thiol-interactor proteins, which can make mixed disulfides with newly made
thiol-containing secretory proteins (53). These compartment-specific
factors may be reconstituted using isolated ER microsomes (54), but so
far such systems are not easily transferred to current biophysical methodologies necessary for detailed analyses of protein structure and
conformational stability.
Before discussing the particular results obtained in the current study,
it is worth considering the relationship of the present analysis of
single-chain insulin and proinsulin to that of insulin-like growth
factor-1 (IGF-1), two homologous proteins that encode similar but
nonhomologous structures (4). One suggestion for the structural difference is that whereas the intra-A-chain disulfide bond of insulin
is very stable, the Cys47-Cys52 pairing of
IGF-1 is an unfavorable high energy bond that is under strain (55).
Like the situation for insulin, IGF-1 bioactivity and normal half-life
requires the presence of all three native disulfide bonds (56-58).
Although disulfide mispairing in vivo has not previously
been reported for single-chain insulin or proinsulin, it is well known
to occur during production of IGF-1 in the secretory pathway (36, 37)
and upon IGF-1 refolding in vitro (55, 59, 60). Mispaired
disulfide isomers of IGF-1 maintain some biological activity and
elements of secondary structure (56, 59). In one report, two such
disulfide isomers were clearly recovered within the same reversed-phase
high-performance liquid chromatography peak (61). Thus, such molecules
may exhibit a sufficiently folded structure to escape ER-based quality
control, resulting in secretion (37). Consistent with the idea that the B-chain provides a template to guide folding of the A-chain (27), during in vitro refolding an insulin B-chain/IGF-1 A-chain
chimera forms stable native disulfides and a single thermodynamically stable tertiary structure, whereas an IGF-1 B-chain/insulin A-chain chimera cannot form/maintain a single set of native disulfide bonds but
instead folds into two distinct disulfide isomers (28, 62). From these
data, it seems reasonable to be concerned about the possibility that
certain mutations in the insulin B-chain sequence might impair the
fidelity of native disulfide bond formation within single-chain
insulins and in proinsulin as well.
In this report, we find that each of the single-chain insulin mutants
produced in yeast is more susceptible to reduction under nondenaturing
conditions (Fig. 2). Although the vps10 mutation has only a
slight effect on the intracellular retention of these mutants (Fig. 1),
the B9D, B10D, and B12E mutants form two different gel bands
representing disulfide isomers (neither of which is appreciably
secreted, although the isomer matching the predominant conformation
assumed by the normal B-chain sequence shows a slight secretion; Fig.
4). For the misfolded isoform of these mutants, which predominates
intracellularly, a second chance at disulfide oxidation in
vitro actually appears to improve the disulfide pairing from that
of the single-chain insulin product originally synthesized in
vivo (Fig. 5). Thus, the trafficking of these three point mutants probably cannot be used to analyze the effects of oligomeric and multimeric assembly on wild-type insulin trafficking in the yeast secretory pathway because, although they do undergo anterograde transport, the bulk of the mutant molecules is not properly folded. By
contrast, the B28K,B29P double mutant does have apparently normal
disulfide maturation (Figs. 3 and 4). However, it is much more poorly
expressed under identical conditions (and needs to be overloaded in our
gels to see the band).
A similar misfolding problem occurs when the B9D, B10D, or B12E point
mutants are introduced into single-chain insulin expressed in mammalian
cells (Fig. 6A). In both yeast and mammalian cells, however,
we were unable to obtain positive evidence of unpaired cysteine
residues for any of the B-chain point mutants studied, suggesting not a
failure to oxidize but a failure to make the proper disulfide pairing
(Figs. 3B and 6B). However, in this case, a major
difference is observed between the two systems. Whereas in yeast,
almost no processed single-chain insulin is secreted (secretion being
composed primarily of the unprocessed ICFP), in mammalian
cells (at least in HEK293 cells), the SCI is efficiently secreted
regardless of the presence of B-chain point mutations or whether the
disulfide pairing is correct or incorrect (Fig. 7). Remarkably, in
these cells, even though single-chain insulins bearing B-chain point
mutations are conformationally distinct from the wild type
(e.g. Fig. 2), the novel conformations that they assume,
including disulfide mispairing, are reasonably well folded (38, 63)
such that they are not recognized by the ER quality control machinery
and they are allowed to be exported from both ER and Golgi
compartments. Nevertheless, it is extremely unlikely that single-chain
insulin isomers bearing improper disulfide pairing maintain full
biological activity (35, 38). Such mispairing will need to be
considered in the further development of SCI constructs for potential
use in gene therapy of type 1 diabetes (64).
Finally, we have examined the effects of the B9D, B10D, and B12E
mutations in the context of the wild-type human proinsulin sequence and
found that these mutations provide a similar tendency toward disulfide
mispairing (Fig. 8), and similar to SCIs, the misfolded forms are
nevertheless secreted from 293 cells (data not shown). It remains
unknown whether such mutations cause the same degree of proinsulin
misfolding when expressed in pancreatic beta cells (19) or in other
specialized secretory cell types. However, it seems clear from these
studies that the possibility of misfolding cannot be excluded simply by
virtue of the fact that the proinsulin/insulin is secreted (43). It is
likely that the content of protein disulfide isomerase, other ER
oxidoreductases, and other ER molecular chaperones may vary between
HEK293 cells and specialized secretory cells, and such activities may
affect the ratio of disulfide isomers in the context of the insulin
sequence (65-67).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
::ADE2 and carries the unmarked pep4-
H3 mutation (23). The VPS10 gene was then
disrupted in this strain (as described above) to produce strain YB136.
In addition, the TRP1 marker of the pMIGLC series of ICFP
expression vectors was replaced by the URA3 marker from a URA3-marked
centromeric plasmid (22) to create a series of vectors called pGMU,
bearing each of the insulin B-chain point mutations. These
vectors, individually, were transformed into YB136 and the AHY63
parental strain.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (71K):
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Fig. 1.
Fate of ICFP and processed single-chain
insulin bearing selected B-chain point mutations expressed in
yeast. All strains were pep4 and were based on
PA11D. Essentially identical results to those shown were obtained in
the AHY63 strain background (see "Experimental Procedures"). Cells
were pulse-labeled and chased, and the cell lysates (C) and
chase media (M) were immunoprecipitated with anti-insulin as
described under "Experimental Procedures." The bands shown
above the position of the ICFP represent nonspecific
background from labeled proteins in the cell lysates. Alternate sets of
two lanes come from vps10 mutant strains. The behavior of
the ICFP and single-chain insulin without additional B-chain point
mutations is shown in the lower panel, left 4 lanes (WT, wild type). KP, B-chain double
point mutant B28K,B29P; Ins, the position of single-chain
insulin. The right-hand panel is an enlargement of the lanes
from the upper left, showing the behavior of the B-chain
point mutant H10D. Note in this case that slightly less single-chain
insulin is recovered intracellularly, and slightly more unprocessed
precursor is secreted.
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[in a new window]
Fig. 2.
Sensitivity of processed single-chain insulin
expressed in yeast to reduction with dithiothreitol under nondenaturing
conditions. PEP4-deficient yeast cells were transformed to
express ICFP bearing either no B-chain mutation (WT) or the
B-chain point mutations as shown (KP, B28K,B29P double
mutant). The cells were pulse-labeled, chased, lysed under
detergent-free conditions, and immunoprecipitated with anti-insulin as
described under "Experimental Procedures." The
immunoprecipitates were exposed to 18 mM DTT for 30 min at
37 °C; recovery of the major single-chain insulin band by SDS-PAGE
(as per Ref. 5, Fig. 10B) is shown. Ins,
insulin.
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Fig. 3.
Anomalous mobility by nonreducing SDS-PAGE of
processed single-chain insulin bearing selected B-chain point mutations
expressed in yeast. PEP4-deficient yeast cells expressing
ICFP bearing either no B-chain mutation (WT, wild type) or
various B-chain point mutations (KP, B28K,B29P
double mutant) were pulse-labeled, chase, lysed, and immunoprecipitated
with anti-insulin. A, the immunoprecipitates were either
boiled in the presence of 100 mM DTT (left) or
nonreduced (right). B, immunoprecipitates were
incubated in a buffer containing (+) or not containing ( ) 20 mM AMS as described under "Experimental Procedures."
Unlike what is shown here, reduction prior to AMS exposure decreased
recovery of the radiolabeled single-chain insulin band consistent with
alkylation of thiol groups (not shown). Ins, insulin.
View larger version (54K):
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Fig. 4.
After processing of ICFP, single-chain
insulins bearing B9D (B9), B10D
(B10), B12E (B12), or
B28K,B29P (KP) are inefficiently
secreted and exhibit anomalous migration by nonreducing SDS-PAGE.
PEP4-deficient yeast were pulse-labeled and chased as
described under "Experimental Procedures," and equal proportions of
cell lysates and media were immunoprecipitated with anti-insulin
(upper panel) and analyzed by nonreducing SDS-PAGE. The
processed single-chain insulin monomers recovered from lysates of cells
expressing B9D, B10D, and B12E are comprised of a form of slow
gel mobility, although in this experiment a very small portion of these
mutants can be detected at a mobility similar to that of the control
single-chain insulin without B-chain point mutations (C).
Although an extremely small percentage of any of these constructs is
secreted (upper panel), upon overexposure of the gel derived
from media, the minor secretion of the B9D, B10D, and B12E single-chain
insulin mutants appears to be enriched in the faster migrating species,
which has a mobility comparable with that of the control protein.
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Fig. 5.
Reduction and partial reoxidation of
single-chain insulin expressed in yeast. PEP4-deficient
yeast were pulse-labeled and chased as described under "Experimental
Procedures," and the cell lysates were immunoprecipitated with
anti-insulin. Parallel immunoprecipitates were then either analyzed by
nonreducing SDS-PAGE (left) or boiled in the presence of 20 mM DTT, cooled, and then treated with 40 mM
iodoacetamide at 37 °C for 5 min before running the samples on
nonreducing SDS-PAGE (right). The carets (<)
correspond to the positions of isomers of the single-chain insulins
bearing different numbers or positions of disulfide bonds.
KP, B-chain double point mutant B28K,B29P. Note that for
B9D, B10D, and B12E mutants, a major species migrates faster than the
original sample by nonreducing SDS-PAGE, suggesting in vitro
disulfide reoxidation rather than alkylation for a least a fraction of
the molecules. WT, wild type.
View larger version (33K):
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Fig. 6.
Anomalous mobility by nonreducing SDS-PAGE of
SCI bearing selected B-chain point mutations expressed in mammalian
cells. A, HEK293 cells were transiently transfected
with empty pcDNA3 vector (V) or vector encoding
SCI (see "Experimental Procedures") without B-chain mutations (+)
or bearing B9D, B12E, or B10D point mutations as indicated.
A, cells were pulse-labeled for 30 min, lysed, and
immunoprecipitated with anti-insulin as described under "Experimental
Procedures" before analysis by nonreducing SDS-PAGE. In the far
left lane, the mobility of iodinated two-chain insulin standard is
shown. Note that a major fraction of the B9D, B10D, and B12E point
mutants migrate with anomalously slow monomer mobility. Additionally, a
very small fraction of the B9D mutant is reproducibly recovered as an
even slower mobility species interpreted as a dimer. All of the
single-chain insulin constructs shown here collapse to a single band of
identical mobility under reducing conditions (not shown). B,
in an experiment similar to that shown in Fig. 3B,
immunoprecipitates were incubated in a buffer containing (+) or not
containing ( ) 20 mM AMS as described under
"Experimental Procedures." However, no band shift indicative of
free thiols was detected for any of the constructs.
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Fig. 7.
Single-chain insulin bearing the B9D point
mutations is efficiently secreted from HEK293 cells. Cells
transiently transfected to express SCI bearing the wild-type B-chain
sequence (+) or mutant sequence were pulse-labeled for 60 min and
chased for 1.5 h. Where indicated, brefeldin A (BFA, 10 µg/ml) was included during the labeling and chase periods. The media
were collected, and cells were lysed. Finally, all samples were
immunoprecipitated with anti-insulin and analyzed by nonreducing
SDS-PAGE. Although the B9D mutant existed as two monomeric isomers with
different SDS-PAGE mobility (marked by carets (>)), both forms
were efficiently released to the medium, and the secretion of all forms
was blocked by brefeldin A treatment. Identical results were
obtained for SCI bearing B10D or B12E mutations (not shown).
C, cell lysate; M, chase medium.
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Fig. 8.
Anomalous mobility by nonreducing SDS-PAGE of
human proinsulin bearing selected B-chain point mutations expressed in
mammalian cells. HEK293 cells were transiently transfected with
pcDNA3 encoding wild-type human preproinsulin (+) or bearing B9D,
B12E, or B10D point mutations as indicated. Cells were pulse-labeled
for 30 min, lysed, and immunoprecipitated with anti-insulin as
described under "Experimental Procedures" before analysis by
SDS-PAGE under nonreducing conditions or after reduction with 20 mM dithiothreitol. Unlike the SCI construct, the presence
of the B9D, B10D, or B12E point mutants causes only a small fraction of
the protein to migrate with anomalously slow mobility (upper two
carets (>)) with a majority migrating in the fast mobility
position equivalent to that of wild-type proinsulin (lower
caret).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are indebted to Drs. Amy Chang (Albert Einstein College of Medicine, NY) and Thomas Kjeldsen (Novo/Nordisk, Bagsvaerd, Denmark) for helpful discussions concerning ICFP expression in yeast, as well as to members of the Arvan laboratory for suggestions made during the course of these studies.
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FOOTNOTES |
---|
* 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.
¶ To whom correspondence should be addressed: Division 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, November 21, 2002, DOI 10.1074/jbc.M209474200
2 M. Liu, J. Ramos-Castañeda, and P. Arvan, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: ICFP, insulin-containing fusion protein; AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; DTT, dithiothreitol; TEMED, N,N,N',N'-tetramethylethylenediamine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SCI, single-chain insulin; ER, endoplasmic reticulum; IGF-1, insulin-like growth factor-1.
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