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Porcine origin of human sputum trypsin?
George H.
Caughey,
Wilfred W.
Raymond, and
Kenneth C.
Fang
Cardiovascular Research Institute and Department of Medicine,
University of California, San Francisco, California 94143-0911
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ABSTRACT |
From purulent cystic fibrosis (CF) sputum,
previous investigators partially purified a trypsinlike protease. A
similar purified enzyme is available commercially as "human sputum
trypsin." To explore the nature and origin of this preparation, we
purified and NH2 terminally
sequenced its major protein component. The resulting sequence,
Ile-Val-Gly-Gly-Tyr-Thr-(Cys)-Ala-Ala-Asn-Ser-Val/Ile-Pro-Tyr-Gln-Val-Ser-Leu-Asn-Ser, differs from known human proteins but is identical to porcine trypsin,
including the Val/Ile polymorphism at residue 12. Specific activity and
electrophoretic and inhibition profiles and immunoreactivity of sputum
and porcine pancreatic trypsin are nearly identical. Because porcine
trypsin is a major ingredient of digestive enzyme supplements taken by
CF patients with pancreatic dysfunction, we propose that one or more
lots of human sputum trypsin derive from enzyme supplements and are of
porcine origin. The path by which trypsin ends up in sputum is unknown.
Because sputum trypsin is active but susceptible to inactivation by
plasma
1-proteinase inhibitor,
it is unlikely to derive from trypsin absorbed into the bloodstream.
However, it may originate from tracheally aspirated stomach contents or
from digestive supplement-contaminated saliva mixed with expectorated
sputum. The imbalance between proteases and antiproteases in CF
bronchial secretions allows trypsin to remain active despite
sensitivity to serpins and secretory leukocyte proteinase inhibitor.
Furthermore, because sputum trypsin activates human progelatinase B, it
may be responsible in part for the reported presence of activated
matrix metalloproteinases in CF sputum.
cystic fibrosis;
1-proteinase inhibitor; gelatinase B; secretory leukocyte proteinase inhibitor
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INTRODUCTION |
THE IDENTIFICATION and purification of an active,
trypsin-like protease from purulent cystic fibrosis (CF) sputum was
first reported by Viscarello and colleagues (11). This enzyme is
distinct from neutrophil elastase, cathepsin G, and proteinase-3 based on its chromatographic behavior and substrate preferences, hydrolyzing an arginine-containing substrate of trypsin but not substrates of
elastase and cathepsin G. It is classified as a serine protease based
on inhibition by soybean trypsin inhibitor and phenylmethylsulfonyl fluoride. Purified from CF sputum, a similar enzyme is sold
commercially as "human sputum trypsin." It is thought to be
distinct from human pancreatic trypsin based on its strong inhibition
by soybean trypsin inhibitor and has been presumed to derive from
leukocytes, which are the predominant cells in the purulent sputum of
patients with CF and the major source of active serine proteinases (4).
However, there has been no definitive identification of cellular
provenance of "sputum trypsin" and no study establishing its
position in the family of human trypsinlike serine proteases. On the
grounds that the presence of an active sputum enzyme, if truly similar to trypsin, could be destructive and a cause of damage in bronchi of CF
patients (2), we decided to explore some properties of human sputum
trypsin. Based on our analysis, we propose that the enzyme is not of
human but of porcine origin, presumably derived from trypsin in
pancreatic enzyme supplements taken by many persons with CF to treat
exocrine pancreatic dysfunction.
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EXPERIMENTAL METHODS |
Measurement of enzyme activity and inhibitor
susceptibility. Amidolytic activity of human sputum
trypsin (lot no. 97451; Elastin, Owensville, MO) and of porcine
pancreatic trypsin (Sigma, St. Louis, MO) was measured
spectrophotometrically by following change in absorbance at 410 nm
during hydrolysis of 0.18 mM
benzoyl-DL-arginine-p-nitroanilide (BAPNA, Sigma) in 50 mM Tris · Cl (pH 7.8) at
37°C. In inhibition studies, absorbance measurements were initiated
immediately after addition of enzyme [1 µg/ml (40 nM) final
concentration] to assay buffer containing 10 µM soybean trypsin
inhibitor (Sigma), 1 µM human
1-proteinase inhibitor (Sigma),
or 4 µM human secretory leukocyte proteinase inhibitor (gift of J. Kramps).
Electrophoresis, blotting, and
sequencing. Human sputum trypsin was subjected to
12.5% SDS-PAGE to separate the major 25-kDa protein band from minor,
faster migrating bands (see Fig. 1). The electrophoresed material was
transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules,
CA) in buffer containing 10% methanol in 10 mM cyclohexylaminopropane
sulfonic acid, pH 11, using an electroblotting apparatus (Idea
Scientific, Minneapolis, MN). The 25-kDa band and the minor 23-kDa band
were visualized with Coomassie blue dye, excised individually, and
subjected to automated
NH2-terminal sequencing at the
University of California, San Francisco Biomolecular Resource Center
with a 470A gas-phase sequenator (Applied Biosystems, Foster
City, CA) with an on-line 120A PTH analyzer. To compare human sputum
trypsin with porcine pancreatic trypsin, both enzymes were subjected to
reducing 12.5% SDS-PAGE along with protein size standards
[phosphorylase B, bovine serum albumin, ovalbumin, carbonic
anhydrase, soybean trypsin inhibitor, and lysozyme (Bio-Rad)] and
then stained with Coomassie blue. To compare immunoreactivity,
electrophoresed aliquots of the two preparations were transferred to
polyvinylidene difluoride membranes as above and subjected to
immunoblotting using polyclonal rabbit antiserum (gift of C. Craik)
raised against rat pancreatic trypsin. After a 1-h incubation with
trypsin antiserum (1:500 dilution), the membrane was washed
and blocked in 20 mM Tris · HCl (pH 7.0)
containing 150 mM NaCl and 0.1% Tween 20. Bound primary antibody was incubated with alkaline phosphatase-conjugated
goat anti-rabbit IgG and detected by incubating the membrane with nitro blue tetrazolium and 5-bromo-4-chloro-indolyl phosphate
(Sigma).
Zymography. Purified human
progelatinase B (matrix metalloproteinase-9; Chemicon International,
Temecula, CA) in 50 mM Tris · HCl (pH 7.5) buffer
containing 20 mM CaCl2 and 150 mM
NaCl was incubated either alone or in the presence of various
concentrations of sputum trypsin at 37°C for 30 min. Reactions were
terminated with 2.5 mM aprotinin and placed at 4°C. To explore
sputum trypsin-mediated processing of progelatinase B, reaction
products were analyzed by gelatin zymography as described (7). Briefly,
8% SDS-polyacrylamide gels containing 1 mg/ml gelatin were subjected
to electrophoresis under nonreducing conditions and washed two times in
2.5% Triton X-100 for 30 min. After incubation at 37°C for
18 h in 40 mM Tris · HCl (pH 7.5) buffer
containing 10 mM CaCl2 and 200 mM
NaCl, gels were stained with Coomassie blue.
Database searches. Computer-assisted
searches of the Protein Databank and of protein sequences
deduced from cDNAs in Genbank (including dbEST) were carried out using
search tools (1) implemented in the BLAST application accessed via the
National Center for Biotechnology Information.
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RESULTS AND DISCUSSION |
Twenty cycles of automated sequencing of the major 25-kDa band in
the sputum trypsin preparation (Fig. 1)
reveal the following NH2-terminal
sequence:
Ile-Val-Gly-Gly-Tyr-Thr-(Cys)-Ala-Ala-Asn-Ser-Val/Ile-Pro-Tyr-Gln-Val-Ser-Leu-Asn-Ser (Table 1). The presence of Cys at residue 7 is inferred because of a blank cycle. Two residues are identified in
cycle 12, indicating a possible polymorphism at this position. The
sequence is similar to that of the mature catalytic domain of many
serine proteases and is distinct from that of the serous cell
trypsinlike protease recently purified from human sputum by Yasuoka et
al. (12), matching the latter enzyme in only 9 of 20 NH2-terminal residues. Protein
sequence database queries reveal no exact matches of the human sputum
trypsin sequence with any human protein. The most similar human
proteins are trypsins, all of which differ by two or more amino acids.
There are no matches with amino acid sequences deduced from any of the
nine trypsinogen genes, pseudogenes, and relic genes intercalated
within human beta T cell receptor loci (10). The only exact protein
match is with porcine trypsin (6). The match includes the
identification of the known porcine Ile/Val polymorphism at position 12 (6). This polymorphism has been reported exclusively in pancreatic
trypsin preparations of porcine origin. Therefore, with high certainty,
the sequencing results assign a porcine origin to human sputum trypsin.

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Fig. 1.
Electrophoretograms of human sputum trypsin (lane
1) and porcine pancreatic trypsin
(lane 2). Both enzymes were
subjected to 12.5% SDS-PAGE and subsequent staining with Coomassie
blue. Size of marker proteins (in kDa) is shown on
left of the electrophoretogram. Arrows
on right indicate positions of ~25-
and ~23-kDa bands subjected to
NH2-terminal amino acid
sequencing. Note similarity in elution position of the major ~25-kDa
band in both enzyme preparations and also the similarity of the
fainter, lower molecular weight bands, which probably derive from
autolytic partial degradation of the major band.
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Sequencing of the minor, 23-kDa band (Fig. 1) reveals the following
sequence, carried out to 10 cycles:
Ile-Val-Gly-Gly-His-Glu-Ala-Gln-Pro-His, which corresponds uniquely to
the sequence of human neutrophil proteinase-3 (9). Thus a minor
component of human sputum trypsin is a second serine protease that is
elastolytic (not tryptic) in specificity and is expected to be present
in neutrophil-rich purulent CF sputum.
As seen in Figs. 1 and 2, the
electrophoretic profile and immunoreactivity of human sputum trypsin
and authentic porcine pancreatic trypsin are highly similar, further
supporting the conclusions derived from
NH2-terminal sequencing. The
25-kDa band in the two preparations is indistinguishable in mobility
and in intensity of binding to antibodies recognizing pancreatic
trypsin. The ~13- and 12-kDa bands seen in both samples in Fig. 1
probably derive from autolysis. The minor ~23-kDa band attributed to
proteinase-3 by NH2-terminal
sequencing is seen only in the sputum-derived material. The specific activity is
similar for sputum trypsin and porcine pancreatic trypsin (1.45 and
2.84 µmol of BAPNA
cleaved · min
1 · mg
enzyme
1, respectively, for
the 2 enzymes). The lower specific activity of the sputum trypsin
preparation may be attributed to a higher proportion of inactive
degradation products or contaminants, as suggested by the SDS-PAGE
profiles of the two preparations.

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Fig. 2.
Immunoblot of sputum trypsin (lane
1) and porcine trypsin (lane
2). After SDS-PAGE, samples (1 µg of protein in
each lane) were transferred to polyvinylidene difluoride membrane and
subjected to immunoblotting using polyclonal antibodies raised against
rat pancreatic trypsin. Arrow indicates elution position of ~25-kDa
band, which exhibits a similar pattern and intensity of immunostaining
in the 2 preparations.
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In further support of the identity of human sputum and porcine
pancreatic enzymes, both preparations are completely inhibited by
soybean trypsin inhibitor, by
1-proteinase inhibitor, and by
secretory leukocyte proteinase inhibitor. The complete inactivation by
soybean trypsin inhibitor distinguishes the enzymes from human trypsins
1 and 2, which are only partially inhibited (5). The finding that
sputum trypsin and porcine pancreatic trypsin are inactivated by
1-proteinase inhibitor has two
implications worthy of note. The first is that it is unlikely that
active porcine trypsin in sputum originates from enzyme absorbed from
the intestine into the bloodstream because any such enzyme will be
quickly inactivated by circulating
1-proteinase inhibitor (and
other serine protease inhibitors). The second implication is that there
must be a shortage of
1-proteinase inhibitor (as well
as secretory leukocyte proteinase inhibitor) in purulent CF sputum;
otherwise, no active trypsinlike activity would have been recovered.
This is consistent with the documented, profound imbalance between
proteases and antiproteases in purulent CF sputum (4). The actual route
by which the porcine trypsin reaches sputum is unclear. The
trypsin could stem from tracheally aspirated gastric contents,
including ingested enzyme supplements, or, more likely, from salivary
contamination of sputum with pharmaceutical trypsin, which is taken
orally in either pill or powdered form.
The results of zymography (Fig. 3) suggest
that sputum/porcine trypsin cleaves and activates human progelatinase B
from the 92-kDa proenzyme form to the active 84-kDa form. Although the 92-kDa proenzyme form of gelatinase B is activated by SDS upon SDS-PAGE
and therefore appears active on zymography, it is inactive in solution.
These findings are similar to those obtained with bovine pancreatic
trypsin, which, among serine proteinases, is the most potent known
activator of progelatinase B (8). Because levels of the 84-kDa
activated form are reported to be greatly elevated in CF sputum (3) but
not in asthmatic sputum, it is possible that porcine trypsin in the
sputum of these patients plays a role in activating gelatinase B.

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Fig. 3.
Activation of progelatinase B by sputum trypsin. Results of SDS-PAGE
gelatin zymography are shown. Enzyme activity is revealed as light
bands against the dark background. Before electrophoresis, the
materials in lanes 1-3 result
from incubation of 0.7 nM human progelatinase B (matrix
metalloproteinase-9) with 0, 5.3, and 53 nM sputum trypsin,
respectively. Migration positions of marker proteins (in kDa) are shown
on left. Electrophoretogram reveals a
concentration-dependent cleavage by trypsin of 92-kDa progelatinase B
(solid arrow) to an active, ~84-kDa form (dashed arrow).
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ACKNOWLEDGEMENTS |
This work supported by National Heart, Lung, and Blood
Institute Grants HL-24136 and HL-03345.
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FOOTNOTES |
Address for reprint requests: G. H. Caughey, Cardiovascular Research
Institute, UCSF, San Francisco, CA 94143-0911.
Received 14 October 1997; accepted in final form 1 April 1998.
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