Proteolysis of Native Proteins
TRAPPING OF A REACTION INTERMEDIATE*
Chenyi
Wu,
Duncan H. L.
Robertson,
Simon J.
Hubbard,
Simon J.
Gaskell
, and
Robert J.
Beynon§
From the Department of Biomolecular Sciences and the
Michael Barber Centre for Mass Spectrometry, University
of Manchester Institute of Science and Technology, Manchester M60 1QD,
United Kingdom
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ABSTRACT |
When limited proteolysis of the mouse major
urinary proteins by trypsin was stopped by rapid denaturation of the
proteinase, a covalent adduct of the two proteins was observed. The
formation of this complex required active trypsin, was favored at low
pH, and could be reversed by the addition of covalent or non-covalent trypsin inhibitors. Electrospray mass spectrometry of the complex demonstrated that it was an acyl-enzyme complex, formed after an
unusual exopeptidase attack on the C-terminal-Arg-Glu-OH sequence by
trypsin. The complex could sequester over 50% of the trypsin in a
digestion mixture, and as anticipated, the protein was an effective
trypsin inhibitor.
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INTRODUCTION |
Proteolysis of native proteins is influenced by higher order
structural features of the interaction between substrate and proteinase, in addition to the restrictions imposed by the primary specificity of the proteinase. Proteolysis can be limited, and the rate
of digestion of peptide bonds with equivalent primary specificity seems
to depend on several parameters, the most important of which is the
capability for local flexibility of the polypeptide chain in the region
of the scissile bond (reviewed recently in Ref. 1). Proteolytic enzymes
can therefore be used as probes of flexible or exposed regions of a
polypeptide chain, to monitor changes in such parameters after
conformational shifts such as those elicited by ligand binding, and to
generate functional subdomains by excision of accessible connecting
regions (2, 3).
We have used limited proteolysis as a structural probe of major urinary
proteins (MUPs).1 MUPs are a
group of closely related proteins that are secreted into mouse urine
and that are members of the lipocalin class of proteins (4-6). They
bind semiochemicals (7, 8) to effect their slow release (9, 10) or to
protect them from, for example, atmospheric oxidation. We have become
interested in the role of these proteins in ligand binding and release
and in the ability of proteolysis to affect the loss of ligands by
disruption of the structure of the native holoprotein. Further, an
N-terminal MUP-derived hexapeptide may be important in communicating
additional chemosignals (11), and we have explored the role of
proteolysis in the generation, or destruction, of this motif. As part
of this investigation we have exposed MUPs to different proteinases and analyzed the products. Digestion by trypsin yielded an unusual pattern
of behavior, specifically, the generation of substantial amounts of a
high mass covalent complex of trypsin and MUP, which we have characterized.
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MATERIALS AND METHODS |
Pooled urine was collected from unanesthetized mice by bladder
massage from mature BALB/c males, housed together in groups of 8-16
using a 12-h dark/12-h light cycle and given free access to food and
water. Immediately after collection, the urine was desalted on 5-ml
"spun columns" of Sephadex G-25, previously equilibrated with
water, and further concentrated with Centricon centrifugal concentrators (Amicon, Stonehouse, Gloucester, United Kingdom) with an
Mr = 10,000 cut-off. The resultant stock
solutions contained MUPs at 40-60 mg/ml
(E2801% = 6.1). This pool comprises a
mixture of very similar allelic variants (12), but when we have
resolved these variants by ion exchange chromatography, we have
observed no difference in behavior during limited
proteolysis2 and we have
routinely used the mixed pool. The mixture of proteins in the
preparation differ in conservative substitutions of only four amino
acids (12). This pool of very similar allellomorphs is referred to in
the singular, as MUP, in this paper.
Trypsin (EC 3.4.21.4) from porcine pancreas (Sigma-Aldrich Chemical Co,
Poole, Dorset, UK) was incubated with MUPs in a volume of 120 µl with
1:1 (by weight) enzyme/substrate ratio. Incubation time, temperature,
and pH were varied in different experiments. The pH was buffered as
follows: chloroacetate, pH 3.0; formate, pH 4.0; benzoate, pH 5.0;
maleate, pH 6.0; Hepes, pH 7.0-8.0; borate, pH 9.0-10.0; and CAPS, pH
11.0. All buffers were 20 mM, and the ionic strength was
adjusted to 0.1 M with NaCl. Buffers were thermodynamically
corrected for temperature and ionic strength effects
(http://www.bi.umist.ac.uk/buffers.html). Where other reactants, such
as soybean trypsin inhibitor and TLCK were included, they were
dissolved in buffer (10 mg/ml) or methanol (5 mM),
respectively. Carboxypeptidase B was added from a 10 mg/ml stock as
supplied by the manufacturer (Boehringer Mannheim, Lewes, East Sussex, UK).
Enzyme reactions were stopped by addition of trichloroacetic acid to a
final concentration of 10% (w/v). Precipitated proteins were pelleted
by centrifugation for 5 min at 12,500 rpm. The pellets were washed 2-3
times with 200 µl of diethyl ether to remove excess trichloroacetic
acid, resuspended in SDS-PAGE sample buffer, and heated at 100 °C
for 5 min prior to separation by SDS-PAGE under reducing conditions in
17.5% acrylamide gels (13). For quantitative analysis, Coomassie
Blue-stained gels were scanned at 300 pixels per inch in an 8-bit
grayscale and analyzed using Quantifier software (Phoretix
International, Newcastle, UK). The relationship between band volume and
amount of protein was linear over the range used in the experiment
0-100 µM MUP (r = 0.98).
In some experiments, proteins separated by SDS-PAGE were transferred to
a 0.45-µm nitrocellulose membrane (Schleicher and Schuell,
Kingston-upon-Thames, Surrey, UK) by electroblotting overnight at a
constant 30 V in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3). The membrane was then
incubated for 1 h in a 1:1000 rabbit anti-MUP serum solution in
phosphate-buffered saline/Tween (10 mM phosphate, 150 mM NaCl, 0.05% Tween (v/v), pH 7.3) and visualized by a
1-h incubation with 1:3000 goat anti-rabbit IgG conjugated to alkaline
phosphatase (Bio-Rad, Hemel Hempstead, Herts, UK) followed by a further
incubation with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and
nitro blue tetrazolium (Sigma). Proteins from SDS-gels were also
blotted on to polyvinylidene difluoride membrane in transfer buffer (10 mM CAPS, 10% methanol, pH 11.0). After a brief staining
with 0.1% (w/v) Coomassie Blue in 40% (v/v) methanol, 1% (v/v)
acetic acid, the desired protein bands were cut from the membrane and
subjected to 5 cycles of Edman degradation on an Applied Biosystems
476A protein sequencer.
For electrospray mass spectrometry (ESI/MS), the pellets recovered
after proteolysis were redissolved with gentle heating for a few
seconds on a boiling water bath in a 100-µl ESI/MS mobile phase (50%
(v/v) acetonitrile, 0.1% (v/v) formic acid). Spectra were acquired on
a Quattro triple quadropole mass spectrometer (Micromass, Manchester,
UK) fitted with an electrospray ionization source and upgraded to
Quattro II specifications. The instrument was tuned and calibrated with
a 20 pmol/µl solution of horse heart myoglobin made up in 50% (v/v)
aqueous acetonitrile, 0.1% (v/v) formic acid. Samples were introduced
into the source at 10 µl/min. Scanning was from
m/z 950 to 1500 at 10 s/scan. Twenty to thirty scans were averaged to produce the final spectra. Data acquisition, instrument control, and post-run processing were by the VG MassLynx software (Micromass). Mass assignments were made from maximum entropy-processed data (14). The protein masses thus calculated were
within 2 Da of the cDNA-inferred masses (12), and repeated measurements over a 6-month period of three of the proteins yielded mass assignments of 18644.2 ± 0.78 Da, 18694 ± 0.84 Da, and
18708.2 ± 1.75 Da (mean ± S.D., n = 10 for
each protein).
Anion-exchange chromatography was on an FPLC system (Amersham Pharmacia
Biotech) fitted with a Resource-Q column (Vt = 6 ml). The column was equilibrated with 20 ml of 50 mM
formate buffer, pH 5.0, before application of 500 µl of the tryptic
digest of MUP (1 mg/ml MUP, 7 mg/ml trypsin, pH 4.0, incubated for 30 min at room temperature in 50 mM formate, pH 5.0). The
column was developed using a 100-ml linear salt gradient (0-1
M NaCl) at a flow rate of 6.0 ml/min. Protein fractions
were collected and reconcentrated with Centricon (Amicon) centrifugal concentrators.
Anhydrotrypsin was prepared by a published method (15) with some
modifications, and purified by affinity chromatography. Porcine
pancreatic trypsin (200 mg) and phenylmethylsulfonyl fluoride (70 mg),
dissolved in 100 ml of 50 mM Tris/HCl, pH 7.0, containing 20 mM CaCl2, were incubated at room temperature
for 30 min with occasional additions of 20 mM NaOH to
maintain the pH at 7.0. Trypsin activity was monitored throughout the
incubation period. The reaction was stopped by adjusting the pH to 3.0 with 1 M HCl. After a brief centrifugation, the supernatant
was dialyzed overnight against 3 liters of 1 mM HCl at
4 °C. The solution of phenylmethylsulfonyl trypsin thus obtained was
brought to 50 mM KOH at 0 °C and incubated for 12 min,
then adjusted to pH 5.0 with formic acid to stop the reaction. The
mixture was finally adjusted to pH 8.0 by the addition of 2 M Tris/HCl. Affinity chromatography of the anhydrotrypsin was performed on immobilized soybean trypsin inhibitor (Pierce & Warriner, Chester, UK) packed in a 5-ml spun column. Typically 2 ml of
gel was used with a capacity of 20 mg of trypsin per ml of gel. The
column was equilibrated with 20 ml of 50 mM Tris/HCl, pH
8.0, before application of 10 ml (20 mg) of anhydrotrypsin. The bound
material was washed with 20 ml of the same buffer and eluted with 3 ml
of 100 mM sodium chloroacetate buffer, pH 2.0. The
anhydrotrypsin thus purified was concentrated/desalted with centrifugal
concentrators. The final concentration was estimated spectrophotometrically (E2801% = 14.7). Tryptic activity at 25 °C toward Bz-Arg-4-nitroanilide was
monitored by following the increase in absorbance at 405 nm. MUP,
MUP1/9-161, and the synthetic peptides, SLQARE and SLQAR,
were tested for trypsin inhibitory properties at low pH in a total
volume of 1 ml of assay solution consisting of 20 mM
formate, pH 4.0, 0.2 mM
N
-benzoyl-DL-arginine-p-nitroanilide,
2.1 µM trypsin. The concentrations of potential
inhibitors ranged from 0 to 25 µM.
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RESULTS AND DISCUSSION |
Purified MUPs comprise over 98% of the proteins in mouse urine
and were used after desalting but without further purification. This
preparation comprises a mixture of MUPs with a high level of sequence
identity, differing in only 4 of 162 amino acids (9), each with a
molecular mass of approximately 18-19 kDa. When the MUP mixture
(referred to subsequently in the singular) was treated with trypsin, a
product band (MUP'), approximately 1 kDa smaller than the MUP, was
formed quite rapidly and was resistant to further proteolysis (Fig.
1a). Thus, proteolysis was
limited and was consistent with removal of short peptide fragments from
the N and/or C termini of the molecule. However, SDS-PAGE analysis also
demonstrated an unexpected outcome of the proteolytic reaction.
Specifically, additional bands, predominantly in the region of 40 kDa
and to a lesser extent at 32 kDa, appeared on the gel. These bands were not present in the starting materials and were not formed when the two
proteins were incubated singly under identical conditions.

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Fig. 1.
Formation of a high mass complex during
proteolysis of MUP by trypsin. a, trypsin (50 µM) was incubated with MUP (65 µM) in 20 mM Hepes buffer, pH 7.5, for between 2 and 60 min. At
various times, samples were removed, and the reaction was terminated by
addition of trichloroacetic acid to a final concentration of 10%
(w/v). The proteins in the reaction mixture were then recovered by
centrifugation, residual acid was washed away with diethyl ether, and
the reaction mixture was analyzed on nonreducing SDS-PAGE.
b, the analysis was conducted in essentially the same
fashion as in a except that the time was fixed at 5 min and
the concentrations of MUP or trypsin were varied. In the first
experiment, trypsin was maintained at 50 µM, and MUP was
varied from 16 to 60 µM. In the second experiment, MUP
was maintained at 65 µM, and trypsin was varied from 12 to 45 µM. MUP' refers, in this and subsequent figures, to
the mixture of products of tryptic attack on MUP at Arg-8 and
Arg-161.
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In this proteolytic reaction, the digestion was stopped by addition of
trichloroacetic acid to a final concentration of 10% (w/v). When the
reaction was stopped using SDS-PAGE sample buffer, without acid
precipitation, no higher molecular weight bands were evinced. These
bands were present in the presence or absence of reducing agents during
SDS-PAGE, precluding the possibility of disulfide-linked complexes. The
amount of the high mass complexes that were formed was dependent on the
concentration of both trypsin and MUP (Fig. 1b).
The precise pattern of limited proteolytic attack on MUP was defined by
electrospray mass spectrometry. We have demonstrated previously that
ESI/MS combined with maximum entropy data analysis is a powerful method
of MUP characterization and is able to identify several gene products
that are consistent (within 2 Da) with cDNA-predicted molecular
masses (12, 16). ESI/MS analysis of the undigested MUP mixture
identified three masses, 18646, 18694, and 18708 Da, each of which is
consistent with a different, but intact, MUP in urine (12). After
tryptic digestion, the overall distribution of peaks remained the same
but the pattern had shifted down the mass scale, to 17699, 17746, and
17760 Da, by measured decrements of 948, 947, and 948 Da, respectively
(Fig. 2). The mass decrements and
SDS-PAGE data are thus consistent and suggest the removal of an
N-terminal or a C-terminal peptide. However, the loss of approximately
948 Da cannot be explained by a simple hydrolytic release of a C- or
N-terminal peptide, generated by cleavage at the first or last tryptic
sites in the sequence. Specifically, cleavage at the first tryptic site
in the sequence: NH2-EEASSTGR
NFNVEF ... would yield
new masses lower by 817 Da. An additional loss of 129 Da, provided by a
second hydrolysis at the C-terminal end of the molecule,
... CLQAR161
E162 yields a loss of 946 Da. N-terminal sequencing would have only revealed the new N terminus,
and the CLQAR161
E162-OH cleavage, an
exopeptidase attack, would not normally be considered a cleavage site
for trypsin. The product of the N- and C-terminal proteolysis on the
same substrate molecule is designated as MUP9-161. Both
the N and C termini of MUP are flexible, and the C terminus cannot be
visualized in the crystal structure (17). Proteolytic attack at the
extrema is therefore explicable in terms of accessibility of the
termini of the protein, although the exopeptidase attack was
unexpected.

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Fig. 2.
Limited proteolysis of MUPs by trypsin.
A mixture of MUPs derived from a single mouse strain (total MUP
concentration = 65 µM) was incubated with trypsin
(50 µM) for 120 min in 20 mM Hepes, pH 7.5. At the end of this time, the reaction was terminated by precipitation
with trichloroacetic acid, as described under "Materials and
Methods." The reaction mixture was then analyzed by electrospray mass
spectrometry, using maximum entropy data processing (14). Panel
a is the transformed spectrum of the undigested mixture.
Panel b is the mass spectrum after digestion with trypsin.
The estimated errors in the mass assignments are derived from maximum
entropy processing of single accumulations of ESI/MS data.
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The 40-kDa band was also unexpected, as there were no species of that
size, or larger, in the initial incubation mixtures. When probed by
Western blotting with a MUP polyclonal antibody it was evident that the
40-kDa species contained MUP (results not shown). The 40-kDa band was
also blotted onto polyvinylidene difluoride membranes and subjected to
automated Edman degradation. Three sequences could be identified
(a) IVGGY ... , at 6-8 pmol; (b)
EEA ... , at 4 pmol; and (c) NFNVE ... , at 4 pmol. The first is attributable to trypsin, the second to the
N-terminal sequence of unproteolyzed MUP, and the third to the
N-terminal sequence of the MUP9 -C terminus. Thus, the
complex contained both MUP and trypsin, and the amounts of each protein
were consistent with a 1:1 stoichiometry, as predicted by the mass of
the complex.
The 40-kDa complex was stable to the conditions of SDS-PAGE and was
likely therefore to be a covalent complex of trypsin and MUP (referred
to hereafter as T-MUP) which is trapped by the virtually instantaneous
denaturation by trichloroacetic acid and concomitant inactivation of
trypsin. If one or the other protein components was first denatured
with trichloroacetic acid before addition of the second protein, the
complex was not formed. Further, the appearance of two MUP-derived
N-terminal sequences in the complex suggested that the complex could
also be formed by MUP9 -C terminus. To test this directly,
the tryptic product MUP9-C terminus was prepared by
tryptic digestion and subsequently purified and separated from trypsin
and unproteolyzed MUP on anion-exchange chromatography (MonoQ FPLC).
This product (MUP9 -C terminus) was then reincubated with
trypsin. Under these circumstances, the T-MUP complex was formed once
again (results not shown).
If trypsin was preincubated with the irreversible inhibitor TLCK (Fig.
3a), no complex could be
generated, demonstrating the requirement for active enzyme. TLCK
alkylates the active site histidyl residue and reduces the
nucleophilicity of the active site seryl residue. TLCK-modified trypsin
is unable to bind to immobilized peptides with a C-terminal argininyl
residue (18), consistent with some element of steric hindrance of the
complex formation as well. In a further experiment, MUP was digested
with trypsin for a fixed period, after which time a stoichiometric excess of soybean trypsin inhibitor was added. The complex could not be
detected after addition of the soybean trypsin inhibitor (Fig.
3b). Thus the strength of the interaction between the
soybean inhibitor and trypsin is sufficient to sequester trypsin, at
the expense of the T-MUP complex. This is good evidence for a
reversible but covalent association of proteinase and substrate. The
need for catalytically active trypsin and the role of the active site nucleophile were further confirmed by experiments in which MUP was
incubated with anhydrotrypsin (Fig. 3c). Under such
conditions, the complex was not formed.

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Fig. 3.
Effect of inhibition of trypsin on complex
formation. a, trypsin (50 µM) was
pretreated with the irreversible inhibitor TLCK for 30 min in 20 mM Hepes buffer, pH 7.6. The proteinase was fully
inhibited, as confirmed by catalytic activity toward
Bz-Arg-4-nitroanilide (see "Materials and Methods"). Subsequently,
the inhibited trypsin was incubated with MUP (conditions as in Fig. 1),
and the complex formation was assessed as described previously (see
legend to Fig. 1). In a further experiment, uninhibited trypsin was
incubated with MUP, and after a 5-min incubation, TLCK was added to a
final concentration of 50 µM. After 30 min, the sample
was precipitated by trichloroacetic acid and analyzed by SDS-PAGE.
b, trypsin (65 µM) was incubated with MUP (40 µM) for up to 15 min, at which point soybean trypsin
inhibitor (STI, 75 µM) was added to the
reaction mixture. Samples were removed before and after addition of
inhibitor and analyzed for complex formation as described previously
(see legend to Fig. 1). c, in a separate experiment, MUP'
(MUP9-161, 80 µM) was incubated with trypsin
(T, 75 µM) or anhydrotrypsin (AHT,
75 µM) in 20 mM formate buffer, pH 4.0, I = 0.1 M, for 5 min before the reaction
was terminated by precipitation with trichloroacetic acid and analyzed
by SDS-PAGE.
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The formation of the T-MUP complex was strongly dependent on pH (Fig.
4a). Trypsin and MUP were
reacted in buffers from pH 4 and pH 11 at a constant ionic strength.
Proteolysis of the MUP, leading to the formation of
MUP9-161, was evident at pH values of 6 and above, in
accordance with the known pH optimum of trypsin. By contrast, complex
formation was most evident at low pH values, and as the pH of the
incubation increased, less of the complex was visible. At pH 4.0, approximately half of the MUP and trypsin was sequestered into the
complex, as estimated by densitometry (Fig. 4b). The complex
is formed preferentially at low pH, under conditions in which the
hydrolytic activity of trypsin would be expected to be minimal. Indeed,
the lack of digestion of MUP at low pH (below pH 6.0) is evident from the SDS-PAGE analysis. However, electrospray mass spectrometry of the
digestion reactions at three pH values indicated that although the
N-terminal endopeptidase cleavage was suppressed at low pH values, the
C-terminal exopeptidase reaction still took place (Table
I). Formation of the complex between MUP
or MUP9-161 and trypsin seems to preclude an intermediate
derived directly from, and as an obligatory outcome of, the hydrolysis
of the N-terminal octapeptide. Another possible explanation for complex
formation would be a transpeptidation reaction, in which
MUP9-161 was transferred to the N terminus of a second
molecule of trypsin or MUP, which acted as an acceptor. This however is
disproved by recovery of both the intact N-terminal trypsin sequence
and the MUP sequence from the complex. Taken together, the data imply that the 40-kDa product is a stoichiometric, probably acyl-enzyme, complex between trypsin and MUP.

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Fig. 4.
pH dependence of complex formation.
a, trypsin (70 µM) and MUP (85 µM) were incubated for 5 min in a series of buffers at
different pH values, all adjusted with NaCl to a constant ionic
strength of 0.1 M (see "Materials and Methods"). The
reaction was terminated with trichloroacetic acid, and the products
were analyzed for complex formation by SDS-PAGE as described
previously. b, the gel was scanned and analyzed by
densitometry as described under "Materials and Methods." The band
volumes for the complex (open squares) and the
total of MUP and MUP digestion products (open
circles) were plotted as a function of pH, expressed as a
percentage of the maximal volume.
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Table I
Effect of pH on tryptic proteolysis of MUPs
Trypsin (70 µM) and MUP (85 µM) were
incubated for 5 min in formate (pH 4.0), Hepes (pH 7.0), or CAPS (pH
11.0) buffers, adjusted with NaCl to a constant ionic strength of 0.1 M (see "Materials and Methods"). The reaction was
terminated by precipitation with trichloroacetic acid, and the MUP
digestion products were analyzed by electrospray mass spectrometry.
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The formation of the complex seemed therefore to be associated with the
unusual cleavage of the C-terminal glutamate residue. The role of the
C-terminal, exposed Arg-161 was elaborated by an additional experiment
in which MUP9-161 was treated with carboxypeptidase B to
remove the C-terminal Arg-161 before incubation with trypsin (Fig.
5). In this instance, formation of the
complex was completely abolished, irrespective of the pH at which the
reaction was conducted. This experiment also suggested that the complex
was formed reversibly, since a C-terminal argininyl residue would not
be susceptible to exopeptidase attack if the MUP was covalently and
irreversibly linked via its C terminus to the catalytic seryl residue
of trypsin. Since the complex is completely destroyed by incubation
with the carboxypeptidase, it is reasonable to assume that the binding
between proteolyzed MUP (with C-terminal arginine intact) and trypsin
is rapid, favoring significant quantities of the complex.

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Fig. 5.
Effect of Arg-161 on complex formation.
a, trypsin (T, 70 µM) and MUP (85 µM) were incubated in 50 mM Hepes buffer, pH
7.5, for 5 min, in the presence or absence of 0.13 µg/ml
carboxypeptidase B (CpB). Control incubations include
carboxypeptidase B incubated alone and MUP incubated with
carboxypeptidase. The reaction was terminated and analyzed for complex
formation as described previously. b, in separate
experiments, incubations of MUP and trypsin, in the presence of
carboxypeptidase B, were conducted at pH 4.0, pH 7.0, and pH 11.0, all
at a constant ionic strength of 0.1 M (see "Materials and
Methods").
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We have made repeated attempts to observe the intact complex by
electrospray mass spectrometry. Previous electrospray mass spectrometric analyses of trypsin (19, 20), however, have indicated
significant heterogeneity, and it has proved impossible to purify a
trypsin variant that yields a single peak. Trypsin was incubated with a
mixture of three MUPs at low pH, and the complex was trapped by
precipitation before redissolution and analysis by ESI/MS. The three
MUPs (starting masses 18646, 18694, and 18708 Da) were all completely
converted into products (18518, 18565, and 18581 Da) that indicated
trypsin-mediated exopeptidase cleavage of the C-terminal Glu-162. In
the 42-kDa region of the mass spectrum, three peaks were also
identified (Fig. 6). Each of these was
within 4 Da of the predicted mass of the (MUP isoform1-161) + trypsin
17 Da. The masses of the three peaks are therefore consistent with formation of an acyl-enzyme complex between
MUP1-161 and trypsin.

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Fig. 6.
Electrospray mass spectrometry of the T-MUP
complex. Trypsin (320 µM) was incubated with MUP (80 µM) in 20 mM formate buffer, pH 4.0, for 5 min. The reaction was terminated with trichloroacetic acid, and the
reaction products were analyzed by electrospray mass spectrometry (see
"Materials and Methods").
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Quantitative estimates of the ratio of complex to unbound MUPs could
not be derived from the ESI/MS data, but SDS-PAGE analysis indicated
that as much as 40% of a stoichiometric mixture of trypsin and MUP is
sequestered as the complex (Fig. 4b). Why such a large proportion of trypsin should be sequestered into this T-MUP complex, particularly at low pH values, is unclear. It might be argued that the
low pH conditions act in two ways. First, suppression of the C-terminal
carboxylate charge might favor enhanced binding of the C-terminal
arginine-exposed peptide to the substrate-binding site of trypsin.
Second, the lowered concentration of the nucleophilic hydroxyl ion
would favor accumulation of a covalent catalytic intermediate that
would normally be attacked by this species. This hypothesis has also
been invoked to explain the effect of lowered pH on the enhanced
peptide synthesis activity of trypsin (18). Indeed, the ability of
trypsin to catalyze peptide bond synthesis is strong support of the
existence of such acyl or tetrahedral complexes. However, these
reaction intermediates are generally thought to be transient, and the
accumulation that is seen in the present example is surprising.
The sequestration of large amounts of trypsin into the T-MUP complex
implies that the MUPs should be capable of acting as trypsin
inhibitors. This was tested by monitoring the ability of MUPs to alter
the rate of hydrolysis of the chromogenic substrate Bz-Arg-4-nitroanilide at low pH values, where the complex is
particularly favored (Fig. 7). As
anticipated, MUPs are weak trypsin inhibitors, but we do not ascribe
any physiological significance to this observation.

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Fig. 7.
Inhibition of amidolytic activity of trypsin
by MUPs and C-terminal peptides. MUP and its tryptic product were
tested for the ability to inhibit the trypsin-catalyzed hydrolysis of
Bz-Arg-4-nitroanilide at pH 4.0, in 20 mM formate buffer.
In separate experiments, peptides derived from the C-terminal sequence
of MUP were also tested for inhibitory activity. MUP, open
circles, MUP9-161, filled
squares; SLQAR, closed circles;
SLQARE, filled triangles.
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The unexpected accumulation of large quantities of a
proteinase·substrate complex raised the possibility that the
structure of MUP was an important contributory factor, and that the
three-dimensional structure of the protein favors the accumulation or
stabilization of the covalent intermediate. The structures of the C and
N termini of MUPs are not visible by x-ray crystallography (17) and are generally held to be mobile segments which would be expected to be
accessible to proteolytic attack (1, 2). Indeed, simple molecular
modeling experiments have shown that a plausible covalent complex can
be formed between trypsin and the MUP C terminus without introducing
any contentious intra- or intermolecular stereochemistry. Furthermore,
this can be achieved without breaking the Cys-64-Cys-156 disulfide
bond. To test the ability of trypsin to bind unstructured C-terminal
peptides, we synthesized two peptides SLQARE and SLQAR. The first is
the full C-terminal hexapeptide sequence of MUP, with the substitution
of a seryl for a cysteinyl residue to obviate peptide dimerization
through disulfide bonds. The second is the C-terminal sequence after
removal of the glutamyl residue and is thus equivalent to the
C-terminal sequence of MUP9-161. Both peptides were weak
trypsin inhibitors (Fig. 7), and the shorter of the two was more
potent. This may reflect the need for exopeptidase attack on the longer peptide.
Trypsin was incubated with the peptides SLQAR or SLQARE, and the
reaction products were analyzed by electrospray mass spectrometry (Fig.
8). Trypsin was resolved as multiple
peaks, and a second set of peaks, offset by approximately 557 Da, was
also observed. These are consistent with addition of the peptide SLQAR
to trypsin and loss of a water molecule as a consequence of formation
of an acyl-enzyme intermediate. The mass difference between trypsin and
the peptide-acyl-enzyme adduct is not sufficient to be visible on
SDS-PAGE, and we have already noted that ESI/MS cannot yield accurate
measurements of the relative abundance of the different peaks. It is
therefore not possible to assess the amount of the complex that was
formed with the short peptides. Complex formation does not appear to be
critically dependent on the three-dimensional structure of MUP,
although the protein/protein interaction might enhance the amount of
complex that is formed (Fig. 9).

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Fig. 8.
Electrospray mass spectrometry of the
peptide-MUP complex. Trypsin (35 µM) and the peptide
SLQARE (600 µM) were incubated at room temperature in a
solution of acetic acid titrated to pH 4.0 for 10 min, prior to
analysis by electrospray mass spectrometry. The incubation mixture was
introduced directly into the source, without additional organic
solvent.
|
|
It has long been recognized that trypsin is able to bind peptides that
have an exposed C-terminal lysine or arginine residue. Indeed, this is
the basis of the methods of recovery of such peptides using
anhydrotrypsin as a trapping matrix (20, 21). It is generally held that
the association reflects non-covalent binding of the C-terminal residue
to the specificity pocket. There has not, to our knowledge, been any
indication previously that the interaction with intact trypsin might be
covalent and reflect a partial reversal of the hydrolytic reaction.
Obviously, the acyl or tetrahedral complex must be formed with the
carbonyl group of one of the substrates when trypsin functions as a
peptide synthetase; the amino group of the N-terminal donor then
attacks this complex (reviewed in Ref. 22). However, this reaction is
relatively weak, and significant accumulation of acylated enzyme would
be surprising. It remains to be seen whether such complexes involving C-terminal basic residues are a generic feature of trypsin catalysis or
even whether the phenomenon can be extended to other serine proteases.
In this respect, the recent structural elucidation of an acyl-enzyme
complex between porcine pancreatic elastase and a heptapeptide, trapped
at low pH values (23), is particularly relevant, as is the formation of
SDS-stable complexes between serine proteases and serpins (24).
However, in the latter example, the evidence pointed to covalent links
additional to that generated in the acyl-enzyme complex. In this
respect, the data presented here are simpler, as they clearly identify
the role of the active site seryl residue.
There may be new applications that can make use of the covalent
trapping, should this association manifest itself as a common occurrence. For example, recovery of peptides that have a C-terminal argininyl residue would be enhanced at low pH values where adventitious tryptic endoproteolysis would be avoided. Such a tool may lend itself
to methods of peptide analysis by mass spectrometry where, for example,
a combined trypsin-peptide complex could be recovered. An
endoproteolytic treatment at neutral pH might be followed by an
acidification, in which case the only free peptide would be the
C-terminal peptide. This additional identification tag might enhance
the identification of the parent protein, which, together with new
methods of N-terminal analysis (25), could reduce the complexity of the
identification problem in proteome analysis.
 |
FOOTNOTES |
*
This work was supported by grants from the Biotechnology and
Biological Sciences Research Council (to R. J. B.) and the award of a
Wellcome Trust Fellowship (to S. J. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: UMIST, P. O. Box 88, Manchester M60 1QD, UK. Tel.: 44-161-200-4221; Fax: 44-161-236-0409; E-mail: r.beynon{at}umist.ac.uk.
The abbreviations used are:
MUP, major urinary
protein; PAGE, polyacrylamide gel electrophoresis; ESI, electrospray; FPLC, fast protein liquid chromatography; TLCK, N
-p-tosyl-L-lysine
chloromethyl ketone; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
2
C. Wu, D. H. L. Robertson, S. J. Hubbard,
S. J. Gaskell, and R. J. Beynon, unpublished observations.
 |
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