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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

    ABSTRACT
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Abstract
Introduction
Methods
Results & Discussion
References

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 alpha 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; alpha 1-proteinase inhibitor; gelatinase B; secretory leukocyte proteinase inhibitor

    INTRODUCTION
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Abstract
Introduction
Methods
Results & Discussion
References

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.

    EXPERIMENTAL METHODS
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Abstract
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Methods
Results & Discussion
References

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 alpha 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.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Methods
Results & Discussion
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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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Table 1.   Comparison of trypsin amino acid termini

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.

In further support of the identity of human sputum and porcine pancreatic enzymes, both preparations are completely inhibited by soybean trypsin inhibitor, by alpha 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 alpha 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 alpha 1-proteinase inhibitor (and other serine protease inhibitors). The second implication is that there must be a shortage of alpha 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).

    ACKNOWLEDGEMENTS

This work supported by National Heart, Lung, and Blood Institute Grants HL-24136 and HL-03345.

    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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results & Discussion
References

1.   Altschul, S. F., W. Gish, W. Miller, E. M. Myers, and D. J. Lipman. Basic local alignment search tool. J. Mol. Biol. 215: 403-410, 1990[Medline].

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3.   Delacourt, C., M. L. Bourgeois, M.-P. D'Ortho, C. Doit, P. Scheinmann, J. Navarro, A. Harf, D. J. Hartmann, and C. Lafuma. Imbalance between 95 kDa type collagenase and tissue inhibitor of metalloproteinases in sputum of patients with cystic fibrosis. Am. J. Respir. Crit. Care Med. 152: 765-774, 1995[Abstract].

4.   Goldstein, W., and G. Doring. Lysosomal enzymes from polymorphonuclear leukocytes and proteinase inhibitors in patients with cystic fibrosis. Am. Rev. Respir. Dis. 134: 49-56, 1986[Medline].

5.   Guy-Crotte, O. M., and C. G. Figarella. Trypsin(ogen) 1 and trypsin(ogen) 2. In: Human Protein Data, edited by A. Haeberli. New York: VCH, 1992.

6.   Hermodson, M. A., L. H. Ericsson, H. Neurath, and K. A. Walsh. Determination of the amino acid sequence of porcine trypsin by sequenator analysis. Biochemistry 12: 3146-3153, 1973[Medline].

7.   Hibbs, M. S., K. A. Hasty, J. S. Seyer, A. H. Kang, and C. L. Mainardi. Biochemical and immunological characterization of the secreted forms of human neutrophil gelatinase. J. Biol. Chem. 260: 2493-2500, 1985[Abstract].

8.   Morodomi, T., Y. Ogata, Y. Sasaguri, M. Morimatsu, and H. Nagase. Purification and characterization of matrix metalloproteinase 9 from U937 and HT1080 fibrosarcoma cells. Biochem. J. 285: 603-611, 1992[Medline].

9.   Rao, N. V., N. G. Wehner, B. C. Marshall, W. R. Gray, B. H. Gray, and J. R. Hoidal. Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties. J. Biol. Chem. 266: 9540-9548, 1991[Abstract/Free Full Text].

10.   Rowen, L., B. F. Koop, and L. Hood. The complete 685-kilobase DNA sequence of the human beta T cell receptor locus. Science 272: 1755-1762, 1996[Abstract].

11.   Viscarello, B. R., R. L. Stein, E. J. Kusner, D. Holsclaw, and R. D. Krell. Purification of human leukocyte elastase and cathepsin G by chromatography on immobilized elastin. Prep. Biochem. 13: 57-67, 1983[Medline].

12.   Yasuoka, S., T. Ohnishi, S. Kawano, S. Tsuchihashi, M. Ogawara, K. Masuda, K. Yamaoka, M. Takahashi, and T. Sano. Purification, characterization and localization of a novel trypsin-like protease found in the human airway. Am. J. Respir. Cell Mol. Biol. 16: 300-308, 1997[Abstract].


Am J Physiol Lung Cell Mol Physiol 275(1):L200-L202
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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