The prohormone convertases PC2 (SPC2)
and PC3/PC1 (SPC3) are the major precursor processing endoproteases in
a wide variety of neural and endocrine tissues. Both enzymes are
normally expressed in the islet beta cells and participate in
proinsulin processing. Recently we generated mice lacking active PC2
due to a disruption of the PC2 gene (Furuta, M., Yano, H., Zhou, A.,
Rouillé, Y., Holst, J. J., Carroll, R. J., Ravazzola,
M., Orci, L., Furuta, H., and Steiner, D. F. (1997) Proc.
Natl. Acad. Sci. U. S. A. 94, 6646-6651). Here we report that
these PC2 mutant mice have elevated circulating proinsulin, comprising
60% of immunoreactive insulin-like components. Acid ethanol
extractable proinsulin from pancreas is also significantly elevated,
representing about 35% of total immunoreactive insulin-like
components. These increased amounts of proinsulin are mainly stored in
secretory granules, giving rise to an altered appearance on electron
microscopy. In pulse-chase experiments, the mutant islets incorporate
lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I
was processed more rapidly to insulin, reflecting the preference of
both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall half-time for
the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4-5-fold greater
elevation of des-31,32 proinsulin, an intermediate that is formed by
the preferential cleavage of proinsulin at the B chain-C-peptide
junction by PC3 and is C-terminally processed to remove
Arg31 and Arg32 by carboxypeptidase E. The
constitutive release of newly synthesized proinsulin from both mutant
and wild-type islets during the first 1-2 h of chase was normal (<2%
of total). These results demonstrate that PC2 plays an essential role
in proinsulin processing in vivo, but is quantitatively
less important in this regard than PC3, and that its absence does not
influence the efficient sorting of proinsulin into the regulated
secretory pathway.
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INTRODUCTION |
The efficient production of insulin in the pancreatic beta cell
requires that it be processed as completely as possible from its
precursor form, proinsulin, into fully active hormone before it is
stored and secreted. Conversion of proinsulin to insulin involves
cleavages at both junctions of the connecting segment that links the B
and A chains in the prohormone to liberate insulin and C-peptide (1).
The recognition sites for cleavage by the converting endoproteases
PC31 and PC2 include
minimally six residues (2) at the B chain-C-peptide junction (residues
29 through 35) and at the C-peptide-A chain junction (residues 62 through 68) of proinsulin. Initial cleavage occurs within these sites
between residues 32 and 33 and residues 65 and 66, respectively, and
the resulting C-terminal basic residue pairs
Arg31-Arg32 and
Lys64-Arg65 are then removed by the action of
carboxypeptidase E to complete the formation of the native beta cell
products. In the islet beta cells, both PC2 and PC3 are present in the
secretory granules, and these two enzymes are believed to cooperate in
processing proinsulin (3-5). Earlier studies from this laboratory
showed that PC3 cleaves preferentially at the B-C junction, while PC2 prefers the C-A junction (4). This was in keeping with the assignment
of these two convertases as the Type 1 and Type 2 calcium-dependent proinsulin processing activities,
respectively, as described by Davidson, et al. (6).
Subsequent studies have shown that either enzyme is capable of cleaving
proinsulin at both junctions to bring about its complete conversion to
insulin (7).2 In addition,
Rhodes, et al. (8) suggested that PC3 might first act at the
B-C junction to produce the intermediate des-31,32 proinsulin, which
they showed was a preferential substrate for PC2 (having an approximate
5-fold faster cleavage rate than intact proinsulin). According to their
scheme, lack of PC2 should lead to the generation of very large amounts
of the partially cleaved intermediate, des-31,32 proinsulin, which
could accumulate from the action of PC3. Since des-31,32 proinsulin is
the major intermediate form found along with proinsulin in the
circulation normally and is increased in the serum of many patients
with type II diabetes (non insulin-dependent diabetes
mellitus), it has been suggested that this indicates a relative lack of
PC2 action (9). We recently generated a PC2 null strain of mice that
lack active PC2 altogether (10). In the present study, we have examined
the effect of this mutation on the biosynthesis and processing of
proinsulin in islets from the mutant mice.
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MATERIALS AND METHODS |
Animals--
The PC2 null mutant mouse line was generated as
described previously (10). For all experiments, 8-12-week-old mutant
mice (PC2
/
) and control litter mates
(PC2+/+) of the same age were used. The care of all animals
used in these studies was in accordance with the National Institutes of
Health and University of Chicago institutional guidelines.
Insulin Biosynthesis--
Islets of Langerhans were isolated as
described (11). Isolated islets were cultured overnight in RPMI medium
containing 11 mM glucose and 10% fetal calf serum. After
overnight culture, islets were labeled in groups of 300-400 in 100 µl of pulse medium (12) containing 27.7 mM glucose, 3 µg/ml glucagon and 500 µCi (1 µCi = 37 GBq) each of
[35S]methionine (1000 Ci/mmol), [3H]leucine
(300 µCi/mmol) (Amersham Corp.). After 30 min of preincubation at
10-15 °C, islet incubations were continued at 37 °C in a water bath for 1 h (pulse). Islets were then washed, divided into four batches and incubated for 1, 2, and 3 h in medium containing 5 mM glucose and 20 µg/ml of unlabeled methionine and
leucine. After the pulse and chase incubations, the islets were washed,
resuspended in immunoprecipitation buffer (0.05 M Tris-HCl,
0.1 M NaCl, 2.5 mg/ml BSA, 1% Triton X-100, pH 7.6)
containing a mixture of protease inhibitors, and sonicated. The
supernatants, after centrifugation for 2 min at 12,000 × g, were then treated with an immunoaffinity absorbent
consisting of guinea pig anti-insulin Ig fraction coupled to Bio-Rad
Affi-Gel 10 agarose beads (Bio-Rad) (13). Insulin and
proinsulin-related immunoreactive proteins were eluted from the beads
with 30% acetonitrile, 1 M acetic acid.
HPLC Procedure--
HPLC was carried out by a modification of
the method of Davidson, et al. (6, 14) on a Waters System
using a Lichrosphere 100 RP18 column, 4.6 × 250 mm, 5 µm
particle size with prefilter (Altech). Buffers were 0.05 M
phosphoric acid, 0.10 M sodium perchlorate, 0.01 M heptanesulfonic acid in water (A) and 90% acetonitrile in water (B). Elution was begun with 42.2% buffer B isocratic for 25 min, followed by a linear gradient increasing buffer B from 42.2 to
48.8% over 112.5 min (total time = 137.5 min). (In some
experiments the gradient was ended after 75 min; total time = 100 min.) Samples were dissolved in 100 µl of 50% acetic acid and
filtered through 0.45 µm Ultrafree-MC filter units (Millipore Corp.).
Proinsulin and Insulin-like Immunoreactive Products in Serum and
Pancreas--
Pancreatic extracts were prepared as described
previously (15). Blood was taken from the retro-orbital sinus. One ml
of serum was pooled and stored at
80 °C until analyzed. Serum was applied to Sep-pak® C18 cartridge (Millipore Corp.), and
extracted protein containing proinsulin and insulin-like products (16) were lyophilized, dissolved in 3 M acetic acid and applied
to a 1 × 50 cm Bio-Gel P-30 column eluted with 3 M
acetic acid containing 50 µg/ml of BSA. Fractions containing
proinsulin or insulin were combined, dried, and resolved with
immunoprecipitate buffer following radioimmunoassay.
Immunocytochemistry--
Mice were fixed by vascular perfusion
with 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Samples of pancreatic tissue were processed for the conventional
electron microscopy by postfixation with OsO4, dehydration
with alcohol, and embedding in Epon. For light microscopic
immunofluorescence, fragments of tissue were prepared as above but by
omitting the postfixation step. Semi-thin (1 µm thick) sections were
treated for Epon removal (17) and incubated for 2 h at room
temperature with mouse monoclonal antiproinsulin antibodies (18, 19)
diluted at 0.1 µg/ml and then for 1 h with goat antimouse IgG
conjugated with fluorescein isothiocyanate. For electron microscopic
immunocytochemistry, islets of Langerhans were dissected from
glutaraldehyde-fixed pancreatic tissue, infused with 2.3 M
sucrose, and processed for cryo-ultramicrotomy (20). Frozen thin
sections were incubated for 1 h at room temperature with
antiproinsulin antibodies followed by 30 min of exposure to goat
antimouse IgG conjugated with 10-nm gold particles. Sections were
stained with 2% uranyl acetate oxalate, pH 7, for 10 min followed by
2% methylcellulose containing 0.5% uranyl acetate for 30 min.
Identification of Mouse Proinsulin-related Components by
HPLC--
Islets isolated from CD3 mice were cultured for 18 h in
RPMI medium (11.1 mM glucose) containing 10% fetal bovine
serum and then were pulse-labeled for 30 min at 37 °C in medium
containing 27.7 mM glucose and 300 µCi/ml each of
3H-leucine and [35S]methionine (12). The
islets were extracted with acid-ethanol (16), and the extracted
proteins were then separated by gel filtration over a 1 × 50 cm
column of Bio-Gel P30 eluted with 3 M acetic acid
containing 50 µg/ml BSA. The proinsulin-containing fractions,
identified by counting of aliquots, were then pooled, dried, and
redissolved in 0.01 M HCl.
In vitro conversion of aliquots of this labeled mouse
proinsulin was carried out using purified recombinant PC2 and PC3
(kindly supplied by Dr. Iris Lindberg, Louisiana State University
Medical Center, New Orleans, LA). Reactions were carried out in 0.01 M sodium acetate, pH 5.5, containing 0.1% Triton X-100, 50 µM dithiothreitol, 5.0 mM CaCl2
(reaction buffer). Five µl of recombinant PC2 or PC3 (0.23 µg) was
incubated in 80 µl of reaction buffer at 37 °C for 1 h. An
aliquot of labeled mouse proinsulin (~80,000 dpm) was then added and
incubation continued for 3 h. The reaction mixtures were
neutralized by addition of 10 µl of 1 M Tris buffer, pH
8.4, an aliquot (1.4 µg) of carboxypeptidase B (CPB) (Boehringer Mannheim) was then added, and the tubes were incubated for an additional 5 min at 37 °C. The reactions were then stopped by acidification with glacial acetic acid to pH ~2.0, and the samples were analyzed by HPLC as described above. The results are shown in
Fig. 1. In this system, mouse proinsulin
II (mP II), which contains both 35S and 3H,
elutes earlier than mouse proinsulin I (m PI) (Fig. 1A).
Fig. 1, B and C, show the effects of recombinant
PC2/CPB. A PC2 prep having very low activity (panel B)
produced only a small amount of des-64,65 mP I (migrating between mP I
and mP II), but not of mP II, indicating the more rapid attack of PC2
on the A-C junction in mP I, very likely due to the presence of the
arginine at the P4 position (fourth residue upstream from cleavage
site). Panel C shows the results using a more active
preparation of PC2; des-64,65 intermediates of both mP I and II are now
evident, but no significant amounts of any other intermediates are
detectable. However, significant amounts of both insulins I and II have
been produced, but relatively more of insulin I, indicating again that
mP I is more rapidly processed by PC2 at both junctions (both junctions
have P4 basic residues in mP I). Panel D shows the effect of
PC3/CPB. No significant production of des-64,65 intermediates or of
insulin is evident, but the expected des-31,32 mP intermediates can be
readily identified, and here again, there is more rapid processing of
mP I at the B-C junction by PC3. These results thus establish the
relative elution positions of all the important mouse proinsulin I and II conversion intermediates and insulins in this HPLC system.

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Fig. 1.
Resolution by HPLC of mouse proinsulins,
intermediates, and insulins generated in vitro by
prohormone convertases PC2 or PC3 and carboxypeptidase B. See
"Materials and Methods" for experimental details. Panel
A shows control pulse-labeled mouse proinsulin fraction;
h = mP I, g = mP II, o = Met oxidized mP II. Panels B and C show
proinsulin incubated with a low (B) or a higher
(C) amount of active recombinant PC2; f = des-64,65 mP I; e = des-64,65 mP II; b = mouse insulin I; and a = mouse insulin II.
Panel D shows proinsulin incubated with recombinant PC3;
d = des-31,32 mP I (as a shoulder on peak
o); c = des-31,32 mP II. Solid line
is 3H; dashed line is 35S
radioactivity.
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RESULTS |
Initial light microscopic studies of the islets of the PC2 null
mice revealed marked hyperplasia of the alpha and delta cells and some
diminution in beta cell mass, as was also indicated by the reduced
pancreatic content of insulin to approximately 40% of normal values
(10). The results of measurements of plasma proinsulin-related and
insulin components in extracts of pancreas or in serum are summarized
in Table I. The proportion of proinsulin was increased to 35% in pancreas, indicating a significant block in
proinsulin maturation in the storage granule compartment. In serum,
proinsulin values were elevated to 60% of total immunoreactivity, but
this greater increase merely reflects the prolonged plasma half-life of
proinsulin, as compared with insulin, in the circulation.
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Table I
Proinsulin/insulin ratio in pancreata and serum from PC2+/+
and PC2 / mice
Whole pancreata and serum pooled from animals of each genotype (age 3 months) were extracted and fractionated by gel filtration followed by
radioimmunoassay of fractions. See "Materials and Methods" for
details.
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We then carried out a series of biosynthetic studies to characterize
proinsulin and insulin-related proteins in the islets and to examine
the kinetics of conversion of the two mouse proinsulins into their
respective insulins. The two mouse proinsulins differ at five
positions, three in the C-peptide and two in the insulin (21). Mouse
proinsulin II contains a single methionine residue substituting for the
more typical lysine at position 29 in the B chain domain, which allows
it to be readily distinguished from mouse proinsulin I in dual labeling
experiments using [35S]methionine and
3H-leucine (see Fig. 3). Our initial studies confirmed the
existence of a partial block in insulin maturation but also revealed a
dramatic difference in the incorporation into
proinsulin/insulin-related components in the islets of the mutant mice.
The overall incorporation of both amino acid precursors on a per islet
basis was reduced to 10% or less of the normal control islet values
(data not shown), a much greater reduction than indicated by the
pancreatic morphometry or the assessments of insulin content, as
mentioned above. This very large decrease in incorporation persisted
after overnight culture of islets in 11.1 mM glucose. One
possible source for this discrepancy may be the very thick mantle of
alpha and delta cells that surrounds the more centrally located beta
cells (10) and that may prevent substrate access. We could partially
circumvent this problem by preincubating islets in the labeling buffer
at low temperature (5-10 °C) for 30 min prior to beginning the
37 °C pulse to allow diffusion of labeled amino acids into the islet core.
The results of a typical pulse-chase study are shown in
Fig. 2, which shows HPLC profiles for
immunopurified proinsulin/insulin components after a 1-h pulse (used to
enhance incorporation) followed by a 3-h chase. Notably in the case of
the wild-type islets, both proinsulins (components g and h) rapidly
disappear and are replaced by the two insulins (components a and b). In
contrast, the proinsulin peaks decrease more slowly in the case of the
mutant islets, and the two insulins appear more slowly. These results,
summarized in Figs. 3
and 4, indicate that mouse proinsulin I
is more rapidly processed to insulin than mouse proinsulin II in both
the wild-type and mutant islets. In the wild-type islets, low levels of
des-31,32 proinsulin intermediates are detectable, but no significant
levels of des-64,65 intermediates. In mutant islets this pattern is
similar, but the levels of both des-31,32 intermediates I and II are
increased about 5-fold to levels comprising up to 25% of the total
insulin-related proteins (Fig. 4). Interestingly, while both
proinsulins are essentially completely processed to insulin in
wild-type islets, conversion slows during the 3rd h of chase so that
about 80% of mP I is converted while only slightly more than half of
the mP II is converted to insulin. These values are in good agreement
with the immunoassay data, which indicated about 35% stored proinsulin
in mutant pancreas (Table I). Since most of this material is in
secretory granules, it is evident that conversion of proinsulin
essentially stops after 4-5 h of granule maturation. Secreted levels
of labeled proinsulin/insulin after 1 h of chase incubation
amounted to less than 2% of total islet proinsulin/insulin
radioactivity in these experiments, indicating that there was no
significant elevation of constitutive secretion from either wild-type
or mutant islets.

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Fig. 2.
Conversion of proinsulin to insulin via
intermediates in a pulse-chase format. Islets from wild-type
(left panels) or mutant (right panels) mice were
labeled with 35S-Met (dashed lines) or
3H-Leu (solid lines) in high glucose for 1 h and then chased in low glucose for a total of 3 h. Proteins were
extracted from islets at the indicated times, immunopurified, and
resolved on HPLC as described under "Materials and Methods." Peaks
are as follows: a = mouse insulin II; b = mouse insulin I; c = des-31,32 mP II; d = des-31,32 mP I; e = des-64,65 mP
II; f = des-64,65 mP I; g = mP II;
h = mP I; and o =Met oxidized mP II. Note
the rapid conversion of mP I and II into insulin in the wild-type
islets and the much slower conversion in the mutant mice associated
with significant accumulations of des-31,32 intermediates
(peaks c and d) and the absence of des-64,65
intermediates.
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Fig. 3.
Kinetics of proinsulin processing in
wild-type and mutant islets. Panel A represents the results
for mP I, and panel B represents the results for mP II from
the experiment shown in Fig. 2. Symbols are as follows: solid
squares = intact proinsulin; closed triangles = des-31,32 intermediate; and open squares = mature
insulin. See "Materials and Methods" for experimental
details.
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Fig. 4.
Time course of accumulation of des-31,32
proinsulin intermediates in wild-type and mutant islets during a 3-h
chase following a 1-h pulse. Note the much greater accumulation of
this intermediate in the mutant (closed symbols)
versus wild-type (open symbols) islets. Also note
that the level of des-31,32 intermediate of proinsulin I
(diamonds) rises and falls faster than that of proinsulin II. Data are compiled from Fig. 3.
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The data from the foregoing experiments are plotted in
Fig. 5. Assuming pseudo-first-order
initial conversion rates (1), half-lives of 0.36, 0.7, 0.8, and
2.5 h can be assigned, respectively, to PC2+/+ mP I,
PC2+/+ mP II, PC2-/- mP I, and
PC2-/- mP II. These data show that the processing of mP I
is approximately 1.9-fold faster than mP II in the PC2+/+
islets, while in the PC2
/
islets, it is about 3.1-fold
faster. However, the rate of proinsulin processing between wild-type
and mutant islets is 2.2-fold slower for mP I and 3.5-fold slower for
mP II. Since mP II makes up ~70% of the total proinsulin in mice,
the overall retardation attributable to the lack of PC2 is
approximately 3-fold.

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Fig. 5.
Semilog plot of the disappearance rates of
proinsulin-like components during a 3-h chase in islets of
PC2+/+ (open symbols) or PC2 /
(closed symbols) mice.
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The subcellular localization of these increased amounts of proinsulin
was examined by light and electron microscopic immunocytochemistry. In
contrast to normal beta cells where proinsulin is localized to the
Golgi apparatus and maturing secretory granules, proinsulin immunostaining in the beta cells of the mutant mice extends to the
majority of the secretory granules (Fig.
6 and 7). The secretory granules are
abundant in these cells, but most of them have a pale, homogeneous
appearance and lack the typical well defined central, dense crystalline
core (Fig. 8). Elevated
proinsulin/insulin ratios have been shown to inhibit the
crystallization of insulin in vitro (22). The formation of
the typical dense crystalline beta granule cores appears to be
similarly inhibited by elevated granule proinsulin in vivo
(for another example, see Ref. 23). All these findings are thus
consistent with the incomplete state of processing of the secretory
granule contents (19), but do not indicate any alteration in the
sorting of proinsulin into secretory granules in the beta cells of the
mutant mice.

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Fig. 6.
Immunofluorescence localization of proinsulin
in pancreatic islets from wild-type (A) and mutant
(B) mice. Proinsulin labeling is represented by
small bright spots located in the perinuclear region in
normal mice (A) and in the mutant (B) by a
granular immunofluorescence that extends throughout the cytoplasm.
Bar = 20 µm.
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Fig. 7.
Immunocytochemical localization of proinsulin
on frozen thin sections of pancreatic beta cells from normal
(A) and mutant mice (B). In normal mice
(A), proinsulin labeling shows the classic location over the
Golgi complex and the maturing secretory granules (msg)
associated with the Golgi apparatus. Mature granules (sg)
appear virtually free of labeling. In mutant mice (B),
proinsulin labeling is found over the Golgi complex and over the vast
majority of secretory granules (sg). Bar = 0.5 µm.
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Fig. 8.
Electron microscopic appearance of secretory
granules in thin sections of beta cells from wild-type (A)
and PC2 mutant (B) mice. Note abundant pale,
immature-appearing secretory granules in the mutant cells.
Bar = 1 µm.
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DISCUSSION |
The data presented here show that despite the lack of active PC2
about two-thirds of the proinsulin is processed to insulin in the
maturing secretory granules of the islet beta cells in the
PC2
/
mice. While earlier studies had indicated that PC3
cleaves preferentially at the B chain-C-peptide junction to generate
des-31,32 proinsulin intermediates, these data confirm that PC3 acting
alone is capable of converting proinsulin completely to insulin by
processing at both junctions (7). However, these results also clearly
demonstrate the preference of PC3 for the B chain-C-peptide junction in
that the processing of both proinsulin I and II in the mutant islets is
accompanied by significant elevations in des-31,32 proinsulin. In the
absence of PC2, there is no detectable generation of des-64,65 intermediates, as would be anticipated (4, 7, 8). The level of PC3 in
the mutant beta cells does not appear to be significantly altered (data
not shown), and thus the results we have obtained reflect the normal
roles of PC2 and PC3 in proinsulin processing in the beta
cells.
It is of interest to compare these results on the generation of
intermediate forms with those reported recently by Sizonenko and Halban
(14) in studies on proinsulin processing in rat islets of Langerhans.
While both rats and mice have two homologous proinsulin genes, there
are differences in sequence surrounding the processing sites that
affect their susceptibility to the convertases. As indicated in
Fig. 9, rat proinsulin I has P4 basic
residues at both positions while rat proinsulin II, like mouse
proinsulin II, lacks the P4 lysine residue at the B-C junction, having
methionine at this position instead. On the other hand, rat proinsulin
II retains an arginine at the P4 position at the C-A junction, while mouse proinsulin II lacks basic P4 residues at both cleavage sites. Interestingly, in their study, Sizonenko and Halban (14) also found a
slower rate of conversion of rat proinsulin II, but this was
accompanied by a marked rise in the level of des-64,65 proinsulin intermediates, in sharp contrast to our finding in normal mouse islets
where the predominant intermediate generated from both proinsulins I
and II was the des-31,32 form (Fig. 4). It is very likely that this
difference between rat and mouse proinsulin II reflects the preference
of PC2 for substrates with a P4 arginine for efficient cleavage of the
A-C junction. The lack of any build up of mouse des-64,65 proinsulin I
in the wild-type islets also indicates that PC3 prefers a P4 basic
residue for cleavage at the B chain junction.

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Fig. 9.
Amino acid sequences surrounding the
processing sites in mouse, rat, and human proinsulins. Critical P4
residues that influence processing rates are
highlighted.
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The foregoing findings are supported by molecular modeling studies of
the catalytic sites of both PC2 and PC3, which indicate that these
enzymes, like furin, contain acidic residues surrounding the P4 subsite
(2). These would be expected to facilitate interactions with basic
amino acid side chains, and hence these prohormone convertases
recognize and cleave sequences having P4 basic residues more
efficiently. The sequence data shown in Fig. 9 also indicate that human
proinsulin is unique in having a P4 basic residue at the B chain
junction (lysine) while it lacks a P4 arginine residue at the A chain
junction (leucine). As a consequence, PC3 would be expected to
efficiently cleave the B chain junction to generate des-31,32
intermediate which, in turn, is a better substrate for PC2 than intact
human proinsulin (8). There would thus be little tendency to generate a
des-64,65 intermediate. A recent study on the processing of proinsulin
in human islets supports these predictions (24). Moreover, the
composition of human plasma proinsulin-related components, which
consist predominantly of des-31,32 intermediate and intact proinsulin,
but with little or none of the des-64,65 intermediate, are in accord
with these considerations (25).
The extent to which prior cleavage at the B chain junction to generate
intermediates that are preferentially cleaved by PC2 plays a role in
the normal sequence of events in proinsulin processing will be best
assessed in future studies using mice lacking PC3, when these become
available. However, a recent report in the medical literature (26) of a
patient who is a compound heterozygote for defects in the SPC3 gene
(27) has indicated that insulin was not detectable among the
circulating plasma immunoreactive insulin-like components, the more
prominent being the des-64,65 proinsulin intermediate and large amounts
of intact proinsulin. These findings taken altogether are consistent
with the likelihood that, in both rodents and man, PC3 plays a
quantitatively more important role in proinsulin processing than PC2.
Even the most severe defect in PC2 production or action in the beta
cell would be expected to produce only a partial block in the
processing of proinsulin, but such a defect could be associated with
diabetes. However, thus far no significant association of the PC2 gene
locus with diabetes has been found although several polymorphisms have been noted (28). Mutations in the PC2 gene leading to lower PC2
expression or activity throughout the neuroendocrine system, on the
other hand, would be less likely to be associated with diabetes in view
of the much more drastic consequences of such mutations on the
production of glucagon, a hormone which normally opposes the action of
insulin. Thus in the PC2 null mice, blood sugar levels are low and
there is no impairment of glucose tolerance despite the high proportion
of circulating proinsulin-like materials (10).
We thank Dianne Ostrega and Kim Biskup for
assistance with insulin radioimmunoassays, Gavin Chiu for assistance
with the development and screening of the null mouse colony, and Hisasi
Furukawa for helpful advice and assistance.