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.

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

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

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

(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 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).

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),

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

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

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

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

(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

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

(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-

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

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

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

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

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

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

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

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

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

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