Vol. 54, No. 3, 1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
N-TERAKINAL SEQUENCE OF SWINE PEPSINOGEN AND PEPSIN. THE: SITE OF PEPSINOGEN ACTIVATION. V.M.Stepanov,
L.A.Baratova, I.B.Pugacheva, L.P.Revina, E.A.Timokhina.
L.P.Belyanova,
Institute of Genetios and Selection of Industrial Mioroorganisms, Post Box 379, Moscow D-182, Laboratory of Bioorganic Chemistry, Moscow State University, Moscow, USSR. Received
August
21,
1973
SUMMARY. The sequence of 119 amino acids of swine pepsinogen comprising the fragment released during the 5ymogen activation as well as the N-terminal part of pepsin is established. The activation of swine pepsinogen is shown to be accompanied by specific cleavage of Leu-Ile bond in the sequence: 41 46 Ala Ala Ala Leu Ile Gly where Ile+ represents the N-terminal residue of pepsin.This sequence is attacked in the course of pepsinogen activation by external enzymes - neutral protelnases and elastase. INTRODUCTION. Although became recently
the mechanism of pepsinogen
an object
of vivid
discussion
of amino aoids of the zymogen preceding which appears Pepsinogen minal acids.
as the N-terminal
activation
fragment
isoleucine
Ong and Perlmann established
the N-terminal
and presumed that
bond which is cleaved
pepsinogen
of one-two
not excluded
(6).
re ( cf.7) rather
Cop.vright AN rights
but it
it
activation
acid preceds
Itzc. reserved.
is Glu-Ile although
was accepted pepsin
1134
was
in the literatu-
has been shown in our laboratory
0 19 73 by Academic Press, of reproductim in any /bun
sequence of
amino acids between these residues
This assumption
than glutamio
of N-ter-
paper 44 amino
41 amino acids of pepsinogen the presence
residue
by the cleavage
as shown in this
during
the sequence
(l-4)
of pepsin was not known.
is accompanied
containing
activation
N-terminal
that
leucine
residue
-
BIOCHEMICAL
Vol. 54, No. 3, 1973
isoleuoine
in pepainogen
ded N-terminal
sequence (4).
in definite
during
pepsinogen
allows
terms the change of primary
AND METHODS. The preparation has been described
acid sequences were determFned Sequenoer,
struoture
of swine pepsinogen elsewhere
40 per cent pyridine.
I-Chlorobutane
by treatment
contained
with
isolated lysates were
chromatography
Pepsinogen as follows.
N-terminal
kept for
To the solution
(Fig.?) (12)
pH 5.0,
20
mg of elastase
At this
was performed in 50 ml in 10 ml 20 h.
were added and the solution time the activity
ensyme. In the control
of elastase
hydro-
was inoubated
showed almost quantitative
active
were
structures
of 300 mg of pepsinogen
then IO mg of elastase
the addition
(PTH)
conversion
elastase
were added. The mixture
of the zymogen into
after
B-2. Their
pancreatic
with
measured by milk-clotting
out
fragment
activation
24 h. at 36'.
in
min. PTH were snalysed
or thermolytic
procedures.
of the same buffer at 36*,
(9-11)
buffer,
dissolved
the extraction
to the sequence 45-119
by aonvent.ional.
of 0.1 88 acetate
370
(8).
from cbymotrgptic of pepsin
with
phenylthiohydantoins
when necessary
derivatives
corresponding
studied
Protein-
5.10W5?d dithiotreitol.Thiaaolinones
HCl at 80' for110
IN
to trimethylsilyl Peptides
used for
to the corresponding
by gas-liquid
Amino
Model 890. The runs were performed snd 300 nmol of pepsinogen
were converted
(4).
using BecbmanSpinco
nmol of leucyl-pepsin of thi~solinones
us to de-
aotivation.
and of leucyl-pepsin Peptide
The study of an exten-
sequence of swine pepsinogen
scribe
MATEIUAI&
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
was
of the mixture conversion mixture
with-
only 2.5 per cent of pepsinogen
were activated. The mixture
was applied
on DEWS-cellulose 1165
column (2 x 40 cm)
Vol. 54, No. 3, 1973
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fia.1. The N-terminal sequence of swine pepsinoaen (P.). The line C. shows the homologous sequences of calf proohymosln (13). 15
P. Leu Val Iga Vsl PRO Ll!XJVsl Arg Lys LYS SER LEU AEG Gln Asn C. Ile Thr Arg Ile PRO LEU !Qr rips Gly LYS SER LEU AZ I.ys Ala 30
P. LEU Ile Iys Asp GLY Lys LEU Iys ASP PHE LEU LYS Thr his Lys C.IlEU~sGlyRisGLY~uLWJGlyASP~LEUUSS 45
P. His Aan Pro Ala Ser Iya Tyr Phe Pro Glu Ala Ala Ala Leu Ile Gly Glu Val C. P. Gly Asp Glu PROLHJ Glu ASN TYP LED ASP Thr Glu TYR PHE & C.AlaSerValPROLEUThrASNTYRLEUASPSerGlnTYRPHXGLY P. Thr ILE Gly Ile
GLY THR PROAla Gln Asp PEE Thr Val Ile %
C.~sILETyrLeuGLYTHRPROProGlyGluPHE 90
P. ASP THR GLY SEE SEEiAsn Leu TRP Val Pro Ser Vsl Tyr CYS Ser C.ASPTRBGLYSERSER AspPhe TRIP CYS Ip 105
P. SER Leu ALA CYS Ser Asp HIS Asn Gln PH3 Aan PROAsp Ser Asp C.SEBAanllWLCY8~sAsn~pSGlnArgPIIEAspPIK)hrg 119
P. Ser Thr Phe Glu Ala Thr Ser Glu Glu Leu Ser Ilr Fia.la.
The internal
homology in swix~ pepainogea.
16
IZJ ILELysASPGlyLysLJW IysAspPhe ~ILEGlyb8PGluProLIWGlyAan~~Asp~~ 44 equilibrated
with 0.1 M acetate buffer,
was achieved by linear (
300
ml ) to
containing
0.5
dex G-25 and lyophilized ensyme was inactivated
LEJ zips %R
pH 5.6.
gradient from the starting
M N&l
the active
Thr Tyr
in the same buffer
ensyme was collected, to give
146.7
The elution buffer
(Fig.2).
The perk
desalted on Sepha-
mg of the protein.
by phenol treatment
The
(4) snd used for
N-terminal assay and the automatic sequenoing. 14 mg of this protein gave after dinitrophenylation, acid hydrolysis and TIC 100 nmol of DNP-Als, 100 nmol of DNP-Leu and 70 nmol of
1166
BIOCHEMICAL
Vol. 54, No. 3, t 973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I,5
’ 70
095
0 200
400
600
800
ml
Pig.2. DEAB-cellulose chromatography of the mixture obtsined by pepsinogen activation with elaetase. Conditions as specified in the text. The peak which shows milk-clotting autivitg is cross-hatched. The peak I contains elastase and the peptides cleaved off in the course of activation. The pesk 2 - pepsinogen . DNP-Ile,
the latter
hydrasinolysis
two derivatives
being identified
and emino acid analysis.
amino acids
of the enzyme obtainsd
equal to that
of pepsin
( Iys,,
that
gave the sequence I-50
of pepsin.
of basic
activation
of the sequenator
(Fig.1)
was
>.
Arg2
RESULTS AND DISCUSSION. Applioation pepsinogen
The content
by elastase
his,,
after
overlapping
The sequence 'l-39 confirmed
to with
the data obtained
by Ong and Perlmann but the sequenoe Gluaa
was found for
residues
by these authors
(6).
40-41 rather
The sequeme
ed in this
sequenaing agreement peptides
41-43
Identification
the struoture of I&u-pepsin with
for
by Ile-45
the first
time revesl-
- the N-terminal
of Leu as residue
of leuayl-pepsin
(4).
emino
44 once more
The automatic
gave the sequence 4W3g which was in
the struotures
of thermolytio
suggested
Ala-Ala-Leu
work was followed
aoid of pepsin. confixmed
than Ala&lu
the
of N-terminsl
hydrolyeate
1167
54-58,
nonapeptide 59-61,
62-70,
(7), 71-
Vol. 54, No. 3, 1973
BIOCHEMICAL
Elaatase
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
~-,
CPepein
Ala Ala Ala Le?i Ile
Gly Asp Glu
from Asp,orysae Fig.3. 82, 87-91
The activation
and of ohymotryptic
The sequence determined of residues
87-119 established
The N-terminal obviously
with
site
of swine
hydrolysate
pe~einogan.
60-71,
the sequenator earlier
overlaps
sin (13)
(Fig.1).
internal
homology that
that
of calf
There is rather
(g-12).
with
that
in t&is laboratory
sequenue of swine pepsinogen
homologous with
84-88
(12).
and pepsin
c-sin
is
and proohymo-
suggestive
indioation
on the
appears when sequences 16-28 and 44-56
are compared (Fig.la). The sequence 74-82 coincides aspartic
acid residue
propane
(14).
Obviously,
Asp-76 as well
as with
ence Ile-Val-Asp Strong
this
I-p-nitrophenolcy-2,3-epoxy
inhibitor
might interaot aoid residue
also with
homology of the corresponding the suggestion
It
is shown that
the hydrolysis
of Leu-Ile
by two hydrophobic main features sider
of pepsin
trait
the cluster
promote oonteining
activation
bond (Fig.3).
with inhibitors.
of abymosin sup-
of Asp-76. is accompanied
specificity.
of alanine of pepsinogen
It
residues sequence.
of amino aoid residues
the binding
of the activating
the %ypersensitive'*
Leu-Ile
1168
by
This bond is flanked
amino acids whinh is in agreement with
the cluster
important
role
the
in the sequ-
diasoacetyl
fragment
on the essential pepsinogen
which contain
with
the aspartic
which reacts
ports
that
reacting
with peptides
is tempting
the
to con-
41-43 as funotionally It might be suggested with
short
side Chains
enzyme to the sequence bond. Peptide
Ala-Ala-
BIOCHEMICAL
Vol. 54, No. 3,1973
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Thr-Leufoundbg
Kas~ellet-- al. in the aotiVa#ngmixture of pepsinogen (15) is homologous with the sequence 4%
bovine
44 of swine pepsinogen. lysis
aocompanies
(la),
that
Apparently,
the point
that
this
part
activation external
of pepsinogen we applied
of this
traits
amino acid of pep-
evokes the suggestion the specificity
with
neutral
proteinases
residues
for
and isoleucine
the activation
1st step Ala+Leu,
resulted
product and Ala-Ala
sin is also present
the mixture
site, at
alanine,
The applica-
of following
amino
3d step Ile+Gly, data ahow that
the
of leucyl-pepsin
as a result
and
of hydrolysis
bonds of the zymogen. "Conventional" although
ding amino acids were oleaved The bond Leu-Ile
as the
far
activation
amino acids.
in cleavage
arise
So
contained
2nd step Leu+Ile,
contains
only trace off
of pep-
amounts of correspon-
during
might be bydrolysed 1169
by
with high yield
product
as N-terminal
attack
at the activation
pepsinogen
The active
alanyl-leucyl-pepsinwhich
cing.
of
by the activation
(Fig.3).
was found
elastase
slowly.
earlier
4th step Gly+Asp, 5th step Asp+Glu.These
Ala-I&u
pepsin
The fact
the proteolytic
had been proved
pancreatic rather
activation
(17)
cannot de-
attack.
the N-terminal
sequence for
of the sequenator
acids:
vari-
- in calf
residue
proteinase
This enzyme performed
leucine
glycine
of the sequence governs
of alanine
although
(16) and chicken
B (lg),
characteristic
prcteinases
pH 5.0.
are remarkably
process.
Availabi1i.Q
cluster
pepsinogen.
the N-terminal
the sequence preceding
bond &ydro-
in human (16) aad bovine
of activating
sin beers rather
tion
is N-terminal
- in swine pepsin
chymosin (20). termine
of bovine
- in human gastricsin
serine
alanine
that Leu-Val
amino acids of pepsins
Thus, valine
pepsins,
appears
the activation
The N-terminal able.
It
the automatic by elastase
sequen-
or
Vol. 54, No. 3, 1973
eventuslly
BIOCHEMICAL
by pepsin
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
derivatives
appearing
in the course
of activation. REFERENCES. I. 2. 3. 4.
5.
6. 7. 8.
9. IO. II. 12.
13. 14.
15. 16.
17.
18. lg. 20.
Bustin AL, Jacobs B.C., J.Biol.Chem.,B, 615 (1971). Funatsu M., Harada P., Hsyasbi K., Kaheda T.,Agriculturel and Biologioal Chemistry (Japan), 36, 305 (1972). Al-Janabi J., Hartsuck J.A., Tsng J., J.Biol.Chem,, x, 4628, (1972). Stepanov V.M., Timokbina E,A., Baratova LA., Belyanova L.P., Korzhenko V.P., Zhukova I.G., Bioohem.Biopbys.Res. comlnun., 43w43.2 (1971). Mc Phie P., J.Biol,Chem., 247, 4277 (1972). 6104 (19Qe). Ong E.Bo, Perlmann G.E., J.Biol.Chem.,m, g, 697 (1970) Tang J., Bioohem.Biopbys.Res~Commu.n., F'isano J., Bromert T.J., J.Biol.Chem.,~, 5597 (1969). Vakhitova LA., Amirkhsxyan M,M,,Stepanov V,M,, Biok.bimiya, Bi, 1164 (1970). Velulis B.A., Stepanov V.M., Biokbimiya, $3, 358 (1971). Pugacheva I,B., Stepanov V.M., Amirkhanyen !&AL, Biokhimiss, z, 993 (1971). Revina L.Po, Vakhitova E.A., Pugacheva I.B., Lapuk Y.I., Stepanov V.M., Biokbimiya, 22, 1074 (1972). Foltmann B., in “Atlas of Protein Sequence and Structure',' v.5, (M.o.Dayhoff, ed.) Silver Spring, Md, 1972, p.D-123. Chen K.C.S., Tang J., J,Biol.Chem.,x, 2566 (1972). Kassell B., Kay J., Urciniszan J.P.,jr., Chemistry and Biology of Peptides, Ann Arbor Science Publ. ,1972,p.713. Tang K.Jo, Tang J., Ped.Proc.,~, 239 (1961). Meitner P,A., Kassell B., Biochem.J.,121, 249 (1971). Bohak L., J.Biol.Chem.,a, 4638 (196z Ryle A.P., Porter R.R., Biochem,J.,jU, 7S (1959). Jirgensons B., Ikenaka T., Gorgyrski V., Macromol.Chem., 28, 96 ('1958).
1170