Human anionic trypsinogenProperties of autocatalytic activation and degradation and implications in pancreatic diseases Zolta´n Kukor, Miklo´s To´th* and Miklo´s Sahin-To´th Department o
Trang 1Human anionic trypsinogen
Properties of autocatalytic activation and degradation and implications
in pancreatic diseases
Zolta´n Kukor, Miklo´s To´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 Ca2+-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
1 mM Ca2+ – 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 Ca2+ 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) genes 1, 2 and 3
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) (for a review on
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: miklos@bu.edu
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)
Trang 2growing 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 cprepar-ationic
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 SacI site were
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
NcoI and EcoRI 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)1carbenicillin and 34 lgÆmL)1chloramphenicol
to a D600 nmof 0.5
1 , induced with 1 mMisopropyl 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 0.1M Tris/HCl (pH 8.0),
5 mM K-EDTA, and disrupted by sonication Inclusion
bodies were pelleted by centrifugation
washed twice with the same buffer Solubilization of
inclusion bodies and in vitro refolding of trypsinogen was
performed as described previously [11–14], in 0.9M guani-dine-HCl, 0.1MTris/HCl (pH 8.0), 2 mMK-EDTA con-taining 1 mM L-cystine and 1 mM L-cysteine Refolded trypsinogens were purified to homogeneity by ecotin-affinity chromatography [18] Both trypsinogens were stable when stored in 50 mMHCl on ice for several weeks Concentra-tions of zymogen soluConcentra-tions were determined from their ultraviolet absorbance at 280 nm using calculated extinction coefficients of 36 160M )1Æcm)1 and 37 320M )1Æcm)1 for cationic and anionic trypsinogens, respectively
Autoactivation of trypsinogens Aliquots of trypsinogens (2 lM final concentrations) were incubated at 37C in 0.1M Tris/HCl (pH 8.0) or 0.1M Na-acetate buffer (pH 5.0), in the absence or presence of indicated concentrations of CaCl2 in a final volume
of 100 lL Where indicated, 100 mM NaCl or 100 mM NaCl and 2 mgÆmL)1BSA 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 mM final concentration) in 200 lL final volume Kinetics of the chromophore release was followed at
405 nm in 0.1MTris/HCl (pH 8.0), 1 mMCaCl2, at 22C 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)1final concentration) in 0.1M Tris/HCl (pH 8.0), 10 mMCaCl2, at 22C for 60 min on separate trypsinogen samples
Autolysis of trypsins Trypsinogens ( 10 lMfinal concentration) were activated with bovine enterokinase ( 1 lgÆmL)1final concentration)
in 0.1M Tris/HCl (pH 8.0), 20 mM CaCl2, at 0C for
120 min and loaded onto an ecotin column Enterokinase, that does not bind to ecotin, was washed away with 20 mM Tris/HCl (pH 8.0), 0.2MNaCl and trypsin was eluted with
50 mM HCl Autocatalytic degradation of trypsin was followed by residual activity measurements at 37C in 0.1M Tris/HCl (pH 8.0) in the presence of the indicated concentrations of CaCl2 Where indicated, 100 mM NaCl was also included At given times, 2.5 lL aliquots were removed and trypsin activity was determined using GPR-pNA (0.14 mMfinal 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 lM 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 lL 2· Laemmli sample buffer Trichloroacetic acid was neutralized with NaOH until the yellow color of the acidified Bromophenol Blue turned blue (1–2 lL of 2MNaOH), and dithiothreitol was added to a final concentration of 100 m Samples
Trang 3were heat-denatured at 95C 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 IMAGEQUANT5.2
(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-expresexpres-sion 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 (KM11 ± 1 lM;
kcat 41 ± 1 s)1) were very similar to those of cationic
trypsin (Km 15 ± 1 lM; kcat 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 0.1MTris/HCl (pH 8.0),
at 37C, both in the physiologically relevant Ca2+
concentration range (0–1 mM, Fig 1A), and in a higher,
unphysiological Ca2+ concentration range (1–20 mM,
Fig 1B), that is frequently used in biochemical assays
Human anionic trypsinogen exhibited minimal
autoactiva-tion in the absence of Ca2+or at Ca2+concentrations up to
0.1 mM (Fig 1A), and significant autoactivation was
observed only at Ca2+ concentrations of 0.5 m and
above The rate of autoactivation increased up to 5 mM
Ca2+, while higher Ca2+concentrations (10 mMand 20 mM) 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 mM Ca2+ and below was a consequence of massive zymogen degradation (Fig 1C) Thus, in 50 lM Ca2+, the trypsinogen band disap-peared completely by 30 min, while a trypsin band was hardly visible The rapid degradation at this low Ca2+
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 lL were withdrawn 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 C in 0.1 M Tris/HCl (pH 8.0), 10 m M Ca2+ (C) Samples were precipitated with 10% trichloroacetic acid (final concentration), run on
a 12% SDS/PAGE minigel under reducing conditions, and stained with Coomassie Blue.
Trang 4concentration 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 mMCa2+,
the trypsinogen band was converted to trypsin and stable
autolysis products were also detected The appearance of
larger peptide fragments at high Ca2+concentration was
due to the significantly slower degradation rate and possibly
the selective protection of certain cleavage sites by Ca2+
Human cationic trypsinogen exhibited characteristic
differences from its anionic counterpart In 0.1M Tris/
HCl (pH 8.0), at 37C, autoactivation was measurable
even in the absence of added Ca2+, and it was significantly
stimulated by Ca2+ concentrations as low as 10 lM
(Fig 2A) Ca2+stimulated autoactivation in a
concentra-tion-dependent manner up to 1 mM, while above this
concentration autoactivation was progressively inhibited
(Fig 2B) As addition of 100 mM NaCl also significantly
decreased the rate of autoactivation
it appears that inhibition by the nonphysiologically high
Ca2+concentrations was caused by ionic strength Analysis
of the Ca2+ dependence of autoactivation revealed a
biphasic activation curve (Fig 2C); a typical saturation
curve with an apparent EC50of 15 lMwas followed by
linear concentration dependence The apparent
half-maxi-mal stimulatory Ca2+concentration (15 lM) was
compar-able to the Ca2+concentration that stabilized cationic trypsin
against autolysis half-maximally (20 lM; see below)
Tryp-sin stabilization by Ca2+is accomplished via binding to the
high-affinity Ca2+binding site composed of five residues,
between Glu75 and Glu85 Consequently, the observation
that low concentrations of Ca2+stimulate autoactivation of
cationic trypsinogen suggest that Ca2+ exerts this effect
through the same high affinity Ca2+ binding site Ca2+
concentrations between 0.1 mM)1 mM further stimulated
autoactivation by binding to the low affinity site in
the activation peptide Determination of an EC50 for the
latter process was not feasible due to the inhibitory effect of
Ca2+concentrations above 1 mM
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 mM Ca2+, the typical banding pattern of
autoactivated cationic trypsinogen is essentially identical to
the picture shown below, which demonstrates
autoactiva-tion of human caautoactiva-tionic 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 )1 m M (B) Inhibition of auto-activation by Ca2+ 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 Ca2+(0 m M in A) was designated as 1.
Trang 5K23Q mutant trypsinogen is resistant to autoactivation,
allowing selective examination of trypsinolytic zymogen
degradation Anionic K23Q-trypsinogen was purified to
homogeneity and 5 lMzymogen was used as substrate in
digestion experiments with 0.5 lM 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 Ca2+(in 1 mMEDTA) cationic trypsin rapidly degraded
anionic K23Q-trypsinogen, and densitometric quantitation
indicated a half-life (t1/2) of 2.25 min (Fig 3C) Addition of
50 lM Ca2+ (final concentration) afforded significant
(fourfold) stabilization, and prolonged the t1/2 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 Ca2+(in 1 mM EDTA) 5 lM cationic K23Q-zymogen was degraded by 0.5 lM trypsin with a t1/2 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 Ca2+-stimulated autoactivation in a concentration dependent manner between 0.5 mM and 5 mM Maximal levels of trypsin generation in 5 mMCa2+did not exceed 30% of the total potential activity, indicating significant zymogen degradation No further stimulation was apparent with 10 mMCa2+, while 20 mMCa2+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 Ca2+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 pH5.0 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.
Trang 6time-course studied (Fig 4B) Addition of 5 mM Ca2+
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 Ca2+, autoactivation of cationic
trypsinogen was more rapid at pH 5.0 (Fig 5A) than at
pH 8.0 (Fig 2) At pH 5.0, Ca2+caused a slight
stimu-lation up to 1 mM, and inhibited autoactivation in a
concentration-dependent manner between 2 m and
20 mM (Fig 5B) Comparing time-courses of autoactiva-tion at pH 5.0 on SDS/PAGE gels confirmed that in the absence of Ca2+anionic trypsinogen was not activated (see Fig 4B), while cationic trypsinogen was fully activated over
Fig 5 Autoactivation of human cationic trypsinogen at pH5.0.
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 Ca2+concentrations up to 1 m M (B)
Inhibition of autoactivation by Ca2+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 C in 0.1 M Tris/HCl (pH 8.0) in the presence of the indicated concentrations of CaCl 2 Aliquots of 2.5 lL were withdrawn from reaction mixtures at indicated times and trypsin activity was deter-mined with 0.14 m 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+ on the relative rate of autolysis in the presence (s) or absence (d) of 100 m M NaCl The rate determined in the absence of added Ca2+was designated as 1.
Trang 7the same time period (Fig 5C) Conversion of cationic
trypsinogen to trypsin was practically quantitative, with no
significant zymogen degradation, and addition of 5 mM
Ca2+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 Ca2+,
anionic trypsin suffered autolysis at a rapid rate (t1/28 min),
and low concentrations of Ca2+ stabilized the enzyme,
with an IC50 of 5 lM (Fig 6A,C) Addition of 100 mM
NaCl to anionic trypsin decreased the rate of autolysis
threefold (in the absence of Ca2+t1/2was 24 min, Fig 6B),
and increased the IC50value for Ca2+stabilization sixfold
(30 lM, Fig 6C) Autolysis of cationic trypsin was
signifi-cantly slower, and in the absence of Ca2+a t1/2of 90 min
was measured (Fig 7A) Thus, in the absence of Ca2+
a >11-fold difference was apparent between the autolysis
rates of the two trypsins (Figs 6A and 7A) Low
concen-trations of Ca2+afforded stabilization with an IC50value of
20 lM(Fig 7A and B) Surprisingly, addition of 100 mM NaCl diminished autolysis of cationic trypsin 14-fold, and even in the absence of Ca2+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 Ca2+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 pH8.0, in 1 m M Ca2+ 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) contained 0.67 l 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)1BSA (C).
Trang 850% 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 degradaactiva-tion 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
and pH 5.0 At pH 8.0, two different Ca2+concentrations
were used, 1 mMor 50 lM The 1 mMCa2+concentration
was selected to model the conditions in the pancreatic juice
or in the duodenum, the physiological site of trypsinogen
activation The 50 lM Ca2+ concentration modeled the
intracellular conditions, where Ca2+concentrations are low
Although true cytoplasmic Ca2+concentrations are below
micromolar levels, we chose to use 50 lMCa2+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 Ca2+ 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 mMNaCl or in the
presence of 100 mMNaCl and 2 mgÆmL)1BSA
As shown in Fig 8A, in 0.1M Tris/HCl (pH 8.0) and
1 mM Ca2+, 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 mM 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 mM 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 0.1MTris/HCl (pH 8.0) with
50 lM Ca2+ 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 0.1M Tris/HCl (pH 8.0) with 50 lM Ca2+, 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 lMCa2+, 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)
Trang 9How 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 pH5.0 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 pH8.0, in 50 l M Ca2+ See Fig 8 for
experi-mental details.
Trang 10cationic 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 mMCa2+or lower At
these Ca2+ concentrations autoactivation was relatively
slow, and could not keep up with pace of trypsin(ogen)
degradation Only in millimolar Ca2+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
Ca2+ (b) Acidic pH stimulated autoactivation of cationic
trypsinogen, but inhibited activation of anionic trypsinogen
(c) Anionic trypsin bound Ca2+ fourfold stronger than
cationic trypsin, as judged by the stabilizing effect of Ca2+
on autolysis Binding of Ca2+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, Ca2+in the
concentration range between 1 mMand 10 mMstimulated 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 Ca2+ concen-trations between 1 mM)20 mM Although this observation
is important for the correct interpretation of autoactivation assays performed under a variety of Ca2+concentrations in the literature; the (patho)physiological significance of such a
Ca2+-mediated inhibition mechanism is questionable (d) Autoactivation of cationic trypsinogen and autolysis of cationic trypsin were markedly inhibited by 100 mMNaCl, 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
Ca2+than reported before [19] Relative to cationic trypsi-nogen, anionic trypsinogen autoactivated faster at pH 8.0 in
20 mM Ca2+ whereas the opposite relationship was described previously [25,26] Finally, in our experiments, high Ca2+concentrations consistently inhibited autoactiva-tion of caautoactiva-tionic trypsinogen, while both stimulaautoactiva-tion 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 Ca2+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
pH8.0 in 50 l M Ca2+(A) and pH5.0 (B and C) Autoactivation
experiments were carried out as in 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.