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AMER. ZOOL., 13:591-604 (1973).
Comparative Aspects of Proinsulin and Insulin Structure and Biosynthesis
D. F. STEINER, J. D. PETERSON, H. TAGER,
Department of Biochemistry, The University of Chicago, Chicago, Illinois 60637
S. EMDIN, Y. OSTBERG, AND S. FALKMER
Kristineberg Zoological Station, S-450 34 Fiskebackskil, Sweden, and Institute of
Pathology, University of Umea, S-901 87 Umea, 6, Sweden
SYNOPSIS. This review summarizes currently available information on the composition
and structure of vertebrate insulins and proinsulins. Consideration is given to the
important structural features of insulin and its precursor that are involved in the
function and formation of the active hormone. Studies on the biosynthesis of insulin
in teleost fishes indicate the existence of larger single chain precursor forms similar
to the mammalian proinsulins. Preliminary results of experiments on insulin biosynthesis in the hagfish (Myxine glutinosa) , which has the most primitive islet parenchyma of all vertebrates, indicate the existence of a similar biosynthetic mechanism.
The major storage product in the B-cells in all the vertebrate species studies thus far
is insulin rather than proinsulin. In fishes an intracellular tryspin-like enzyme may
suffice to convert proinsulin to insulin, while in mammals a more complex mechanism
involving both an endopeptidase and an exopeptidase is probably required. Conversion
occurs within the Golgi apparatus and newly formed secretory granules in the B-cells.
The similarity to the higher vertebrates in the biosynthesis and molecular structure
of insulin in the primitive hagfish indicates that the properties and biological role
of this hormone have remained fairly constant throughout several hundred million
years, or that its evolution has followed the same pattern in most extant organisms
despite considerable differences in their origin and living conditions. A hypothesis
for the evolution of insulin and of the B-cells based on the biosynthetic mechanism involving proinsulin and its conversion to insulin is briefly considered.
INTRODUCTION
which serve to integrate and modulate the
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The fact is frequently ignored that all t h e
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organisms are continuously undergoing ^
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bi
thetic
evolutionary change and that the biochem- m e c h a n i s m s a l s Q b h a v e e v o l v e d i n '
nd
ical and morphological organization of ^
theS£ c ha i
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various tissues and metabolic pathways re- -r,,
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fleets not only an adaptation of present , „
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hormone structure and function is not iust
environmental demands but also the whole a c h a l ] e n ^
a n d fascinati
blem! it
evolutionary history of a particular orga- o f f m m a * ^ s s i b i H t i e s for b * o : T c l e n i n g o u r
nism or group
of organisms. Endocrine
J ^ I I T
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understanding ofr metabolic regulation, of
systems provide particularly fruitful areas t .
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the molecular mechanisms of action of hortor comparative studies because the vicis,
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developmental and funcsitudes of life have necessitated continual
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tional disorders which occur in endocrine
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systems and give rise to distinctive diseases,
lar and hormonal regulatory mechanisms
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T h e aim of
this report is to provide
Portions of this work were supported by grants an overview of the evolution of insulin
from ihe U.S.P.H.S. (AM 13914), the Swedish production in the vertebrates. After reviewMedical Research Council (Projects No. B73-12X- • »i
^ » ci
i i •
-710 non
A »,„ ,ot> 9 M ! u IVT J- T iing the present state of knowledge in mam718-08B and B72-12R-3863, the Nordic Insulin
,
,
Fund, and the Board for Medical Research of m a l s a n d s o m e teleost fishes concerning inSwedish Life Insurance Co.
sulin structure, the biosynthesis of insulin,
591
592
STEINER, PETERSON, TAGER, EMDIN, OSTBERG, AND FALKMER
1
2
3
A
5
6
7
S
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Phe.Val.A£n.Gln.Hi8.Uu.Cy8.Gly.Ser.Hl«.Leu.Val.Glu.Ala.Lcu.Iyr.Leu.V«l.Cy».01y.Clu.Arg.Gly.Phe.Phe.Tyr.Thr.Pro.Ly».Thr
1 2
3
4
5
6
8
9
10 11 12 13 14 15 16 17 18 19 \
21
Gly.Ilu.Val.Glu.Gln.Cys.Cys.Thr.Ser.Ilu.Cys.Ser.Leu.Tyr.Gln.Leu.Glu.Asn.Tyr.Cya.Aan.
Human I n a u l l n
FIG. 1. Primary structure of human insulin (Smith, 1966) .
and the structure and properties of proinsulin, preliminary observations on insulin
and proinsulin in the Atlantic hagfish,
Myxine glutinosa are reported.
Tihe hagfish is one of the two extant
Orders of the Cyclostomes which represent
a sister group to all the other vertebrates,
viz., the Gnathostomes. The other Order
of the Cyclostomes is the lampreys. The
hagfishes and the lampreys are of particular
interest in the comparative endocrinology
of the endocrine pancreas as they appear
to represent an evolutionary link between
the presumably gut-connected dispersed insulin-producing parenchyma of Deuterostomian invertebrates and the pancreatic
islets of vertebrates (Falkmer and Patent,
iy72). The Cyclostomes have attracted the
attention of comparative endocrinologists
in general—and that of scientists working in the field of diabetes research in
particular—since the hagfish and the
lamprey are the highly specialized survivors
of the earliest vertebrates, the Ostracoderms
(Falkmer et al., 1973), and may possibly
have some precambrian ancestor in common with the Gnathostomes (Jarvik, 1964).
Thus, in these organisms the production
of an insulin with some "primitive" features may be anticipated.
INSULIN STRUCTURE
Insulin is an unusual small protein consisting of two chains linked together by
two pairs of disulfide bonds (Fig. 1). The
A-chain usually contains 21 and the Bchain, 30 amino acid residues. The primary
structures of insulins isolated from a
variety of vertebrate species have been
determined in the interval since Sanger
used this hormone as the prototype in his
pioneering studies of amino acid sequence
(Ryle et al., 1955; Smith, 1966). These results, shown in Figure 2, indicate that
amino acid substitutions can occur at many
A CHAIN
G l y Pro
Arg Val
Ala Lys Thr
Thr lie
Asp Arg Phe
Substitutions
-
- (His) Asp
-
-
-
His Asn Arg - Asn Lys His Asp
-
G i n Ser
-
-
-
Human
Glylle'Val-Glu-Gln-Cys-Cys-Thr-Ser-lle'Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15
16 17 18 19 20 21
Hagfish
Glylle-Val-Glu-Gln-Cys-Cys.His.Lys-Arg-Cys-Ser.lle.Tyr.Asx-Leu
B CHAIN
Substitutions
Human
Del
Val
M
Meel t
0
Hagfish
-
Ala
Thr Ser G l y
Arg Ala Lys (Pro)
Ala Pro
Pro Arg
Arg Ala
Pro Pro
-
-
Pro Asp
Lys Asn
Asn Lys
-
Asn
Asp Thr
Thr Asp
- Ser
Ser
-
G i n Asp Asp -
-
Me
Del
Asn
Ser
(Gin)
Mel Asp
- Ser Ser Del Ala
Phe- Val- Asn-Gln-His-Leu-Cys-Gly Ser- His-Ley V a l - G l u - Ala-Leu-Tyr-Leu- V a l - C y i - G l y Glu- Arg-GlyPhe-Phe-Tyr-Thr-Pro- Lys-Thr
|
2
3
4
5
6
7
8
9
10 11 12 13
14 15 16 17
18 19
20
21
22 23 24
25 26 27 28 29 30
Arg-Thr- X • Gly-His-Leu-Cys-Gly-Lys-Asp-Leu-Val-Asn-Ala
FIG. 2. Compilation of known amino acid suhstiunions in the insulin molecule, including partial
sequences of the A- and B-rhains of hav-fish insu-
lin. Invariant positions are indicated by dashes,
Del = deletion. (After Smith, 1966, and Humbcl
et al., 197!i.)
593
INSULIN STRUCTURE AND BIOSYNTHESIS
positions within either chain without
greatly altering the biological effectiveness
of the hormone as measured in various
bioassay systems. On the other hand, certain structural features have been well
preserved throughout vertebrate evolution
including the positions of the three
disulfide bonds, the N-terminal and Cterminal regions of the A-chain, and the
hydrophobic residues in the C-terminal
region of the B-chain, as well as others
(Smith, 1966; Humbel et al., 1972). Since
chemical modifications in any of these
regions tend to markedly reduce or abolish
biological activity, these evidently play
important roles in maintaining the secondary and tertiary structure necessary for
activity (Carpenter, 1966; Humbel et al.,
1972). The C-terminal hydrophobic sequence of the B-chain (residues 23-27) also
plays an important role in the formation
of insulin dimers (vide infra).
Preliminary results in the characterization and amino acid sequence of hagfish
(Myxine glutinosa) insulin in our laboratories (Peterson et al., 1973) indicate that
although more than half the amino acid
residues differ from those found in the
mammalian insulins (Table 1) (see also
Weitzel et al., 1967), structural conservation is found in the important regions of
the molecule described above (Fig. 2). One
interesting difference is the substitution
of aspartic acid for histidine at position
10 of the B-chain, an important residue
for zinc binding in the formation of insulin
hexamers as described below. Hagfish insulin crystallizes under conditions similar
to those required for the crystallization of
mammalian insulin but zinc or other divalent metal ions are not needed (Fig. 3).
The biological activity of hagfish insulin
has been reported to be 2 I.U./mg (i.e., 8%
of mammalian insulin) as determined by
the fat pad assay (Weitzel et al., 1967).
Within the last three years the threedimensional structure of crystalline porcine
insulin at a high resolution has been determined successfully by means of X-ray diffraction analysis (Blundell et al., 1971).
The results have proven invaluable in
interpreting much of the available chemical data on the properties of insulin. Detailed knowledge of the spatial organization
of the molecule also promises to provide
further insight into the mechanism of
action of insulin at a molecular level. The
hexameric unit cell of crystalline zinc insulin (Fig. 4) consists of three dimers
arranged around a m,ajor three-fold axis
which passes through two zinc atoms each
of which is coordinated with three B ]0
histidine side chains located just above
or just below the plane of the hexamer
TABLE 1. CompONtiion of iovinc (S) and hagfixh (H) inxulin.
A-Chain
Lvs
His
Arg
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
He
Leu
Tvr
Piie
B-Oliain
Insulin
B
H
B
H
B
H
1
2
1
2
1
—
1
1
1
1
2
1
3
2
3
3
2
2
4
3
—
1
3
3
1
3
7
1
4
3
5
—
1
6
4
3
(i
51
6
2
1
4
1
5
2
2-3
1
3
4
4
2
1
1
2
—
1
2
2
—
1
—
1
2
1
2
—
1
1
1
3
1
3
2
3
—
—
4
2
3
4
21
4
21
2
3(1
9
1
1
4
2
1-2
1
1
3
2
2
2
30-31
6
51-52
594
STEINER, PETERSON, TAGER, EMDIN, OSTBERG, AND FALKMER
I—.'
».»
*•
'*
,
* ft
<3
FIG. 3. Tetragonal crystals of hagtish insulin. Crystallization was carried out at 20 C in sodium cit-
rate buffer at pH 6.0 in the absence of metal ions.
(Blundell et al., 1971). The insulin dimers
are held together in die crystals by hydrogen bonds between the peptide groups of
residues 24 and 26 within the C-terminal
segments of the B-chain forming an antiparallel pleated-sheet structure (Blundell
et al., 1971). The locations in space of the
known invariant amino acids within the insulin monomer are shown in Figure 5.
As might be anticipated from the extensive amino acid substitutions that occur
between mammalian and piscine insulins,
it is not surprising that the immunological
cross-reactivity between diese proteins is
rather weak. For a more extensive consideration of insulin antigenicity in relation to structure, several recent discussions
may be consulted (Humbel et al., 1972;
Arquilla et al., 1972).
derived in biosynthesis from a larger single
chain precursor protein, proinsulin (Fig. 6)
(Steiner and Oyer, 1967; Nolan et al., 1971;
Steiner et al., 1972). When islets of Langerhans isolated from rat pancreas are incubated with labeled amino acids, proinsulin is synthesized first and is subsequently
transformed to insulin by proteolysis
within the cells (Steiner et al., 1967; Clark
and Steiner, 1969). Several kinds of evidence
summarized in detail elsewhere (Steiner
et al. 1970, 1972; Kemmler et al., 1971) indicate that newly syndiesized proinsulin is
transferred from the cisternal space of the
ix>ugh endoplasmic reticulum to secretory
granules via the Golgi apparatus in a sequence similar to that known to occur
in many other secretory cells. The conversion of proinsulin to insulin is a slow
process having a half-time of about 1 hour
in rat islets in vitro (Steiner et al., 1969).
Conversion evidently begins at about the
same time that the newly synthesized pro-
THE BIOSYNTHESIS OF INSULIN
Recent studies have shown that insulin is
INSULIN STRUCTURE AND BIOSYNTHESIS
595
FIG. 4. View of the zinc-insulin hexamer along
the threefold axis showing three dimers arranged
around two zinc atoms which lie on the threefold
axis. (Reproduced with permission of Blundell et
al., 1971.)
insulin reaches the Golgi apparatus and it
continues for a relatively long time after
new secretion granules have been formed.
Crude secretion granule fraction isolated from rat islets that have been incubated for a short time with labeled
amino acids to allow these to be incorporated into proinsulin retain the
ability to convert this endogenous labeled
substrate to insulin during incubation in
vitro, but they do not convert proinsulin
that is added externally (Kemmler and
Steiner, 1970). Disruption of the particles
by sonication, freeze-thawing or detergents
destroys their ability to convert the proinsulin. These results suggest that the converting enzymes are localized within the
newly formed secretion granules or Golgi
vesicles. After most of the proinsulin has
been converted to insulin, the insulin
596
STEINER, PETERSON, TAGER, E M DIN, OSTBERG, AND FALKMER
o2
B9
FIG. 5. Locations of invariant side chains in the
insulin monomer. This view is oriented along the
threefold axis. (Reproduced with permission of
Blundell et al., 1971.)
evidently combines with zinc ions to form
small crystalline inclusions which are vis-
ible with the transmission electron microscope as the central core of the mature
80
\
FIG. 6. Structure of bovine proinsulin. (Reproduced from Xolan et al., 1071.)
INSULIN STRUCTURE AND BIOSYNTHESIS
597
correct proportions of A- and B-chains,
as well as the necessary chemical determinants for appropriate folding of the
polypeptide chain in a configuration that
is conducive to the formation of the correct disulfide bonds and tertiary structure.
However, recent studies of the biosynthesis
of several other peptide hormones which
do not contain disulfide bonds also have
indicated the existence of larger precursor
forms (Noe and Bauer, 1971; Cohn et al.,
1972; Gregory and Tracy, 1972). Clearly,
in these instances, other explanations must
exist for the occurrence of these precursors,
and we may anticipate that additional reasons for the existence of proinsulin eventually may emerge as more information
\
accumulates regarding these biosynthetic
systems.
As a consequence of the sequestration
FIG. 7. Diagrammatic representation of the insu- of the proteolytic conversion process within
lin biosynthetic mechanism of the /9-cell. (R.E.R.
the secretion granules of the B-cells, the
= rough endoplasmic reticulum, M.V. = microremainder of the proinsulin interchain
vesicles.)
connecting segment, which we have desigsecretion granules. This scheme for the nated the C-peptide, is also retained in the
biosynthesis, conversion, and intracellular secretory granules of the B-cells and distransport of new secretory products in the charged along with insulin in essentially
equivalent amounts during active granule
beta cell is summarized in Figure 7.
The role of proinsulin seems to be con- extrusion by exocytosis (Rubenstein et al.,
fined mainly to the biosynthetic process 1969). The C-peptide contains all the
since most of it is converted to insulin be- additional amino acids of proinsulin aside
fore secretion occurs. However, small from the pairs of basic residues located at
amounts of proinsulin are secreted into either end through which it is joined to the
the blood under normal conditions in man insulin chains in the intact polypeptide
and other species, and additional physio- (see Fig. 6). Methods have been developed
logic roles for proinsulin have not been in one of our laboratories for the isolation
excluded (Rubenstein et al., 1972). In of the C-peptide from fresh mammalian
terms of biosynthesis, proinsulin appears pancreas, and the amino acid sequences
to function to promote the formation of of nine mammalian C-peptides have now
the correct disulfide bonds of the insulin been elucidated (Fig. 8). This region of the
molecule. Tims, after full reduction and proinsulin molecule is far more variable
denaturation in 8 M urea at a slightly than the insulin portion. Thus, while acalkaline pH, the single peptide chain of cepted point mutations occur at a rate of
proinsulin rapidly reoxidizes to its original approximately four per hundred residues
disulfide bond structure in high yield when per million years in insulin (Dayhoff, 1972),
diluted, while insulin chains or partly this figure for the C-peptide is about 60.
cleaved intermediate forms of proinsulin Only the fibrinopeptides have undergone
give very low yields under similar condi- as rapid an evolutionary change, suggesting
tions (Steiner and Clark, 1968). Proinsulin that this portion of the proinsulin molethus ensures the efficient formation of in- cule has fewer highly specific structural resulin by providing the stoichiometrically quirements that must be conserved. MoreBETA GRANULE FORMATION
T
PROINSULIN'
(S-S Bond
formation)
1 TRANSFER STEP
STEP 3
(Enargy <tep*nd nt
Cfl*» (topandtnt)
SECRETED PRODUCTS
INSULIN
j . . .
C-PEPTIDE
I941
PROINSULIN
|.
INTERMEDIATES!
598
STEINER, PETERSON, TAGER, EMDIN, OSTBERG, AND FALKMER
1
2
3
4
5
6
7
8
9
10 11
12
13
14 15
NH 5 + - Glu - Alo - Glu - Asp - Leu - Gin - Vol - Gly - Gin - Vol - Glu - Leu - Gly - Gl£ - Gly -
MAN
NH j * - Glu - Alo - Glu - Asp - Pro - Gin - Vol - Gly - Gin - Vol - Glu - Leu - Gly - Gl^ - Gly -
MONKEY
N H 3 * - Glu'- Alo - Glu - Asp - Pro - Gin - Vol - Gly - Glu - Vol - Glu - Leu - Gly - Gly_ - Gly -
HORSE
N H , * - G l u - V o l - Glu - Asp-Pro - Gin-Vol-Pro-Gin-Leu-Glu-Leu-Gly - G l y - G l y -
RAT I
NH S * - Glu - Vol - G l u - A s p - P r o - G i n - V o l - A l o - G i n - L e u - G l u - L e u - Gl*-Gly_-Gly-
RAT I
NH S * -Glu-Alo - G l u - A s n - P r o - G i n - A l o - G l y - A l o - V o l - G l u - L e u - G l y - G l ^ - G l y -
PIG
N H S + - Glu-Vol -GJu_-Gly - Pro - Gin -Vol -Gly- Alo - Leu-Glu - L e u - Alo- Gly - Gly -
COW, LAMB
+
N H S - Asp-Vol - Glu -
16
17
18
19
-Leu-Alo-GI^-Alo-
20
21 22 23
24 2 5 26
27
28
29
30
DOG
31
- Pro-Gly- Alo- Gly - Ser - Leu - Gin - Pro - Leu - Alo •Leu-Glu-Gly-Ser-Leu-GJn-COiT MAN
- Pro-Gly - A l o - Gly - Ser - Leu - Gin - Pro -Leu - Alo •Leu - Glu - Gly - Ser - Leu -Gin - C02~ MONKEY
- Pro-Gly- Leu-Gly -Gly - L e u - G i n - P r o - L e u - A l a Leu-Alo-Gly-Pro-Gln-Gln_-CO 2 ~ HORSE
- Pro-Glu- Alo-Gly - Asp-Leu-Gin-Thr-Leu-Ala Leu-Glu-Vol-Ala-Arg-Gin-C0 2 ~ RAT I
- P r o - G l y - Alo-Gly - Asp - Leu - Gin - Thr - Leu- Alo • Leu-Glu-Vol-Alo-Arg-Gin-C0 2 ~ RAT I
-Leu-Gly-
-Gly - L e u - G i n - Alo-Leu- Alo- Leu-Glu-Gly-Pro-Pro-Gin-C0 2 ~ PIG
- P r o - G i y - Alo-Giy - Giy - L e u -
- Glu - Gly - Pro - Pro -Gin - C0 2 ~ COW, LAMB
- Pro-Gly -Glu -Gly - Gly - L e u - G i n - P r o - L e u - A l o Leu-Glu-Gly-Alo-Leu-Gin-C0 2 ~ DOG
FIG. 8. Amino acid sequences of several mammalian C-peptides. (These sequences do not include
the basic residues at each end that link the C-pep-
tide to the insulin chains in the proinsulins of
these species.)
over, this variability implies that the Cpeptide probably does not function as an
endocrine substance in a physiological
sense, even though it is secreted into the
bloodstream with insulin (Rubenstein et
al., 1972). Nevertheless, from a comparison
of the available structures as well as the
known compositions of the C-peptides of
the anglerfish and codfish (Table 2), it does
appear that considerable structural conservatism has occurred. This is reflected in
the unusual and restricted composition of
these peptides, in the presence of a glycdnerich central region surrounded by hydrophobic regions, and in the presence of
more hydrophilic character in the regions
near the cleavage sites (Fig. 8). These features may play important roles in dictating
the folding of the peptide cliain necessary
TABLE 2. Amino acid composition of cod and
angltrfish proinsulin- connecting polypeptides.
Ood*
Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
Leu
He
Pro
Lys
Arg
Total
Anglerfish t
1
2
3
9
1
4
4
3
5
2
3
1
3
2
1
4
2
30
7
6
33
* Data from Grant and Eeid (1968).
t Data from Traketellis and Schwartz (1970).
INSULIN STRUCTURE AND BIOSYNTHESIS
for correct disulfide bond formation, and
they may serve also to direct the specific
cleavage of proinsulin by the converting
enzymes.
PROPERTIES OF PROINSULIN
The unique composition of the C-peptide
undoubtedly also confers important properties on proinsulin. The isoelectric pH of
mammalian proinsulins ranges from about
5.1 to 5.45 and is thus very close to that
of insulin (5.3). It also has similar stability,
solubility, and self association properties.
Sedimentation studies indicate that proinsulin forms dimers in dilute acid solutions
and hexamers at neutral pH in the presence
of zinc ions (Frank and Veros, 1968).
Spectral (Frank and Veros, 1970) as well
as immunological (Rubenstein et al., 1972)
studies indicate that the insulin moiety
of proinsulin must have nearly the same
conformation as native insulin, and there
is no spectral evidence for the existence of
ordered secondary structure within the
connecting peptide region. However, immunological studies in guinea pigs and
rabbits indicate that the connecting segment in proinsulin contains strongly antigenic and specific determinants (Rubenstein et al., 1972). In contrast to the lack
of reactivity of isolated insulin chains
against antibodies to native insulin, antibodies to proinsulin or to C-peptide generally react well with both of these forms of
antigen; no definite conclusions regarding
the conformation of this region can be
deduced from these results, however.
Many of the properties of proinsulin described above can be readily understood in
terms of the known three-dimensional
structure of porcine insulin. In the insulin
hexamer it is noteworthy that the Ctermini of the B-chains and the N-termini
of the A-chains, where the connecting
peptide is attached in proinsulin, lie near
the external surface of the hexamer,
oriented away from the three-fold axis
(see Fig. 4). Thus, in proinsulin hexamers,
the connecting peptide may be located
around the periphery, on the outside of
599
the polymer, where it would not obstruct
the regions involved in dimer or hexamer
formation. The ability of proinsulin to
aggregate like insulin could account for
its tendency to co-crystallize with insulin
during the commercial preparation of insulin (Steiner and Oyer, 1967; Nolan et
al., 1971). Although pure proinsulin is
less readily crystallized than insulin, preliminary X-ray studies have been carried
out and these indicate that the asymetric
unit is a dimer (Fullerton et al., 1970).
Further analysis by these techniques may
eventually provide definitive information
regarding the conformation of the insulin
moiety, as well as the C-peptide region,
in proinsulin.
BIOSYNTHESIS OF PROINSULIN AND INSULIN
IN THE HAGFISH IN VITRO
The biosynthesis of proinsulin and insulin in the hagfish is at present being
studied in vitro (Emdin et al., 1973).
Batches of 5-6 hagfish islet organs were incubated under a variety of conditions in a
medium containing glucose (3 mg/ml) and
(3H)-leucine as a tracer. The composition
of the medium was similar to that of hagfish plasma in terms of salts and amino
acids. The individual batches were then
extracted with acid ethanol, and the extracts were partially purified before gel
filtration on BioGel P-30 columns (0.9 X
100 cm) equilibrated with 3 M acetic acid.
The positions in the elution profiles and
the relative purities of hagfish proinsulin
and insulin were then determined by
means of immunoprecipitation and polyacrylamide gel electrophoresis. The rate of
incorporation of (3H)-leucine into proinsulin and insulin was found to be a slow
process, requiring 12-15 times more time
at 11 C than for rat islets at 37 C. Also, the
rate of proinsulin synthesis and conversion
was shown to be temperature dependent
(Fig. 9). At 30 C essentially no incorporation into proinsulin occurred.
Tn order to establish a precursor-product
relationship between proinsulin and insulin, several pulse-chase experiments were
600
STEINER, PETERSON, TAGER, EMDIN, OSTBERG, AND FALKMER
PR0INSULIN
500-
^•400-
300-
j= 200o
o
I iooH
25
30
35
FRACTION NO.
40
FIG. 9. Elution patterns from columns of Bio-Gel
P-30 showing labelling of proinsulin with (3H) labelled leucine at various temperatures (6, 11 and
18 C) for 48 hours. The results have been normalized, and the radioactivity of unrelated proteins
has been deleted.
carried out (Fig. 10). From these experiments the approximate half-time of conversion of proinsulin to insulin could be
calculated; these were 12 hours at 11 C and
9 hours at 18 C. The corresponding halftime for conversion in the rat is approximately 1 hour at 37 C.
protein has been isolated which consists of
the anglerfish insulin bearing an additional
tripeptide sequence, GlyThr-Lys, at the
amino-terminus of the A-chain and presumably representing a residuum of the connecting region of anglerfish proinsulin
(Yamaji et al., 1972). These workers also
have shown that this intermediate form
can be transformed to insulin by trypsin
treatment. Grant and coworkers have presented evidence suggesting that tryptic
activity alone can account for the conversion of codfish proinsulin to insulin (Grant
and Coombs, 1971; Grant et al., 1971).
They have identified a trypsin-like enzyme
in codfish islets which also appears to exist
in a zymogen or inactive form. The enzyme
can be inhibited by NEP (O-ethyl-O(p - nitrophenyl) - phenylpropylphosphonate),
an inhibitor of trypsin-like enzymes and by
DFP (di-isopropyl-fluorophosphate) (Reid
et al., 1968). In many of the fish insulins,
including the cod and the anglerfish, a Cterminal basic residue is present on the Bchain which corresponds to the penultimate lysine residue at position B-29 in most
mammalian insulins (Reid et al., 1968;
—•— 48hours pulse
in
^
2
— o - 4 8 hours pulse
+ 2 4 hours chase
3000 PROINSULIN
|
I
A\l(\
it
u
^ 2000XCTI
Several studies have now indicated that
insulin biosynthesis in teleost fishes (Grant
and Reid, 1968; Trakatellis and Schwartz,
1970; Grant and Coombs, 1971) as well as
in cyclostomes, as decribed above, proceeds
via a precursor that is similar to mammalian proinsulin. Labeled amino acids were
incorporated into proinsulin in incubated
principal islets from the cod and angler
fish (Grant and Reid, 1968; Trakatellis and
Schwartz, 1970). Insulin began to appear
later during incubation, and several intermediate forms also could be identified. In
the anglerfish an interesting intermediate
+11 C
a.
o
0.
COMPARATIVE ASPECTS OF PROINSULIN
BIOSYNTHESIS AND CONVERSION TO INSULIN
INSULIN
4000-
1000-
4
<£
25
30
35
FRACTION NO.
40
45
FIG. 10. Elution patterns from columns of Bio-Gel
P-30 obtained in a pulse-chase experiment with
hagfish islets incubated at 11 C. When the fH) leucine-medium was removed after 48 hours of incubation and replaced with a medium containing
non-labelled leucine for 24 hours, the radioactivity
of the proinsulin fell, whereas that associated with
insulin correspondingly increased. The results are
normalized essentially as described in Figure 9.
INSULIN STRUCTURE AND BIOSYNTHESIS
601
stituted the primitive "converting enzyme"
and that in the evolution of terrestrial
NHj-Ph«- L y j • Ala- Arg*Arg• Glu — - — Gin-Lys-Arg-Gly—y
Asn
forms modifications in insulin structure
-(S-S)j
1
and in the converting enzyme system were
E, I Trypsin-like enzyme
gradually added.
-Lyl-AkrArg-Vg
NH -Gly-4
Asn
NH-j-PheIt is possible that the pairs of basic
1
residues found at the cleavage sites in the
mammalian proinsulins allow for greater
NH -GltiGln'i-ys'Arg
specificity and for more rapid rates of
cleavage by the trypsin-like enzyme in the
Carboxyptldase
B-like enzyme
B-cells. The product of this kind of cleavNH -Pheage alone would be insulin bearing two
additional residues of arginine at the C3Arg
terminus of the B-chain. This form has
NH -Glu
— Gin
somewhat lower biological activity than
C- Peptlds
insulin (Chance, 1971) and is less soluble
FIG. 11. The cleavage of a mammalian proinsulin
near neutral pH due to its higher isoto insulin and C-peptide by ithe combined action
of trypsin-like and carboxypeptidase B-like prote- electric point. Removal of the arginine
residues by a carboxypeptidase-B-like
ases.
enzyme may thus be required to circumvent
Humbel et al., 1972). These lysine residues difficulties in storage or secretion that may
evidently provide the necessary basic sites arise from these altered properties.
for tryptic cleavage o£ the fish proinsulins.
It may be concluded that, despite some
The presence of an additional C-term- differences in the details, the major bioinal residue of alanine, threonine or serine chemical pathways involved in insulin bioin the mammalian insulins beyond the synthesis, as well as the molecular structure
lysine residue at B-29 (Fig. 2) requires a of insulin in a wide sampling of the vertemore complex cleavage mechanism for con- brates, ranging from the hagfish through
version of the mammalian proinsulins. The man, are strikingly similar. It is tempting
mammalian system cleaves the proinsulin to speculate that some of the protein horat the pairs of basic residues at either end mones, perhaps especially those associated
of the connecting segment and releases with the gastrointestinal tract and certain
these basic residues as free amino acids, basic metabolic functions, can remain rethus giving rise to insulin and the free markably constant even though evolution
C-peptide as the major products of conver- over several hundred million years has
sion (Fig. 11). We have shown that pan- evoked extensive changes in many other
creatic trypsin combined with an excess of processes and organ systems.
carboxypeptidase B, an exopeptidase that
cleaves C-terminal basic residues from
SOME SPECULATIONS ON THE EVOLUTION
peptides, can quantitatively convert bovine
OF INSULIN AND THE BETA-CELLS
proinsulin to insulin and C-peptide in
vitro (Kemmler et al., 1971). No degradaAs indicated elsewhere in this symposium
tion of the insulin occurred under the (Falkmer et al., 1973), there is evidence
conditions used in these model experi- that insulin-producing cells are located in
ments. Studies with isolated crude secretion the intestinal mucosa in certain invertegranule fractions from rat islets have pro- brate species and that these cells possibly
vided evidence for the existence of trypsin- were arranged similarly in the ancestral
like and carboxypeptidase-B-like activities vertebrates. Likewise, during the developin these particles (Kemmler et al., 1972), ment of the pancreas in mammals, B-cells
but the enzymes have not yet been isolated first appear in the endoderm of the gut in
or characterized. These results suggest that the region of the pancreatic anlage (Pictet
trypsin or a similar enzyme may have con- and Rutter, 1972). Whether these cells inPROINSULIN CLEAVAGE
2
2
2
+
2
602
A
STEINER, PETERSON, TAGER, EMDIN, OSTBERG, AND FALKMER
PRIMITIVE
MECHANISM
OF
INSULIN
FORMATION
>> B CELL
EVOLUTION
CIRCULATION
FIG. 12. Hypothetical mode of evolution of insulin and the B-cell system. The primitive mucosal
cells of the digestive tract may have elaborated a
proinsulin-like protein along with other digestive
hydrolases. During digestion this protoproinsulin
could be degraded to give rise to fragments having
insulin-like properties. This process might then
have been internalized in specialized cells (B-cells)
restricted to this function in order to provide more
precise regulation of the synthesis, storage, and release of the hormone.
deed arise from the endoderm of the gut
has been questioned; however, a definitive
answer to this question is not as yet available (see Epple et al., 1973).
The existence of a zymogen-like proinsulin and the presence in the B-cells of a
proteolytic converting enzyme system
which has components having modes of
cleavage similar to certain exocrine pancreatic proteases is consistent with a close
evolutionary relationship between the
acinar and B-cells. These relationships
prompt the hypothesis that in the most
primitive form the secretory cells forming
the mucosa of the intestine discharged a
number of digestive hydrolytic enzymes
into the gut, among which was a protein
resembling proinsulin (Steiner et al., 1969,
1972). This primitive proinsulin, or protoproinsulin, may have had some kind of
hydrolytic activity that has since been
lost. In the gut during the digestion of
food, the protoproinsulin may have been
degraded by the digestive proteases with
the production of small amounts of intermediate insulin-like proteins that were absorbed into the blood (Fig. 12). The close
temporal association between the appear-
ance of this insulin-like protein in the
blood and the influx, of nutrients such as
amino acids and sugars would have enhanced the possible evolution of an endocrine role for this protein. This would, of
course, be especially likely if the insulinlike protein enhanced the utilization of
these nutrients by the tissues of the organism in some way, perhaps by interacting
in a favorable manner with the plasma
membranes of the tissue cells, perhaps even
by hydrolyzing certain critical bonds in the
membranes. This cyclic absorption and interaction could have constituted the basis
for a rudimentary regulatory system which
conferred a selective advantage to these
organisms. In the course of time this primitive endocrine system may have been refined by the gradual specialization of some
of the mucosal cells for the unique role
of making and storing the insulin-like protein and releasing it in judicious amounts
at appropriate times. These specialized
cells also eventually began to discharge
the finished hormone directly into the
bloodstream, and thus retained a close
association with the vascular system even
though their direct association with the intestinal cells was lost.
Although this is an attractive hypothesis,
many gaps in our knowledge of the origin
and function of the B-cells must be filled
in before we can determine whether it is
correct. Information is especially needed
regarding the existence of proinsulin-like
proteins in invertebrates and in more primitive vertebrates. Modern methods of protein purification and the use of powerful
immunological tools should enable us to
carry out these studies in the near future.
As more structural information on many
different classes of proteins accumulates,
unsuspected relationships may suddenly
emerge. Thus, a recent study of the salivary
gland nerve growth factor has suggested
that this protein is closely related to proinsulin and that its gene may have arisen
from the gene for proinsulin by the process
of gene duplication (Frazier et al., 1972).
This observation is especially interesting in
view of the many developmental and func-
INSULIN STRUCTURE AND BIOSYNTHESIS
tional similarities between the salivary
glands and the pancreas. Likewise, secretin
from the intestine and glucagon from the
pancreatic alpha cells clearly are closely
related proteins derived from a common
ancestral gene (Mutt et al., 1970). Further
study of many exocrine pancreatic and intestinal protein sequences may reveal important evolutionary relationships between
these and some of the proteins of the ftcells. Only time and much more patient
study can slowly fill in the gaps in this
fascinating but incomplete picture, but
these efforts will surely be richly rewarded both in terms of practical as well
as theoretical gains.
REFERENCES
Arquilla, E. R., P. V. Miles, and J. W. Morris. 1972.
Immunochemistry of insulin, p. 159-173. In D. F.
Steiner and N. Freinkel [ed], Handbook of physiology. Vol. 1. The endocrine pancreas. Williams
and Wilkins Co., Baltimore.
Blundell, T. L., G. G. Dodson, E. Dodson, D. C.
Hodgkin, and M. Vijayan. 1971. X-ray analysis
and the structure of insulin. Recent Progr. Hormone Res. 27:1-40.
Carpenter, F. H. 1966. Relationship of structure to
biological activity of insulin as revealed by degradative studies. Amer. J. Med. 40:750-758.
Chance, R. E. 1971. Chemical, physical, biological
and immunological studies on porcine proinsulin and related polypeptides, p. 292-305. In Proc.
7th Congr. Int. Diabetes Fed., Buenos Aires, 1971.
Excerpta Med. Int. Congr. Ser. No. 231, Amsterdam.
Clark, J. L., and D. F. Steiner. 1969. Insulin biosynthesis in the rat: demonstration of two proinsulins. Proc. Nat. Acad. Sci. U.S.A. 62:278-285.
Colin, D. V., R. R. MacGregor, L. L. Chu, J. R.
Kimmel, and J. W. Hamilton. 1972. Calcemic
fraction-A: biosynthetic peptide precursor of
parathyroid hormones. Proc. Nat. Acad. Sci.
U.S.A. 69:1521-1525.
Dayhoff, M. O. [ed.]. 1972. Atlas of protein sequence and structure 5:50.
Emdin, S., J. D. Peterson, C. L. Coulter, Y. Ostberg, S. Falkmer, and D. F. Steiner. The structure and biosynthesis of insulin in a primitive
vertebrate, the cyclostome, myxine glutinosa. Abstract submitted to 9th Int. Congr. Biochem.,
Stockholm, 1973.
Epple, A., and T. L. Lewis. 1973. Comparative histophysiology of the pancreatic islets. Amer.
Zool. 13:567-590.
Falkmer, S., S. Emdin, N. Havu, G. Lundgren, M.
Marques, Y. Ostberg, D. F. Steiner, and N. W.
Thomas. 1973. Insulin in invertebrates and cyclo-
603
stomes. Amer. Zool.
Falkmer, S., and G. J. Patent. 1972. Comparative
and embryological aspects of the pancreatic isiets, p. 1-28. In D. F. Steiner and N. Freinkel
[ed.], Handbook of physiology. Vol. 1. The endocrine pancreas. Williams and Wilkins Co.,
Baltimore.
Frank, B. H., and A. J. Veros. 1968. Physical studies on proinsulin-association behavior and conformation in solution. Biochem. Biophys. Res.
Comm. 32:155-160.
Frank, B. H., and A. J. Veros. 1970. Interaction of
zinc with proinsulin. Biochem. Biophys. Res.
Comm. 38:284-289.
Frazier, W. A., R. H. Angeletti, and R. A. Bradshaw. 1972. Nerve growth factor and insulin.
Science 176:482-488.
Fullerton, W. W., R. Potter, and B. W. Low. 1970.
Proinsulin: crystallization and preliminary X-ray
diffraction studies. Proc. Nat. Acad. Sci. U.S.A.
66:1213-1219.
Grant, P. T., and T. L. Coombs. 1971. Proinsulin,
a biosynthetic precursor of insulin. Essays Biochem. 6:69-92.
Grant, P. T., T. L. Coombs, N. W. Thomas, and
J. R. Sargent. 1971. The conversion of ("C) proinsulin to insulin in isolated subcellular fractions
of fish islet preparations. Mem. Soc. Endocrinol.
19:481-495.
Grant, P. T., and K. B. M. Reid. 1968. Biosynthesis of an insulin precursor by islet tissue of
cod (Gadus callarias) . Biochem. J. 110:281-288.
Gregory, R. A., and H. J. Tracy. 1972. Isolation of
two "Big Gastrins" from Zollinger-Ellison tumor
tissue. Lancet 2:797-799.
Humbel, R. E., H. R. Bosshard, and H. Zahn.
1972. Chemistry of insulin, p. 111-132. In D. F.
Steiner and N. Freinkel [ed.], Handbook of
physiology. Vol. 1. The endocrine pancreas. Williams and Wilkins Co., Baltimore.
Jarvik, E. 1964. Specializations in early vertebrates.
Ann. Soc. Roy. Zool. Belg. 94:11-95.
Kemmler, W., J. D. Peterson, A. H. Rubenstein,
and D. F. Steiner. 1972. On the biosynthesis, intracellular transport and mechanism of conversion of proinsulin to insulin and C-peptide. Diabetes 21:572-582.
Kemmler, W., J. D. Peterson, and D. F. Stiener.
1971. Studies on the conversion of proinsulin to
insulin: I. Conversion in vitro with trypsin and
carboxypeptidase B. J. Biol. Chem. 246:67866791.
Kemmler, W., and D. F. Steiner. 1970. Conversion
of proinsulin to insulin in a subcellular fraction
from rat islets. Biochem. Biophys. Res. Comm.
41:1223-1230.
Mutt, V., J. E. Jorpes, and S. Magnussen. 1970.
Structure of porcine secretin. Eur. J. Biochem.
15:513-519.
Noe, B. D., and G. E. Bauer. 1971. Evidence for
glucagon biosynthesis involving a protein intermediate in islets of the anglerfish (Lophius
Americanus). Endocrinology 89:642-651.
604
STEINER, PETERSON, TAGER, EMDIN, OSTBERG, AND FALKMER
Nolan, C, E. Margoliash, J. D. Peterson, and D. F.
Steiner. 1971. The structure of bovine proinsulin. J. Biol. Chem. 246:2780-2795.
Peterson, J. D., D. F. Steiner, S. O. Emdin, Y. Ostberg, and S. Falkmer. 1973. Isolation, composition and amino acid sequence of the insulin
from a primitive vertebrate (hagfish; Myxine
glutinosa) . Fed. Proc. 32:577.
Pictet, R., and W. J. Rutter. 1972. Development of
the embryonic endocrine pancreas, p. 25-66. In
D. F. Steiner and N. Freinkel [ed.], Handbook of
physiology. Vol. 1. The endocrine pancreas. Williams and Wilkins Co., Baltimore.
Reid, K. B. M., P. T. Grant, and A. Youngson.
1968. The sequence of amino acids in insulin
isolated from the islet tissue of the cod (Gadus
callarias) . Biochem. J. 110:289-296.
Rubenstein, A. H., J. L. Clark, F. Melani, and D.
F. Steiner. 1969. Secretion of proinsulin C-peptide by pancreatic /3-cells and its circulation in
blood. Nature (London) 224:697-699.
Rubenstein, A. H-, F. Melani, and D. F. Steiner.
1972. Circulating proinsulin: immunology, measurement, and biological activity, p. 515-528. In
D. F. Steiner and N. Freinkel [ed.], Handbook of
physiology. Vol. 1. The endocrine pancreas. Williams and Wilkins Co., Baltimore.
Ryle, A. P., F. Sanger, L. F. Smith, and R. Kitai.
1955. The disulfide bonds of insulin. Biochem.
J. 60:541-556.
Smith, L. F. 1966. Species variation in the amino
acid sequence of insulin. Amer. J. Med. 40:662666.
Steiner, D. F., and J. L. Clark. 1968. The spontaneous reoxidation of reduced beef and rat proinsulins. Proc. Nat. Acad. Sci. U.S.A. 60:622-629.
Steiner, D. F., J. L. Clark, C. Nolan, A. H. Rubenstein, E. Margoliash, B. Aten, and P. E. Oyer.
1969. Proinsulin and the biosynthesis of insulin.
Rec. Progr. Hormone Res. 25:207-282.
Steiner, D. F., J. L. Clark, C. Nolan, A. H. Rubenstein, E. Margoliash, F. Melani, and P. E. Oyer.
1970. The biosynlhesis of insulin and some speculations regarding the pathogenesis of human diabetes, p. 123-132. In E. Cerasi and R. Luft [ed.],
The pathogenesis of diabetes mellitus. Proc. 13th
Nobel Symp. Almqvist and Wiksell, Stockholm.
Steiner, D. F., D. D. Cunningham, L. Spigelman,
and B. Aten. 1967. Insulin biosynthesis: evidence
for a precursor. Science 157:697-700.
Steiner, D. F., W. Kemmler, J. L. Clark, P. E. Oyer,
and A. H. Rubenstein. 1972. The biosynthesis
of insulin, p. 175-198. In D. F. Steiner and N.
Freinkel [ed.], Handbook of physiology. Vol. 1.
The endocrine pancreas. Williams and Wilkins
Co., Baltimore.
Steiner, D. F., and P. E. Oyer. 1967. The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma. Proc. Nat.
Acad. Sci. U.S.A. 57:473-480.
Trakatellis, A. C, and G. P. Schwartz. 1970. Biosynthesis of insulin in anglerfish islets. Nature
225:548-549.
Weitzel, V., W. Stratling, J. Hahn, and O. Martini. 1967. Insulin vom Schleimfisch (Myxine
glutinosa; Cyclostomata). Hoppe-Seyler's Z.
Physiol. Chem. 348:525-532.
Wilson, S. 1969. The antigenic loci in insulin, p.
403-405. In J. Ostman and R. D. G. Milner [ed.].
Diabetes. Proc. 6th Congr. Int. Diabetes Fed.,
Stockholm, 1967. Excerpta Med. Int. Congr. Ser.
No. 172, Amsterdam.
Yamaji, K., K. Tada, and A. C. Trakatellis. 1972.
On the biosynthesis of insulin in anglerfish islets.
J. Biol. Chem. 247:4080-4088.