Báo cáo khoa học: Human anionic trypsinogen Properties of autocatalytic activation and degradation and implications in pancreatic diseases potx

Human anionic trypsinogen
Properties of autocatalytic activation and degradation and implications
in pancreatic diseases
Zolta
´
n Kukor, Miklo
´
sTo
´
th* and Miklo
´
s Sahin-To
´
th
Department of Molecular and Cell Biology, Goldman School of Dental Medicine, Boston University, Boston, USA
Human pancreatic secretions contain two major trypsinogen
isoforms, cationic and anionic trypsinogen, normally at a
ratio of 2 : 1. Pancreatitis, pancreatic cancer and chronic
alcoholism lead to a characteristic reversal of the isoform
ratio, and anionic trypsinogen becomes the predominant
zymogen secreted. To understand the biochemical conse-
quences of these alterations, we recombinantly expressed
and purified both human trypsinogens and documented
characteristics of autoactivation, autocatalytic degradation
and Ca
2+
-dependence. Even though the two trypsinogens
are  90% identical in their primary structure, we found that
human anionic trypsinogen and trypsin exhibited a signifi-
cantly increased (10–20-fold) propensity for autocatalytic
degradation, relative to cationic trypsinogen and trypsin.

Furthermore, in contrast to the characteristic stimulation of
the cationic proenzyme, acidic pH inhibited autoactivation
of anionic trypsinogen. In mixtures of cationic and anionic
trypsinogen, an increase in the proportion of the anionic
proenzyme had no significant effect on the levels of trypsin
generated by autoactivation or by enterokinase at pH 8.0 in
1m
M
Ca
2+
– conditions that were characteristic of the
pancreatic juice. In contrast, rates of trypsinogen activation
were markedly reduced with increasing ratios of anionic
trypsinogen under conditions that were typical of potential
sites of pathological intra-acinar trypsinogen activation.
Thus, at low Ca
2+
concentrations at pH 8.0, selective
degradation of anionic trypsinogen and trypsin caused
diminished trypsin production; while at pH 5.0, inhibition
of anionic trypsinogen activation resulted in lower trypsin
yields. Taken together, the observations indicate that
up-regulation of anionic trypsinogen in pancreatic diseases
does not affect physiological trypsinogen activation, but
significantly limits trypsin generation under potential
pathological conditions.
Keywords: anionic trypsin; cationic trypsin; autoactivation;
autolysis; alcoholic pancreatitis.
The human pancreas secretes three isoforms of trypsinogen,
encoded by the protease, serine (PRSS)genes1,2and3.

On the basis of their relative electrophoretic mobility, the
three trypsinogen species are commonly referred to as
cationic trypsinogen (product of PRSS1, OMIM 276000),
anionic trypsinogen (product of PRSS2, MIM 601564),
and mesotrypsinogen (product of PRSS3)(forareviewon
human trypsinogen genes and proteins see [1] and references
therein). While individual variations may be considerable,
normally the cationic isoform constitutes about 2/3 of the
total trypsinogen content, and anionic trypsinogen makes
up approximately 1/3 [2–4]. Mesotrypsinogen is a minor
species, accounting for less than 5% of trypsinogens in
human pancreatic juice [5,6]. The evolutionary rationale for
the existence of several isoforms has not been clarified yet,
but it is believed that differences in inhibitor sensitivity may
be advantageous in digestion of foods containing trypsin
inhibitors.
A characteristic feature of human pancreatic diseases
as well as chronic alcoholism is the relatively selective
up-regulation of anionic trypsinogen secretion [3,4]. In
chronic pancreatitis, the total trypsinogen content of the
pancreatic juice may be unchanged or decreased while
in chronic alcoholism an increase in total trypsinogen
secretion was demonstrated. In these conditions, the pro-
portion of anionic and cationic isoforms becomes reversed,
and anionic trypsinogen dominates pancreatic secretions. In
acute pancreatitis, the ratio of trypsinogen isoforms in the
pancreatic juice has not been investigated so far, but a
preferential increase in immunoreactive anionic tryp-
sin(ogen) in the serum was documented by several studies
[7–10]. It is unclear whether or not elevated anionic

trypsinogen secretion might cause or predispose for pan-
creatitis. Alternatively, increased anionic trypsinogen secre-
tion might be innocuous or even protective in pancreatic
physiology.
Recently, methodology has been developed for the
recombinant expression, in vitro refolding and purification
of human cationic trypsinogen [11–13]. This type of
recombinant trypsinogen preparation has been used in a
Correspondence to M. Sahin-To
´
th, Department of Molecular and Cell
Biology, Goldman School of Dental Medicine, Boston University,
715 Albany Street, EVANS-4; Boston, MA 02118, USA.
Fax: +1 617 414 1041; Tel.: +1 617 414 1070;
E-mail:
Abbreviations: GPR-pNA, N-CBZ-Gly-Pro-Arg-p-nitroanilide; Hu1,
human cationic trypsinogen; Hu2, human anionic trypsinogen.
*Present address: Department of Medical Chemistry, Molecular
Biology and Pathobiochemistry, Semmelweis University, Budapest,
Puskin Street 9, Hungary, H-1088.
(Received 13 January 2003, revised 23 February 2003,
accepted 18 March 2003)
Eur. J. Biochem. 270, 2047–2058 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03581.x
growing number of studies that investigated the effects of
hereditary pancreatitis-associated mutations [11–17]. Here
we report the successful production of recombinant human
anionic trypsinogen in a pure and stable form. Activation
and degradation characteristics of this recombinant prepar-
ation was documented and compared to those of cationic
trypsinogen. Furthermore, interactions between the two

isoforms were studied and the results indicated that an
increase in the proportion of the anionic proenzyme had no
significant effect on physiological trypsinogen activation,
but resulted in decreased trypsin generation under condi-
tions that mimicked the potential milieu(s) of intracellular
pathological trypsinogen activation.
Experimental procedures
Materials
Reagent grade bovine serum albumin was purchased from
Biocell Laboratories (Rancho Dominguez, CA, USA),
N-CBZ-Gly-Pro-Arg-p-nitroanilide (GPR-pNA) was from
Sigma, and bovine enterokinase was from Biozyme Labor-
atories (San Diego, CA, USA).
Plasmid construction
The coding cDNA for human anionic trypsinogen was
PCR-amplified from a commercial plasmid (pcDNA3.1/GS
harboring GeneStorm clone no. H-M27602M, Invitrogen)
and cloned in place of the cationic trypsinogen gene in the
pTrap-T7/Hu1 expression vector using the flanking NcoI
and SacI restriction sites (pTrap-T7/Hu2). The activation
peptide sequence of recombinant anionic trypsinogen in
pTrap-T7/Hu2 was Met-Ala-Pro-Phe-(Asp)4-Lys. One of
the native EcoRI sites and the internal SacIsitewere
removed by introducing silent mutations into the codons
for Leu41 and Glu209 (numbering starts with Met1 of the
native pretrypsinogen sequence). Mutation K23Q was
introduced by linker mutagenesis. A synthetic oligonucleo-
tide linker encoding the mutation was ligated between the
NcoIandEcoRI sites of pTrapT7/Hu2. Construction of the
pTrap-T7 expression plasmid harboring the wild-type

cationic trypsinogen gene was described previously [11,14].
Expression and purification of trypsinogen
Small scale expression and in vitro refolding of human
trypsinogens was carried out as reported previously [11–13].
In a typical experiment, 200 mL cultures of Rosetta(DE3)
(Novagen) cells harboring pTrap-T7/Hu1 or pTrap-T7/
Hu2 plasmid were grown in Luria–Bertani medium with
50 lgÆmL
)1
carbenicillin and 34 lgÆmL
)1
chloramphenicol
to a D
600 nm
of 0.5
1
, induced with 1 m
M
isopropyl thio-b-
D
-
galactoside, and grown for an additional 5 h. Rosetta(DE3)
host strains are BL21(DE3) derivatives designed to enhance
the expression of eukaryotic proteins that contain codons
rarely used in Escherichia coli. Cells were harvested by
centrifugation, re-suspended in 0.1
M
Tris/HCl (pH 8.0),
5m
M

K-EDTA, and disrupted by sonication. Inclusion
bodies were pelleted by centrifugation
2
(5 min, 16 000 g)and
washed twice with the same buffer. Solubilization of
inclusion bodies and in vitro refolding of trypsinogen was
performed as described previously [11–14], in 0.9
M
guani-
dine-HCl, 0.1
M
Tris/HCl (pH 8.0), 2 m
M
K-EDTA con-
taining 1 m
ML
-cystine and 1 m
ML
-cysteine. Refolded
trypsinogens were purified to homogeneity by ecotin-affinity
chromatography [18]. Both trypsinogens were stable when
stored in 50 m
M
HCl on ice for several weeks. Concentra-
tions of zymogen solutions were determined from their
ultraviolet absorbance at 280 nm using calculated extinction
coefficients of 36 160
M
)1
Æcm

)1
and 37 320
M
)1
Æcm
)1
for
cationic and anionic trypsinogens, respectively.
Autoactivation of trypsinogens
Aliquots of trypsinogens (2 l
M
final concentrations) were
incubated at 37 °Cin0.1
M
Tris/HCl (pH 8.0) or 0.1
M
Na-acetate buffer (pH 5.0), in the absence or presence of
indicated concentrations of CaCl
2
in a final volume
of 100 lL. Where indicated, 100 m
M
NaCl or 100 m
M
NaCl and 2 mgÆmL
)1
BSA was included in the activation
mixtures. At given times, 2.5 lL aliquots were removed for
trypsin activity assays. Trypsin activity was determined
using the synthetic chromogenic substrate, GPR-pNA

(0.14 m
M
final concentration) in 200 lL final volume.
Kinetics of the chromophore release was followed at
405 nm in 0.1
M
Tris/HCl (pH 8.0), 1 m
M
CaCl
2
,at22°C
using a Spectramax Plus 384 microplate reader (Molecular
Devices). Trypsin activity was expressed as percentage of the
potential maximal activity, that was determined by entero-
kinase activation (400 ngÆmL
)1
final concentration) in 0.1
M
Tris/HCl (pH 8.0), 10 m
M
CaCl
2
,at22°Cfor60minon
separate trypsinogen samples.
Autolysis of trypsins
Trypsinogens ( 10 l
M
final concentration) were activated
with bovine enterokinase ( 1 lgÆmL
)1

final concentration)
in 0.1
M
Tris/HCl (pH 8.0), 20 m
M
CaCl
2
,at0°Cfor
120 min and loaded onto an ecotin column. Enterokinase,
that does not bind to ecotin, was washed away with 20 m
M
Tris/HCl (pH 8.0), 0.2
M
NaCl and trypsin was eluted with
50 m
M
HCl. Autocatalytic degradation of trypsin was
followed by residual activity measurements at 37 °Cin
0.1
M
Tris/HCl (pH 8.0) in the presence of the indicated
concentrations of CaCl
2
. Where indicated, 100 m
M
NaCl
was also included. At given times, 2.5 lL aliquots were
removed and trypsin activity was determined using GPR-
pNA (0.14 m
M

final concentration) in 200 lL final volume.
Trypsin activity was expressed as percentage of the initial
activity measured at the beginning of the incubation.
SDS/PAGE analysis of trypsinogens
Autoactivation and degradation of trypsinogens was also
visualized by gel electrophoresis and staining. Typically,
samples containing 2 l
M
trypsinogen in 100 lL volume
were precipitated with trichloroacetic acid (10% final
concentration), the precipitate was pelleted in an Eppendorf
microcentrifuge, and solubilized in 20 lL2· Laemmli
sample buffer. Trichloroacetic acid was neutralized with
NaOH until the yellow color of the acidified Bromophenol
Blue turned blue (1–2 lLof2
M
NaOH), and dithiothreitol
was added to a final concentration of 100 m
M
.Samples
2048 Z. Kukor et al. (Eur. J. Biochem. 270) Ó FEBS 2003
were heat-denatured at 95 °C for 5 min, and loaded onto
12% mini-gels. Gels were run at 30 mA, and stained for
30 min with a 0.5% Brilliant Blue R (Acros Organics, New
Jersey, NJ, USA) solution containing 40% methanol and
10% acetic acid, followed by overnight de-staining with
30% methanol, 10% acetic acid. Where indicated, densito-
metric quantitation of bands was also carried out. Gels were
dried between two layers of cellophane according to the
instructions of the Gel-Dry gel drying kit (Invitrogen).

Dried gels were scanned at 600 d.p.i. resolution in gray-scale
mode, and images were saved as TIFF files. Quantitation
of gel bands was carried out with the
IMAGEQUANT
5.2
(Molecular Dynamics) software. Rectangles were drawn
around each band of interest, and an identical rectangle was
used in each lane for background subtraction.
Results
Recombinant expression of human anionic trypsinogen
The gene for human anionic trypsinogen was cloned under
the control of the T7 promoter into the pTrap-T7 expression
vector [11,14], that was developed originally for the expres-
sion of human cationic trypsinogen. Over-expression of
anionic trypsinogen was achieved in E. coli strains carrying
an inducible T7 RNA polymerase gene, as described in
Experimental procedures. Inclusion bodies containing
denatured trypsinogen were isolated, solubilized with guani-
dine-HCl and subjected to in vitro refolding [11–13]. To
ensure that only trypsinogen that regained native confor-
mation was used in the following experiments, the refolded
material was purified to homogeneity via inhibitor-affinity
chromatography on immobilized ecotin [18]. A single peak
eluted from the ecotin column, suggesting that homogenous
trypsinogen was obtained. Homogeneity was further con-
firmed by anion-exchange chromatography (MonoQ) and
size-exclusion chromatography (Superose 6), where the
preparation also yielded single peaks (not shown). Further-
more, analysis of the purified samples by native PAGE or
SDS/PAGE revealed single bands, excluding the presence of

multiple forms or oligomerization (not shown). Catalytic
parameters of recombinant anionic trypsin (K
M
11 ± 1 l
M
;
k
cat
41 ± 1 s
)1
) were very similar to those of cationic
trypsin (K
m
15 ± 1 l
M
; k
cat
50 ± 1 s
)1
), as determined
with the chromogenic peptide substrate GPR-pNA. The
turnover number of anionic trypsin was also comparable to
values reported previously for native trypsins on small
synthetic substrates [13,19]. Finally, anionic trypsin was
inhibited with a 1 : 1 stoichiometry by human pancreatic
secretory trypsin inhibitor (not shown).
Autoactivation of human trypsinogens at pH 8.0
Autoactivation was measured in 0.1
M
Tris/HCl (pH 8.0),

at 37 °C, both in the physiologically relevant Ca
2+
concentration range (0–1 m
M
, Fig. 1A), and in a higher,
unphysiological Ca
2+
concentration range (1–20 m
M
,
Fig. 1B), that is frequently used in biochemical assays.
Human anionic trypsinogen exhibited minimal autoactiva-
tion in the absence of Ca
2+
or at Ca
2+
concentrations up to
0.1 m
M
(Fig. 1A), and significant autoactivation was
observed only at Ca
2+
concentrations of 0.5 m
M
and
above. The rate of autoactivation increased up to 5 m
M
Ca
2+
, while higher Ca

2+
concentrations (10 m
M
and 20 m
M
)
slightly inhibited the activation rate, but still resulted in
higher levels of trypsin (Fig. 1B). Analysis of anionic
trypsinogen samples by SDS/PAGE revealed that the lack
of autoactivation at 0.1 m
M
Ca
2+
and below was a
consequence of massive zymogen degradation (Fig. 1C).
Thus, in 50 l
M
Ca
2+
, the trypsinogen band disap-
peared completely by 30 min, while a trypsin band was
hardly visible. The rapid degradation at this low Ca
2+
Fig. 1. Autoactivation of human anionic trypsinogen. Approximately
2 l
M
trypsinogen (final concentration, in a final volume of 100 lL)
was incubated at 37 °C, in 0.1
M
Tris/HCl (pH 8.0) with the indicated

concentrations of CaCl
2
. (A,B) Aliquots of 2.5 lLwerewithdrawn
from reaction mixtures at indicated times and trypsin activity was
determined with 0.14 m
M
(final concentration) GPR-pNA. Activity
was expressed as percentage of the potential total activity, as deter-
mined on similar zymogen samples activated with enterokinase at
22 °Cin0.1
M
Tris/HCl (pH 8.0), 10 m
M
Ca
2+
.(C)Sampleswere
precipitated with 10% trichloroacetic acid (final concentration), run on
a 12% SDS/PAGE minigel under reducing conditions, and stained
with Coomassie Blue.
Ó FEBS 2003 Human anionic trypsinogen (Eur. J. Biochem. 270) 2049
concentration resulted only in faintly visible bands of larger
peptide fragments, as the bulk of the protein was digested to
small peptides. In contrast, in the presence of 5 m
M
Ca
2+
,
the trypsinogen band was converted to trypsin and stable
autolysis products were also detected. The appearance of
larger peptide fragments at high Ca

2+
concentration was
due to the significantly slower degradation rate and possibly
the selective protection of certain cleavage sites by Ca
2+
.
Human cationic trypsinogen exhibited characteristic
differences from its anionic counterpart. In 0.1
M
Tris/
HCl(pH8.0),at37°C, autoactivation was measurable
even in the absence of added Ca
2+
, and it was significantly
stimulated by Ca
2+
concentrations as low as 10 l
M
(Fig. 2A). Ca
2+
stimulated autoactivation in a concentra-
tion-dependent manner up to 1 m
M
, while above this
concentration autoactivation was progressively inhibited
(Fig. 2B). As addition of 100 m
M
NaCl also significantly
decreased the rate of autoactivation
3

or autolysis (see below),
it appears that inhibition by the nonphysiologically high
Ca
2+
concentrations was caused by ionic strength. Analysis
of the Ca
2+
dependence of autoactivation revealed a
biphasic activation curve (Fig. 2C); a typical saturation
curve with an apparent EC
50
of  15 l
M
was followed by
linear concentration dependence. The apparent half-maxi-
mal stimulatory Ca
2+
concentration (15 l
M
) was compar-
able to the Ca
2+
concentration that stabilized cationic trypsin
against autolysis half-maximally (20 l
M
;seebelow).Tryp-
sin stabilization by Ca
2+
is accomplished via binding to the
high-affinity Ca

2+
binding site composed of five residues,
between Glu75 and Glu85. Consequently, the observation
that low concentrations of Ca
2+
stimulate autoactivation of
cationic trypsinogen suggest that Ca
2+
exerts this effect
through the same high affinity Ca
2+
binding site. Ca
2+
concentrations between 0.1 m
M
)1m
M
further stimulated
autoactivation by binding to the low affinity site in
the activation peptide. Determination of an EC
50
for the
latter process was not feasible due to the inhibitory effect of
Ca
2+
concentrations above 1 m
M
.
SDS/PAGE analysis of autoactivation of human cationic
trypsinogen at pH 8.0 was described in our previous studies

(e.g. see Figs 3,4 in [11] and Fig. 1 in [16]). At pH 8.0, in the
presence of 1 m
M
Ca
2+
, the typical banding pattern of
autoactivated cationic trypsinogen is essentially identical to
the picture shown below, which demonstrates autoactiva-
tion of human cationic trypsinogen at pH 5.0. A notable
feature of human cationic trypsin(ogen) is that it exists as an
equilibrium mixture of single-chain and double-chain forms.
The double-chain form, that in every functional aspect
appears to be identical to the single-chain form, is generated
by autocatalytic cleavage of the Arg122-Val123 peptide
bond. The dynamic equilibrium between the two forms is
maintained by continuous trypsin-dependent cleavage and
resynthesis of the Arg122-Val123 bond. On reducing SDS/
PAGE gels, that dissociate the two chains, double-chain
trypsinogen appears as a 15-kDa band, containing both the
N- and C-terminal chains that are identical in size (band A).
Activation of double-chain trypsinogen to double-chain
trypsin results in the appearance of band B, which
corresponds to the N-terminal chain of double-chain
trypsin. For a more detailed description of the unique
properties of double-chain trypsin(ogen) the reader is
referred to our recent study [16].
Trypsinolytic degradation of human trypsinogens
at pH 8.0
One of the striking observations from the comparative
autoactivation studies of human trypsinogens at pH 8.0 was

the marked susceptibility of anionic trypsinogen to auto-
catalytic degradation. To get a more accurate comparison
for the rates of zymogen degradation between the two
trypsinogens, Lys23 in the activation peptide was replaced
with Gln in human anionic trypsinogen. The resulting
Fig. 2. Autoactivation of human cationic trypsinogen. Experimental
conditions are given in Fig. 1. (A) Stimulation of autoactivation in the
Ca
2+
concentration range 0.01 m
M
)1m
M
. (B) Inhibition of auto-
activation by Ca
2+
in the concentration range 1 m
M
)20 m
M
.(C)
Relative rates of autoactivation were plotted against the Ca
2+
con-
centration between 0.01 m
M
)0.5 m
M
. The rate of autoactivation
without any added Ca

2+
(0 m
M
inA)wasdesignatedas1.
2050 Z. Kukor et al. (Eur. J. Biochem. 270) Ó FEBS 2003
K23Q mutant trypsinogen is resistant to autoactivation,
allowing selective examination of trypsinolytic zymogen
degradation. Anionic K23Q-trypsinogen was purified to
homogeneity and 5 l
M
zymogen was used as substrate in
digestion experiments with 0.5 l
M
cationic trypsin as
enzyme. Cationic trypsin was used, because it remained
stable without significant loss of activity during the time
course studied. Figure 3A demonstrates that in the absence
of Ca
2+
(in 1 m
M
EDTA) cationic trypsin rapidly degraded
anionic K23Q-trypsinogen, and densitometric quantitation
indicated a half-life (t
1/2
) of 2.25 min (Fig. 3C). Addition of
50 l
M
Ca
2+

(final concentration) afforded significant
(fourfold) stabilization, and prolonged the t
1/2
to 10 min
(Fig. 3B,C). Using the same strategy, in a recent study we
determined the degradation of a K23Q-mutant of cationic
trypsinogen by cationic trypsin [16]. At pH 8.0, in the
absence of Ca
2+
(in 1 m
M
EDTA) 5 l
M
cationic K23Q-
zymogen was degraded by 0.5 l
M
trypsin with a t
1/2
of
45 min. Thus, cationic trypsinogen is 20-fold more resistant
to trypsinolytic degradation than anionic trypsinogen is.
For comparison, densitometric quantitation data for K23Q
cationic trypsinogen were also included in Fig. 3C.
Autoactivation of human trypsinogens at pH 5.0
In contrast to the rapid autoactivation at pH 8.0, anionic
trypsinogen autoactivated much slower at pH 5.0 (Fig. 4A),
and Ca
2+
-stimulated autoactivation in a concentration
dependent manner between 0.5 m

M
and 5 m
M
. Maximal
levels of trypsin generation in 5 m
M
Ca
2+
did not exceed
30% of the total potential activity, indicating significant
zymogen degradation. No further stimulation was apparent
with 10 m
M
Ca
2+
, while 20 m
M
Ca
2+
inhibited the rate of
autoactivation and yielded somewhat higher trypsin levels.
SDS/PAGE analysis of anionic trypsinogen samples
revealed that the lack of autoactivation at pH 5.0 in the
absence of Ca
2+
was not due to rapid zymogen degrada-
tion, as observed at pH 8.0 (see Fig. 1). Instead, zymogen
activation was inhibited by the acidic conditions, and a
stable trypsinogen band was observed over the 120 min
Fig. 3. Degradation of K23Q-anionic trypsinogen (Hu2) by human

cationic trypsin. Approximately 5 l
M
trypsinogen (final concentration,
in a final volume of 100 lL) was digested with 0.5 l
M
cationic trypsin
at 37 °C, in 0.1
M
Tris/HCl (pH 8.0) in 1 m
M
EDTA (A) or in 50 l
M
Ca
2+
(B). Reactions were terminated at indicated times by trichloro-
acetic acid precipitation, and analyzed by reducing SDS/PAGE and
Coomassie Blue staining. In the 0 min samples, trichloroacetic acid
was added before trypsin. (C) Densitometric quantitation of gels
(n ¼ 3, error less than 15%). Also shown are data from ref [16], where
trypsinolytic degradation of the K23Q mutant of human cationic
trypsinogen (Hu1) was determined under identical conditions.
Fig. 4. Autoactivation of human anionic trypsinogen at pH 5.0.
Approximately 2 l
M
trypsinogen (final concentration, in a final vol-
ume of 100 lL) was incubated at 37 °C, in 0.1
M
Na-acetate buffer
(pH 5.0) with the indicated concentrations of CaCl
2

.(A)Trypsin
activity was determined and expressed as described in Fig. 1. (B)
Samples (2 l
M
zymogen in 100 lL) were trichloroacetic acid-precipi-
tated and analyzed by reducing SDS/PAGE (12%) and Coomassie
Blue staining.
Ó FEBS 2003 Human anionic trypsinogen (Eur. J. Biochem. 270) 2051
time-course studied (Fig. 4B). Addition of 5 m
M
Ca
2+
stimulated conversion of trypsinogen to trypsin, as a faint
trypsin band became apparent at 60 min, and more
significant trypsin generation was detectable by 120 min.
In the absence of Ca
2+
, autoactivation of cationic
trypsinogen was more rapid at pH 5.0 (Fig. 5A) than at
pH 8.0 (Fig. 2). At pH 5.0, Ca
2+
caused a slight stimu-
lation up to 1 m
M
, and inhibited autoactivation in a
concentration-dependent manner between 2 m
M
and
20 m
M

(Fig. 5B). Comparing time-courses of autoactiva-
tion at pH 5.0 on SDS/PAGE gels confirmed that in the
absence of Ca
2+
anionic trypsinogen was not activated (see
Fig. 4B), while cationic trypsinogen was fully activated over
Fig. 5. Autoactivation of human cationic trypsinogen at pH 5.0.
Approximately 2 l
M
trypsinogen (final concentration, in a final vol-
ume of 100 lL) was incubated at 37 °C, in 0.1
M
Na-acetate buffer
(pH 5.0) with the indicated concentrations of CaCl
2
. (A,B) Trypsin
activity was determined and expressed as described in Fig. 1. (A) Slight
stimulation of autoactivation by Ca
2+
concentrations up to 1 m
M
.(B)
Inhibition of autoactivation by Ca
2+
concentrations above 1 m
M
.(C)
Samples (2 l
M
zymogen in 100 lL) were trichloroacetic acid-precipi-

tated and analyzed by reducing SDS/PAGE (12%) and Coomassie
Blue staining. Bands A and B correspond to the two chains of double-
chain trypsin(ogen), see text for more explanation.
Fig. 6. Autocatalytic degradation (autolysis) of human anionic trypsin.
Trypsinogen was activated by enterokinase and purified on an ecotin-
column, as described in Experimental procedures. Approximately
2 l
M
aliquots of anionic trypsin (final concentration) were incubated at
37 °Cin0.1
M
Tris/HCl (pH 8.0) in the presence of the indicated
concentrations of CaCl
2
. Aliquots of 2.5 lLwerewithdrawnfrom
reaction mixtures at indicated times and trypsin activity was deter-
minedwith0.14m
M
GPR-pNA (final concentration). Residual
activities were expressed as percentage of trypsin activity measured at
the beginning of the incubation. (B) Autolysis in the presence of
100 m
M
NaCl. (C) Effect of Ca
2+
ontherelativerateofautolysisinthe
presence (s) or absence (d) of 100 m
M
NaCl. The rate determined in
the absence of added Ca

2+
was designated as 1.
2052 Z. Kukor et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the same time period (Fig. 5C). Conversion of cationic
trypsinogen to trypsin was practically quantitative, with no
significant zymogen degradation, and addition of 5 m
M
Ca
2+
had only a minor effect on the rate of autoactivation
(Fig. 5C).
Autolysis of human trypsins at pH 8.0
Previous experiments using purified native human trypsins
indicated that human anionic trypsin was less stable and
underwent faster autolysis than cationic trypsin [19,20]. To
characterize the autolytic process of the recombinant trypsin
preparations in more detail, we purified human anionic
and cationic trypsin after enterokinase activation of the
respective recombinant zymogens. In the absence of Ca
2+
,
anionic trypsin suffered autolysis at a rapid rate (t
1/2
8min),
and low concentrations of Ca
2+
stabilized the enzyme,
with an IC
50
of 5 l

M
(Fig. 6A,C). Addition of 100 m
M
NaCl to anionic trypsin decreased the rate of autolysis
threefold (in the absence of Ca
2+
t
1/2
was 24 min, Fig. 6B),
and increased the IC
50
value for Ca
2+
stabilization sixfold
(30 l
M
, Fig. 6C). Autolysis of cationic trypsin was signifi-
cantly slower, and in the absence of Ca
2+
a t
1/2
of 90 min
was measured (Fig. 7A). Thus, in the absence of Ca
2+
a >11-fold difference was apparent between the autolysis
rates of the two trypsins (Figs 6A and 7A). Low concen-
trations of Ca
2+
afforded stabilization with an IC
50

value of
20 l
M
(Fig. 7A and B). Surprisingly, addition of 100 m
M
NaCl diminished autolysis of cationic trypsin 14-fold, and
even in the absence of Ca
2+
it took almost 21 h to observe a
Fig. 7. Autocatalytic degradation (autolysis) of human cationic trypsin.
(A) See Fig. 6 for experimental details. (B) Effect of Ca
2+
on the
relative rate of autolysis. The rate determined in the absence of added
Ca
2+
was designated as 1.
Fig. 8. Autoactivation of physiological and pathological mixtures of
human trypsinogens at pH 8.0, in 1 m
M
Ca
2+
. Autoactivation experi-
ments were carried out as described in Fig. 1. Hu1 (h), human cationic
trypsinogen (2 l
M
); Hu2 (s), human anionic trypsinogen (2 l
M
).
Physiological mixtures (j) contained 1.33 l

M
(67%) Hu1 and 0.67 l
M
(33%) Hu2 trypsinogen. Pathological mixtures (d)contained0.67l
M
(33%) Hu1 and 1.33 l
M
(67%) Hu2 trypsinogen. Experiments were
carried out under three conditions, in buffer only (A), in 100 m
M
NaCl
(B) and in 100 m
M
NaCl with 2 mgÆmL
)1
BSA (C).
Ó FEBS 2003 Human anionic trypsinogen (Eur. J. Biochem. 270) 2053
50% loss of activity (not shown). Thus, there is a
remarkable difference in salt sensitivity between the two
human trypsins with respect to autolysis.
Interactions between anionic and cationic trypsinogens
and trypsins during trypsinogen activation
The experiments presented in Figs 1–7 provided a detailed
biochemical characterization of the autocatalytic activa-
tion and degradation of the two major human trypsino-
gens. The notably different behavior of the two zymogens
suggested that changes in their ratio should have
profound effects on the overall stability of the pancreatic
trypsinogen pool and its susceptibility to autoactivation.
To model these changes in vitro, we examined the effect of

increasing anionic trypsinogen proportions in different
mixtures of the two trypsinogens. In these experiments,
the two human trypsinogens were mixed at two different
ratios, 2 : 1 (physiological mixture; 67% cationic trypsinogen
and 33% anionic trypsinogen) or 1 : 2 (pathological mixture,
33% cationic trypsinogen and 67% anionic trypsinogen).
Autoactivation experiments were carried out at pH 8.0
andpH5.0.AtpH8.0,twodifferentCa
2+
concentrations
were used, 1 m
M
or 50 l
M
.The1m
M
Ca
2+
concentration
was selected to model the conditions in the pancreatic juice
or in the duodenum, the physiological site of trypsinogen
activation. The 50 l
M
Ca
2+
concentration modeled the
intracellular conditions, where Ca
2+
concentrations are low.
Although true cytoplasmic Ca

2+
concentrations are below
micromolar levels, we chose to use 50 l
M
Ca
2+
because at
this concentration autoactivation was somewhat faster
and full time-courses could be analyzed within reasonable
time limits. Qualitatively identical results were obtained
when experiments were repeated at pH 8.0 without any
added Ca
2+
. Finally, experiments at pH 5.0 modeled
conditions in acidic intracellular vesicular compartments,
that are known sites of pathological trypsinogen activation
[21,22]. In addition, it was also important to demonstrate
that any differences observed also existed in the presence of
salts or other proteins, as the routinely used in vitro
biochemical system obviously lacked the variety of salts
and proteins present in the intra-acinar environment or in
pancreatic secretions. Therefore, in addition to experiments
performed in buffer only, autoactivation of mixtures was
also compared in the presence of 100 m
M
NaCl or in the
presence of 100 m
M
NaCl and 2 mgÆmL
)1

BSA.
AsshowninFig.8A,in0.1
M
Tris/HCl (pH 8.0) and
1m
M
Ca
2+
, autoactivation of the two trypsinogens
proceeded at comparable rates, but resulted in a twofold
difference in final trypsin levels (Fig. 8A, white symbols).
As demonstrated above (see Figs 1,2), this difference is due
to the more rapid degradation of anionic trypsin(ogen)
during autoactivation. Interestingly, when the two trypsino-
gens were mixed either in a physiological or in a
pathological mixture, rates of autoactivation did not
change appreciably and final trypsin levels differed only
by 20% (Fig. 8A, black symbols). Similarly, the physiolo-
gical activator, enterokinase, generated approximately
identical amounts of trypsin from both mixtures (not
shown). Addition of 100 m
M
NaCl drastically reduced
the rate of autoactivation by cationic trypsinogen, while
anionic trypsinogen was much less affected (Fig. 8B, white
symbols). Mixtures of the two trypsinogens, however,
exhibited not too different activation rates and yielded
essentially identical trypsin levels (Fig. 8B, black symbols).
Finally, in the presence of 100 m
M

NaCl and 2 mgÆmL
)1
BSA autoactivation of the two trypsinogen mixtures
exhibited rates and final trypsin levels that showed a
 20% difference only (Fig. 8C, black symbols). Interest-
ingly, the BSA preparations used noticeably inhibited
autoactivation of anionic trypsinogen (compare Figs 8B,C,
white circles), while cationic trypsinogen was not affected.
Although this problem was not investigated any further,
this effect was in all likelihood due to the contaminating
presence of a serum trypsin inhibitor in some of the
commercial BSA preparations.
A different picture emerged when experiments were
performed in the presence of 0.1
M
Tris/HCl (pH 8.0) with
50 l
M
Ca
2+
. Under all three conditions tested, cationic
trypsinogen autoactivated to significant levels, while essen-
tially no trypsin generation was detectable with anionic
trypsinogen (Fig. 9A–C, white symbols), due to practically
total degradation (see Fig. 1). Accordingly, autoactivation
of mixtures of the two trypsinogens was proportional to the
cationic trypsinogen content, and pathological mixtures
consistently exhibited activation rates and final trypsin levels
that were at least twofold lower compared to physiological
mixtures (Fig. 9A–C, black symbols).

Experiments at pH 5.0 showed similar differences in the
autoactivation characteristics of the two types of trypsino-
gen mixtures. Once again, rates of autoactivation seemed
to reflect the cationic trypsinogen content and autoactiva-
tion rates of pathological mixtures were markedly sup-
pressed (Fig. 10A–C, black symbols). Clearly, this
difference was caused by the inability of anionic trypsino-
gen to autoactivate at this acidic pH (Fig. 10A–C, white
circles; also see Fig. 4). Due to the extended time-courses,
final trypsin levels were not determined accurately, but it
appeared that pathological mixtures should yield at least
twofold less trypsin than physiological mixtures
(Fig. 10A).
The anomalous and distinct migration of the two human
trypsinogens on SDS/PAGE gels allowed the visualization
of both species present in the mixtures (Fig. 11). In 0.1
M
Tris/HCl (pH 8.0) with 50 l
M
Ca
2+
, both mixtures
contained only active cationic trypsin by the end of the
60 min incubation (Fig. 11A). Both the single-chain form
and the double-chain form (denoted by bands A and B in
Fig. 11) were observed. In agreement with the activity
assays, the stronger intensity of the cationic trypsin band in
the physiological mixture was noticeable. Furthermore,
rapid disappearance of the anionic trypsinogen band
without the appearance of a clearly detectable anionic

trypsin band was also evident in both mixtures. Taken
together, the observations confirmed that under these
conditions (pH 8.0, 50 l
M
Ca
2+
, Fig. 11A) selective degra-
dation of anionic trypsinogen resulted in lower trypsin
generation in pathological mixtures of the two human
trypsinogens. Finally, at pH 5.0 cationic trypsinogen was
completely activated to trypsin in the physiological mix-
ture, while significant amounts of anionic trypsinogen
remained unactivated in the pathological mixture, reflect-
ing the resistance of this trypsinogen species to activation
at acidic pH (Fig. 11B,C).
2054 Z. Kukor et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Discussion
How do the two major trypsinogen isoforms of the human
pancreas interact with respect to autocatalytic activation
and degradation? What are the biochemical consequences
of the up-regulation of anionic trypsinogen in pancreatic
secretions of patients with pancreatic diseases or chronic
alcoholism? To address these questions, we recombinantly
produced human anionic trypsinogen and purified it in a
stable form. Although recombinant expression of anionic
trypsin activity per se was reported in a few studies [6,13],
pure and stable zymogen preparations were difficult to
achieve, due to the notoriously unstable nature of this
trypsinogen isoform. In this respect, methodology devel-
oped earlier for the recombinant production of human

Fig. 10. Autoactivation of physiological and pathological mixtures of
human trypsinogens at pH 5.0. Autoactivation experiments were car-
ried out in 0.1
M
Na-acetate buffer (pH 5.0). See Fig. 8 for other
experimental details.
Fig. 9. Autoactivation of physiological and pathological mixtures of
human trypsinogens at pH 8.0, in 50 l
M
Ca
2+
. See Fig. 8 for experi-
mental details.
Ó FEBS 2003 Human anionic trypsinogen (Eur. J. Biochem. 270) 2055
cationic trypsinogen was critical [11–13], including the use of
immobilized ecotin for the final purification step [18].
To understand the behavior of trypsinogens in more
complex mixtures, first we documented their properties
individually, under the typical experimental conditions used
in recent literature. At least four major differences were
observed. (a) Trypsinolytic degradation of anionic trypsi-
nogen or trypsin was 10 to 20-fold faster. As a consequence
of their highly different stability, the two trypsinogens
exhibited distinct autoactivation profiles. Thus, essentially
no trypsin activity was detectable during autoactivation of
anionic trypsinogen at pH 8.0 in 0.1 m
M
Ca
2+
or lower. At

these Ca
2+
concentrations autoactivation was relatively
slow, and could not keep up with pace of trypsin(ogen)
degradation. Only in millimolar Ca
2+
concentrations was
significant autoactivation detected, when the rate of auto-
activation exceeded the rate of degradation. In contrast,
because degradation of cationic trypsin(ogen) was much
slower, autoactivation resulted in the development of
significant trypsin activity even in the absence of added
Ca
2+
. (b) Acidic pH stimulated autoactivation of cationic
trypsinogen, but inhibited activation of anionic trypsinogen.
(c) Anionic trypsin bound Ca
2+
fourfold stronger than
cationic trypsin, as judged by the stabilizing effect of Ca
2+
on autolysis. Binding of Ca
2+
to the high-affinity site also
stimulated autoactivation of cationic trypsinogen, while this
effect was either absent in anionic trypsinogen or it was
masked by the rapid degradation. Interestingly, Ca
2+
in the
concentration range between 1 m

M
and 10 m
M
stimulated
autoactivation of anionic trypsinogen, almost in a manner
that was observed for bovine trypsinogen [23] or rat anionic
trypsinogen [24]. In contrast, autoactivation of cationic
trypsinogen was progressively inhibited by Ca
2+
concen-
trations between 1 m
M
)20 m
M
. Although this observation
is important for the correct interpretation of autoactivation
assays performed under a variety of Ca
2+
concentrations in
the literature; the (patho)physiological significance of such a
Ca
2+
-mediated inhibition mechanism is questionable. (d)
Autoactivation of cationic trypsinogen and autolysis of
cationic trypsin were markedly inhibited by 100 m
M
NaCl,
while anionic trypsin(ogen) was significantly less sensitive to
this salt effect. In physiological terms, this observation
would suggest that anionic trypsinogen can autoactivate

much faster than cationic trypsinogen under conditions
prevailing in the pancreatic juice.
Trypsinogen autoactivation and degradation were studied
previously with purified native trypsinogen preparations
[19,20,25–27]. Although experimental conditions (pH, tem-
perature, salt and buffer concentrations) were frequently
varied in these studies, some of the results regarding the
characteristic differences between the two human tryp-
sin(ogen)s were similar to our findings. Thus, compared to
the other isoform, anionic trypsin exhibited much more rapid
autolysis, and cationic trypsinogen autoactivated more
prominently at acidic pH. On the other hand, our observa-
tions disagree with previous results in some detail. In our
study, cationic trypsin was more stable in the absence of
Ca
2+
than reported before [19]. Relative to cationic trypsi-
nogen, anionic trypsinogen autoactivated faster at pH 8.0 in
20 m
M
Ca
2+
whereas the opposite relationship was
described previously [25,26]. Finally, in our experiments,
high Ca
2+
concentrations consistently inhibited autoactiva-
tion of cationic trypsinogen, while both stimulation and
inhibition was found in early studies [25–27].
Characterization of the individual trypsinogens set the

stage for the analysis of their mixtures. These experiments
sought to answer one question: what happens to trypsino-
gen activation and degradation when the normal ratio of
cationic and anionic trypsinogen is reversed, as seen in
pancreatic diseases or chronic alcoholism? The results
indicated that trypsin generation by autoactivation or
enterokinase activation was not affected significantly by
the ratio of the two isoforms, under conditions that were
typical of the pancreatic juice. This observation suggests
that the primary trypsin functions, i.e. activation of other
zymogens and digestion of ingested proteins; are unaffected
by up-regulation of anionic trypsinogen. In contrast,
trypsinogen activation was markedly diminished by an
increased ratio of anionic trypsinogen under conditions that
mimicked potential intracellular sites of pathological tryp-
sinogen activation, such as the cytoplasm or acidic vesicles.
Increasing the ratio of anionic trypsinogen resulted in
decreased overall trypsin generation at pH 8.0 in the
presence of low Ca
2+
concentrations, due to the selective
degradation of anionic trypsin(ogen). Similarly, total trypsin
formation was suppressed at pH 5.0, where the acidic pH
selectively inhibited activation of anionic trypsinogen.
Under both conditions, the concentration of cationic
trypsinogen seemed to determine the rate of autoactivation
and the final levels of trypsin generated. Consequently, an
Fig. 11. Autoactivation of physiological (67% Hu1–33% Hu2) and
pathological (33% Hu1–67% Hu2) mixtures of human trypsinogens at
pH 8.0 in 50 l

M
Ca
2+
(A) and pH 5.0 (B and C). Autoactivation
experiments were carried out asin Fig. 9A (A) and 10(B and C). Samples
(2 l
M
total zymogen in 100 lL) were trichloroacetic acid-precipitated
and analyzed by reducing SDS/PAGE (12%) and Coomassie Blue
staining. Panel C is an enlargement from the 120 min lanes of panel B,
demonstrating the resolution of the four human trypsin(ogen) species
in the gel. Tg, trypsinogen; bands A and B correspond to the two
chains of double-chain trypsin(ogen), see text for more explanation.
2056 Z. Kukor et al. (Eur. J. Biochem. 270) Ó FEBS 2003
increase in the proportion of the anionic trypsinogen
resulted in a Õloss of trypsinogen functionÕ,astheratioof
cationic trypsinogen was decreased.
Although this study clarified the biochemical conse-
quences of increased anionic trypsinogen secretion, the
(patho)physiological sequelae have remained contentious.
The most straightforward interpretation of the results is to
suggest that anionic trypsinogen plays a protective role in
pancreatic physiology. As a defensive mechanism, acinar
cells increase secretion of the anionic isoform in pancreatic
diseases or toxic conditions, thereby decreasing the chance
for premature trypsinogen activation inside the pancreas,
while maintaining acceptable trypsin function in the duo-
denum. In other words, increased anionic trypsinogen levels
do not cause or predispose to pancreatitis; instead, they
represent a protective response by the diseased organ. One

obvious caveat of this simplistic model is that it assumes that
total trypsinogen levels remain constant in these patholo-
gical states, only the ratio of the two isoforms changes. In
reality, this is almost never the case. In chronic pancreatitis,
trypsinogen secretion is usually somewhat depressed, while
in chronic alcoholism secreted levels of total trypsinogen can
be significantly elevated [3,4]. It is possible, that under the
latter conditions the increased trypsinogen synthesis may
render the pancreas more susceptible to inappropriate
zymogen activation, despite the protective effects of anionic
trypsinogen.
In contrast to a possible safeguard role, chronically
increased anionic trypsinogen levels and ensuing lower
intrapancreatic trypsin concentrations may also be regarded
as a disease-causing factor. This interpretation relies on the
theory that in some cases a loss of trypsin(ogen) function
might be associated with the development of pancreatitis. A
loss-of-function theory of pancreatitis pathogenesis was
proposed by Halangk et al. (2002) who in an elegant series
of experiments with isolated rat acini and lobuli demon-
strated that during caerulein-induced pancreatitis, trypsin
might play a protective role inside the acinar cells by
degrading trypsin(ogen) and possibly other proteases thus
preventing the escalation of intra-acinar digestive enzyme
activation [28]. More recently, a genetically engineered
mouse deficient in one of the zymogen granule membrane
proteins (integral membrane-associated protein-1, Itmap-1)
was shown to develop more severe secretagogue- and diet-
induced experimental pancreatitis, but with diminished
intrapancreatic trypsinogen activation, documenting the

possible association of decreased trypsinogen function with
the development of pancreatitis [29].
Acknowledgement
This work was supported by NIH grant DK58088 to M. S T. Special
thanks to Lan Guan for her help with the densitometry.
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