Proc. Natt Acad. Sci.
USA
Vol. 79, pp. 4575-4579, August 1982
Biochemistry
Human somatostatin I: Sequence of the cDNA
(recombinant DNA/polypeptide hormone structure/protein processing/homology)
LU-PING SHEN*, RAYMOND L. PICTETt, AND WILLIAM J. RUTTER
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143
Communicated by 1. S. Edelman, April 28, 1982
ABSTRACT RNA has been isolated from a human pancreatic
somatostatinoma and used to prepare a cDNA library. After pre-
screening, clones containing somatostatin I sequences were iden-
tified by hybridization with an anglerfish somatostatin I-cloned
cDNA probe. From the nucleotide sequence of two ofthese clones,
we have deduced an essentially full-length mRNA sequence, in-
cluding the preprosomatostatin coding region, 105 nucleotides
from the 5' untranslated region and the complete 150-nucleotide
3' untranslated region. The coding region predicts a 116-amino
acid precursor protein (Mr, 12,727) that contains somatostatin-14
and -28 at its COOH terminus. The predicted amino acid sequence
of human somatostatin-28 is identical to that of somatostatin-28
isolated from the porcine and ovine species. A comparison of the
amino acid sequences of human and anglerfish preprosomatostat-
in I indicated that the COOH-terminal region encoding somato-
statin-14 and the adjacent 6 amino acids are highly conserved,
whereas the remainder of the molecule, including the signal pep-
tide region, is more divergent. However, many of the amino acid
differences found in the pro region of the human and anglerfish
proteins are conservative changes. This suggests that the propep-
tides have a similar secondary structure, which in turn may imply
Downloaded from https://www.pnas.org by 38.43.142.174 on November 13, 2023 from IP address 38.43.142.174.
a biological function for this region of the molecule.
Somatostatin is a 14-amino acid polypeptide that inhibits the
secretion of other polypeptides and hormones, including
growth hormone, insulin, glucagon, and gastrin (reviewed in
ref. 1). It occurs in the pancreas, stomach, and small intestine,
as well as in the central nervous system. Its presence in neuronal
tissue (2) and its apparent neurophysiological action (3) has led
to the postulate that somatostatin is also a neurotransmitter.
Higher Mr forms of somatostatin immunoreactivity have
been detected (4). Studies (5) of the biosynthesis of somatostatin
in cultured rat pancreatic islets have suggested that the larger
immunoreactive species are somatostatin precursors. A 28-
amino acid peptide containing the somatostatin moiety has been
isolated from porcine intestine (6) and hypothalamus (7) and
more recently from ovine hypothalamus (8).
We recently have cloned and sequenced somatostatin cDNA
from the endocrine pancreas of anglerfish (9). The nucleotide
sequence of the coding region predicted a 121-amino acid poly-
peptide containing somatostatin at its COOH terminus. This
polypeptide is presumed to be the first somatostatin precursor.
We also discovered another cDNA from anglerfish that encoded
a 125-amino acid polypeptide that contained a somatostatin-like
moiety at its COOH terminus. This somatostatin differs from
the former somatostatin in two of the 14 amino acids (Tyr in
place of Phe-7 and Gly in place of Thr-10). Therefore, we
termed the classical somatostatin sequence somatostatin I and
the novel sequence somatostatin II. Somatostatin II has been
chemically synthesized and shown to inhibit selectively insulin
release with no detectable effect on glucagon release. Thus, the
activity is qualitatively different from somatostatin I. This jus-
The publication costs ofthis article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertise-
ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
4575
tifies the assertion that these represent two functional forms of
the hormone (unpublished observations). Subsequently, others
have demonstrated the presence of two somatostatin forms in
catfish (10-12).
This report describes the cloning and sequence determina-
tion of a cDNA coding for human preprosomatostatin I. As yet
we have been unable to detect cDNA sequences coding for a
sanatostatin II. The predicted amino acid sequence of the pu-
tative somatostatin precursor contains- segments; identical to
those of somatostatin-14 and somatostatin-28, which have been
isolated from pig and sheep, suggesting that these molecules
are derived from a common precursor. We also have compared
the amino acid sequence derived from human and anglerfish
preprosomatostatin I. An analysis of the conserved structures
provides evidence of functional regions in the molecule.
MATERIALS AND METHODS
cDNA Synthesis and Construction of Recombinant Plas-
mids. Thirty milligrams of human pancreatic somatostatinoma
tissue embedded for freeze sectioning, provided by D. M.
McCarthy, was cleaned of its embedding compound, lyophi-
lized, and then homogenized in 6 ml of 4 M guanidine thio-
cyanate (13). The RNA was sedimented through a CsCl cushion
(14), extracted with phenol/chloroform, and precipitated with
ethanol. The total yield of RNA was 480 mg. The first-strand
cDNA was synthesized in a reaction mixture (150 ml) containing
25 mg of RNA, 10 mM Tris chloride (pH 8.3), 70 mM KCI, 8
mM MgCl2, 0.05% 2-mercaptoethanol, 1 mM dGTP, dCTP,
and dTTP, 0.5 mM dATP, 40 units of reverse transcriptase
(RNA-dependent DNA nucleotidyltransferase; J. Beard, Na-
tional Cancer Institute), and 100 ,uCi (1 Ci = 3.7 X 1010 becque-
rels) of [a-32P]dATP. After 30 min at 43°C, the RNA was base-
hydrolyzed, and the first-strand cDNA molecules were tailed
with 30 dC residues by using terminal deoxynucleotidyltrans-
ferase. The second strand was synthesized as described by
Cooke et aL (15) except that DNA polymerase I replaced reverse
transcriptase for extension of the oligo(dG) primer (Collabora-
tive Research). Approximately 70 ng of dC-tailed double-
stranded cDNA was hybridized with an equimolar portion of
pBR322 that had been tailed with dG in the Pst I site. The re-
sulting hybrid plasmids were used to transform Escherichia coli
strain HB101 in compliance with the National Institutes of
Health guidelines (P3/HV1).
Library Screening. Tetracycline-resistant transformants grown
on Whatman 541 paper were screened by using probes com-
prising either the first-strand cDNA prepared from tumor RNA
(specific activity, 5 x 108 cpm/liter per mg) or a nick-translated
insert derived from cloned anglerfish or human preprosoma-
tostatin cDNA. The hybridizations were carried out at 68°C for
*
Present Address: Shanghai Institute of Biochemistry, Chinese Acad-
emy of Sciences, Shanghai, People's Republic of China.
t Present Address: Institute for Research in Molecular Biology, Uni-
versity of Paris 7, Tour 43, 2 place Jussieu, 75251 Paris, France.
 ~i
4576 Biochemistry: Shen et al.
20 hr in 0.75 M NaCV0.075 M Na citrate, pH 7/0.1 M sodium
phosphate, pH 7/0.1% polyvinylpyrrolidone/0. 1% Ficoll/1%
bovine serum albumin in the presence of sonicated, denatured
salmon sperm DNA (100 tkg/ml) and labeled DNA (105 cpm/
ml). The filters were washed twice with 0.3 M NaCV0.03 M
Na citrate, pH 7/0.1% NaDodSO4 at room temperature for 30
min with continual agitation, followed by two 15-min washes
in 0.015 M NaCl/0.0015 M Na citrate, pH 7/0.1% NaDodSO4
at 50'C. For conditions of lower stringency, the latter washing
step was eliminated. Hybridizing colonies were identified by
autoradiography for 20 hr at -70'C with a single Dupont Light-
ning Plus intensifying screen.
Characterization of Recombinants. The size of the recom-
binant plasmid was estimated as follows: a portion of the colony
was removed, and the cells were lysed; the DNA was electro-
phoresed in 1% agarose gel and transferred to nitrocellulose
filters. The hybridization and washing conditions used were as
described above.
The sequence of the cDNA inserts was determined by the
procedure of Maxam and Gilbert (16) with the G+A modifi-
cation of Cooke et aL (17).
RESULTS
Cloning and Identification of Somatostatin cDNA. Total
RNA was isolated from a portion of a human pancreatic soma-
tostatinoma that contained 100 times as much somatostatin
immunoreactivity as a normal pancreas contains (18). A cDNA
library was constructed in the Pst I site of pBR322, and 933
tetracycline-resistant, ampicillin-sensitive colonies were ob-
tained. Assuming that somatostatin mRNA was an abundant
species in the somatostatinoma, we screened for cDNA clones
derived from frequent mRNAs by in situ hybridization with
Downloaded from https://www.pnas.org by 38.43.142.174 on November 13, 2023 from IP address 38.43.142.174.
[32P]cDNA prepared from the tumor RNA; 71 such colonies
were identified, but these did not give detectable cross-hy-
bridization with anglerfish somatostatin I or II [32P]cDNA
probes. To provide a more sensitive test for hybridization,
DNAs prepared from the 71 recombinants were electropho-
resed and bound to nitrocellulose filters, which were incubated
with the two anglerfish somatostatin cDNA probes and then
washed under conditions of low hybridization stringency. Sev-
enteen recombinants now hybridized with the anglerfish so-
matostatin I cDNA probe; none hybridized with the somato-
statin II probe. Two recombinant plasmids, pHSl-68 (283 base
pairs) and pHS8-90 (382 base pairs), were shown by DNA se-
quence analysis to contain a segment encoding somatostatin.
A
Amino acids 51
4
24 78
pro- .,-
Nucleotides 105 72 234
B gions coding for somatostatin-14
Nar I Hinf I Pst I
pHS8-86
97 193 211
Hinfl
Nar I
Pst I Pst I
i -N 0 -
pHS3-16
Pst I
I '
Proc. Nati Acad. Sci. USA 79 (1982)
The cDNA library was rescreened with probes prepared from
pHSl-68 and pHS8-90 insert DNA, yielding 73 positives, 59
of which were found among the 71 "abundant" clones. The large
number of positive clones (8% of the total) is in agreement with
the high level of somatostatin found in the tumor. The restric-
tion maps of 18 of the larger recombinants were compared with
pHSl-68 and pHS8-90, allowing the selection of two plasmids
with inserts extending furthest into the 5' (pHS8-86, 581 base
pairs) and 3' (pHS3-16, 340 base pairs) regions, respectively.
These plasmids were used to derive the essentially full-length
somatostatin mRNA sequence.
Sequence of Human Somatostatin I cDNA. The general
strategy of sequence determination used for pHS8-86 and
pHS3-16 is indicated in Fig. 1. All restriction enzyme sites used
to initiate the determination were confirmed by assaying the
sequence through them from adjacent sites. In this instance,
it was important to determine the sequence of both strands
because several sequence compression artifacts were observed.
The sequence was checked against portions of sequence deter-
mined from four additional independently isolated clones. By
this method, the entire sequence was confirmed except for the
first two 5' and the last 11 3' nucleotides, which are absent from
the four extra clones. Further, comparison of the partial re-
striction maps of 17 of the larger recombinants revealed no se-
quence heterogeneity.
The 603 nucleotides of human preprosomatostatin I mRNA
sequence derived from the two clones is shown in Fig. 2, along
with the predicted amino acid sequence of the somatostatin
precursor; the size is shown in Fig. 3. The mRNA comprises
105 bases of 5' untranslated sequence, 348 bases of coding se-
quence, and the entire 150 bases of the 3' untranslated region,
which is rich in A and U and contains the classical A-A-U-A-A-
A sequence terminating 17 bases before the site of poly(A) ad-
dition. The first AUG in the sequence (nucleotides 106-108)
is likely to be the initiation codon because the region upstream
from it contains two translation termination codons in phase
with the proposed reading frame. The only region ofthe mRNA
that may be incomplete is the 5' untranslated region. However
preliminary data obtained from the DNA sequence of a genomic
clone encoding human somatostatin I, in combination with in
vitro transcription experiments, indicate that the mRNA initi-
ation site is located no more than five bases beyond the end of
the sequence presented here (unpublished results).
Structure of Human Preprosomatostatin L, The nucleotide
sequence predicts that human preprosomatostatin I is a protein
SS-28
1 14 m
3'
Poly(A)
m - FIG. 1. Organizationofhumanpre-
42 15S prosomatostatinIcDNA. (A) Structure
of preprosomatostatin and its mRNA.
The rectangle represents the trans-
lated portion of the mRNA. The re-
Bgl 11 Pst I Bgl 11 (i), somatostatin-28 (SS-28), and
the signal peptide (pre-) are indicated.
The sizes of the pre-, pro-, and SS-14
317 364 366 portions of preprosomatostatin are in-
Bgl 11 Bgl 11 dicated in terms of the numbers of
amino acids and nucleotides. The sizes
of the 5' and 3' untranslated regions
deduced from these cDNA clones are
also indicated. (B) Structures of pHS8-
86 and pHS3-16 and the strategy for
determining their sequences. The re-
striction sites at which sequence de-
Poly(A) terminations were initiated and the
Pst I Pst I direction and extent of the sequence
* ---I determinations are shown.
 Biochemistry: Shen et d Proc. NatL Acad. Sci. USA 79 (1982) 4577
ACACAAGCCGCUUUAGGAGCGAGGUUCGGAGCCAUCGCUGCUGCCUGCUGAUCCGCGCC
-102 -100
met leu ser
UAGAGUUUGACCAGCCACUCUCCAGCUCGGCUUUCGCGGCGCCGAG. AUG CUG UCC.
-90
cys arg leu gln cys ala leu. ala ala leu ser ile val leu ala
UGC CGC CUC CAG UGC GCG CUG GCU GCG CUG UCC AUC GUC CUG GCC
-80 -70
leu gly cys. val thr gly ala pro ser asp pro arg leu. arg gln
CUG GGC.UGU GUC ACC GGC.GCU CCC UCG GAC CCC AGA CUC CGU CAG'
-60
phe leu gln lys ser leu ala ala ala ala. gly lys gln glu leu
UUU CUG CAG AAG UCC CUG GCU GCU.GCC GCG GGG AAG'CAG GAA CUG
-50 -40
ala lys tyr phe leu ala glu. leu leu ser glu pro asn gln thr
GCC AAG UAC UUC.UUG GCA GAG CUG'CUG UCU.GAA CCC AAC CAG ACG
-30
glu asn asp ala leu glu pro glu asp leu ser gln ala ala glu
GAG.AAU GAU GCC CUG GAA CCU GAA GAU CUG UCC CAG GCU GCU GAG
-20 -10
gln asp glu met arg leu glu le-u gln arg ser ala asn ser asn
CAG GAU GAA AUG AGG CUU GAG CUG CAG AGA UCU GCU AAC UCA AAC
FIG. 2. The sequence .of human
preprosomatostatin mRNA deduced
1
pro ala met ala pro arg glu arg lys ala gly cys lys asn phe. from that of the composite sequences
CCG GCU AUG GCA CCC CGA GAA CGC AAA GCU GGC UGC AAG AAU UUC of the cDNA clones. The predicted
amino acid sequence is indicated and
numbered by designating the first res-
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10 14 idue of somatostatin-14 as 1. The

phe trp lys thr phe thr ser cy s AM amino acids towards the NH2 terminus
UUC UGG AAG ACU UUC ACA UCC UGU UAG CUUUCUUAACUAGUAUUGUCCAUA and COOH terminus of the peptide are
numbered negatively and positively,
UCAGACCUCUGAUCCCUCGCCCCCACACCCCAUCUCUCUUCCCUAAUCCUCCAAGUCUUC respectively. A possible signal peptide
extends from Met at position -102 to
AGCGAGACCCUUGCAUUAGAAACUGAAAACUGUAAAUACAAAAUAAAAUUAUGGUGAAAU Gly at -79; the propeptide extends
from Ala at -78 to Lys at -1; and so-
matostatin-28 extends from Ser at -14
UAU(A)n to Cys at 14.

of 116 amino acid residues with Mr 12,727. The code for the the rat precursor contains no cysteine residues. The predicted
somatostatin tetradecapeptide is located between- nucleotides amino acid sequencefrom residue 1 to 14 is identical to that of
411 and 453, followed immediately by the termination codon
UAG. The signal peptide, which is part of the primary trans-
lation product of all secreted hormones, can be recognized at
the NH2 terminus by typical structural features, including a
positively charged residue (arginine at -98) four residues from
the NH2 terminus and an internal hydrophobic core. The length
of the signal peptide varies among secreted proteins so that it
is not possible to define the start of the prosomatostatin moiety
precisely. The clipping of the prepeptide sequence in other
proteins usually occurs immediately after a small neutral amino
acid (glycine, serine, cysteine, or alanine). Because alanine is
most frequently found at the clipping site and because proline
is found occasionally in the second position of mature secreted FIG. 3. The size of the human prepro-
proteins (see compilations in refs. 21 and 22), the cleavage could -_ 750 somatostatin mRNA. Human pancreatic
somatostatinoma RNA was electropho-
occur at position -78, resulting in a 92-amino acid prosoma- resed in a 1.5% methylmercury(l) hy-
tostatin molecule (mass, 10,348 daltons). However, cleavage at droxide agarose gel (19), transferred to
aspartic acid at position -75 or at another site alsoseems pos- nitrocellulose paper (20), and hybridized
sible. Assuming one of these is correct, then three cysteines with 32P-labeled pHS1-68. The hybridiz-
would exist in the signal peptide (at positions -99, -95, and ing mRNA molecules were detected by au,
-82) and two cysteines in the somatostatin moiety, but no cys- toradiography. The size of the mRNA, in
bases, is indicated. The molecular length
teine residues would be present in the pro region. This agrees standards were HindmI-digested bacterio-
with the observation of Patzelt et aL (5) that the pro region of phage A DNA and Hae m-digested 0X174.
 4578 Biochemistry: Shen et al Proc. Natl. Acad. Sci. USA 79 (1982)
somatostatin-14, whereas that from residue -14 to 14 is iden- the sequence of somatostatin-14 at its COOH terminus. The
tical to the somatostatin-28 sequence determined for other predicted sequence agrees precisely with the known sequence
mammalian species (6-8). Presumably both peptides are re- of somatostatin-14 isolated from other mammals. Human pre-
leased from a prohormone by a trypsin-like peptidase reacting prosomatostatin I, like other secretory proteins, presumably
with basic residues at the cleavage site. Somatostatin-14 is pre- contains a signal (pre-) peptide that targets the molecule for
ceded by the basic dipeptide arginine-lysine whereas somato- secretion. We have tentatively identified residues -102 to
statin-28 is preceded by a single basic residue, arginine at po- -79 as the signal peptide on the basis of structural similarities
sition -15. The above structures would account for the Mr with other signal peptides. This leaves a pro region of 92 amino
12,000, 3,000, and 1,600 proteins having somatostatin-like im- acids beginning with alanine -78. The general organization of
munoreactivity detected in the human pancreatic somatostati- the human preprosomatostatin I molecule (summarized in Fig.
noma (18). 2) is essentially the same as that previously described for the
precursors for somatostatin I and II from anglerfish (9).
DISCUSSION Somatostatin-28 (which contains somatostatin-14 at its COOH
We isolated a somatostatin I cDNA from a human somatostat- terminus) has been described in porcine (6, 7) and ovine (8) spe-
inoma. The DNA sequence predicts the amino acid sequence cies. This identical sequence is present in the last 28 amino acids
of the primary translation product of the mRNA for preproso- of human preprosomatostatin I. Therefore, we envisage that,
matostatin I. This 116-amino acid molecule (Mr 12,727) contains after secretion of the preprosomatostatin molecule and removal
a. ::: ::: ATG CTG ::: TCC TGC CGC CTC CAG TGC GCG
*** * *** * *** *** * ***
CTG
**
GCT GCG CTG TCC ATC
* *** *
b. ATG AAG ATG GTC TCC TCC TCG CGC CTC CGC TGC CTC CTC GTG CTC CTG CTG TCC
-102 -100 -90
c. Met Leu Ser Cys Arg Leu Gln s Ala Leu Ala Ala Leu Ser Ile
d.
e
Met Lys Met Val Ser Ser Ser Arg Leu Arg Cys Leu Leu Val Leu Leu Leu Ser
n I
-107 -100 -90

GTC CTG GCC ::: CTG GGC TGT GTC ACC GGC GCT CCC TCG GAC CCC AGA CTC CGT
*T ** * ** ** * * ** * * **.* ** * * *** **
CTG ACC GCC TOO ATC AGC TGC TCC TTC GCC GGA CAG AGA GAC TOO AAA CTC CGC
-80
Val Leu Ala Leu Gly Cys Val
' t ' Thr
' Gly
-'
Ala Pro
I -
Ser Asp Pro Arg Leu Arg
Leu Thr Ala Ser Ile Ser Cys Ser Phe Ala Gly Gin Arg Asp Ser Lys LeuI
Asp
Arg
Gln Se r
Leu Arg Lys

-80

CAG TTT CTG CAG AAG TCC ::: CTG GCT GCT GCC GCG GGG AAG CAG GAA CTG GCC
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* * * *** ** * * .* *** * ** *** ** ** *

CTG CTG CTG CAC CGG TAO COG CTG:: : CAG GGC TCC AAA CAG GAC ATG ACT
-70 -60
Gln Phe Leu Gln Lys Ser Leu Ala Ala Ala Ala Gly Lys Gln Glu Leu Ala
Leu Leu Leu His Arg Tyr -Pro "'eu Gln Gly Ser Lys Gln Asp Met Thr
-70 -60

AAG TAC TTC TTG GCA GAG CTG CTG ::: TCT GAA CCC AAC CAG ACG GAG AAT
**
GAT
**
* * * *** ** *** *** ** ** ** * * *** * **-*
CGC TCC GCC TTG GCC GAG CTG CTC CTG TCG GAC CTC CTG CAG GGG GAG AAC GAG
-50 -40
Lys Tyr Phe Leu Ala Glu Leu Leu Se r Glu Pro Asn Gln Thr Glu Asn Asp
I
Arg Ser Ala Leu Ala Glu Leu Leu Leu Ser Asp Leu Leu Gln 'Gly Glu Asn Glu
-50 -40

GCC CTG GAA CCT GAA GAT CTG TCC CAG GCT GCT GAG ::: ::: CAG GAT 'GAA ATG
** *** ** G** * * * *C*C** * * ** **
GOT CTG GAG:: GAG GAG AAC TTC OCT CTG GCC GAA GGA GGA CCC GAG GAC GCC
-30 -20
Ala Leu Glu Pro Glu Asp Leu
I Ser Gln Ala Ala Glu Gln Asp Glu Met
I
Glu .Glu Asn Phe Pro Leu Ala Glu Gly Gly Pro Glu Asp Ala
Ala Leu Glu
-30
FIG. 4. Comparison of the human
and anglerfish preprosomatostatin I
AGG CTT GAG CTG CAG AGA TCT GCT AAC TCA AAC CCG GCT ATG GCA CCC CGA GAA proteins and mRNAs. Homology be-
-**
tween the sequences was maximized
* ** * * ** .* *** ** **
** ** ** *

CAC GCC GAC CTA GAG CGG GCC GCC AGC GGG GGG CCT CTG CTC GCC COO OGG GAG
-10 by inserting gaps. In nucleotide se-
Arg Leu Glu Leu Gin Arg Ser Ala Asn Ser Asn Pro Ala Met Ala Pro Arg Glu quences of human preprosomatostatin
I mRNA (line a) and anglerfish pre-
His Ala Asp Leu Glu Arg Ala Ala Ser Gly Gly Pro Leu Leu Ala Pro Arg Glu
-10
prosomatostatin I mRNA (line b), as-
-20 terisks indicate homologous nucleo-
tides. In amino acid sequences of human
CGC AAA'GCT GGC TGC AAG AAT TTC TTC TGG AAG ACT TTC ACA TCC TGT preprosomatostatin I protein (line c)
* ** ** .*** *** *** ** *** .*** *** ** ** *** ** *** **
and anglerfish preprosomatostatin I
AGA AAG GCC GGC TGC AAG AAC TTC TTC TGG AAA ACC TTC ACC TCC TGC
14
protein (line d), vertical bars indicate
1 10 homologous amino acids. The amino
Arq Lys Ala Gly COs Lys Asn Phe Phe Trp Lys Thr Phe Thr Se Cs
Arg Lys Ala Gly Cys Lys Asn Phe Phe Trp Lys Thr
I
Phe Thr
I Ser
TS
Cys
acid sequence is numbered by desig-
nating the first residue of somatostat-
1 10 14 in-14 as 1.
 Biochemistry: Shen et al.
ofthe signal peptide, somatostatin-28 and -14 may be generated
by processing of the prohormone. Indeed, a basic dipeptide
(arginine-lysine) typical of structures found at cleavage sites of
other peptide hormones exists appropriately in positions -1
and -2 just upstream from the somatostatin-14 sequence.
There is only a single basic residue (arginine) at position - 15,
directly prior to the start ofsomatostatin-28. We-assume, there-
fore, that the processing enzymes involved require basic resi-
dues and may be related to the trypsin family of proteases. It
is not yet clear whether somatostatin-28 is an obligatory pre-
cursor to somatostatin-14 or whether the cleavages are inde-
pendent. The human preprosomatostatin I sequence, together
with the proposed processing steps, account satisfactorily for the
various forms of somatostatin immunoreactivity that have been
detected. A species of Mr 12,000-12,500, detected in rat pan-
creatic islets (5) or in human somatostatinoma (18), seems larger
than expected for the Mr 10,348 prohormone. This difference
might arise because of glycosylation of the prohormone. In-
spection of the sequence indicates a possible glycosylation site
at positions asparagine-glutamine-threonine (positions -42 to
-40) in the prosomatostatin moiety (23), although there is no
experimental evidence to support this idea. Somatostatin-re-
lated species of Mrs 3,000 and 1,600 presumably represent so-
matostatin-28 and -14, respectively.
The major question raised by the characterization ofthe pre-
prosomatostatin sequence involves the function ofthe NH2 ter-
minal region of the prosomatostatin moiety. Is this simply a
"connecting peptide" linking the functional somatostatin moiety
with the signal peptide? Or do these amino acids serve another
biological function, such as is found in the multifunctional pro-
opiocortin (24)? A comparison of the structures of the human
and anglerfish somatostatin I may provide insight with respect
to the functional regions of the molecule because they should
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be rather conserved during the evolutionary process. This com-
parison is presented in Fig. 4; appropriate insertions have been
made to maximize homology. The amino acids display 45% ho-
mology overall, compared to 53% homology at the nucleotide
level. Inspection of the two structures shows that somatostatin-
14 is conserved precisely. The somatostatin-28 sequences are
79% homologous between the two species, with most of the
additional homology coming from a block of six residues im-
mediately preceding somatostatin-14. In addition, there is an-
other block of conserved amino acids surrounding the cleavage
site for somatostatin-28 (positions - 15, - 17, and - 18). These
conserved regions could provide specified sites for enzymatic
cleavage. More persuasive evidence in favor of a function for
somatostatin-28 is provided by the 100% conservation of this
peptide among mammals. Further, recent experiments show
that somatostatin-28 and somatostatin-14 bind with different
affinities to somatostatin receptors in various cells (25). The rea-
son for the divergence between anglerfish and mammalian so-
matostatin-28 at the amino terminus is not clear.
The remainder of the protein sequence shows greater diver-
gence. There is clearly no region conserved to the degree ofthe
somatostatin-14 and -28 sequences. The signal peptides of the
two species are probably of somewhat different lengths and are
about 38% homologous in amino acid sequence. Both putative
prepeptides display the type of sequence features (e.g., a hy-
drophobic core) thought to be important for the secretion func-
tion. The proregions of the prosomatostatin moiety are also of
different lengths in the two species but still show 38% sequence
homology overall; interspersed, within this area are regions of
high homology (e.g., amino acids -32 to -41 and -45 to -60).
Further, there is persuasive conservation of acidic, basic, hy-
drophobic, and hydrophilic amino acids typical of related func-
tional structures. Charged residues occur at 22 different posi-
tions within-the pro regions: 9 of these positions are homologous,
Proc. Natl. Acad. Sci. USA 79 (1982) 4579
whereas a further 11 positions represent conservative changes
(see Fig. 4). The degree of conservation of structure seen in this
region of prosomatostatin is sufficiently strong to suggest that
this region may have a biological role beyond simply connecting
the signal peptide with the functional somatostatin moiety. This
region could provide the necessary protein conformation to fa-
cilitate processing of somatostatin-28 or somatostatin-14, or
both. Alternatively prosomatostatin moiety may possess so-
matostatin-like activity different from either somatostatin-28 or
-14. Finally, a distinct biological function for a portion of pro-
somatostatin is not ruled out.
By using the somatostatin I cDNA sequences, it should be
possible to produce in an alternate host sufficient quantities of
prosomatostatin to test its biological activity and prepare anti-
bodies against this region of the molecule. This should allow
decisive tests on its fate and function.
We thank Dr. Peter Hobart for helpful advice during the course of
the experiments. We also are indebted to Dr. Graeme Bell, who pro-
vided important technical advice during the research, and to Dr.
Graeme Bell and Dr. David Standring for valuable advice during the
preparation of the manuscript. We acknowledge the assistance ofLeslie
Spector in typing the manuscript and Sonja Bock in computer analyses.
The human somatostatinoma tissue was provided by Dr. Denis Mc-
Carthy. This research was. supported by a grant from the National In-
stitutes of Health (AM21344).
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