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Infection and Immunity, January 2002, p. 419-421, Vol. 70, No. 1
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.1.419-421.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Isolation and Properties of Stachyrase A, a Chymotrypsin-Like Serine Proteinase from Stachybotrys chartarum

Tomasz Kordula,¹ Agnieszka Banbula,² Jeremy Macomson,³ and James Travis^3,2*

Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio 44115,¹ Institute of Molecular Biology, Jagiellonian University, 31-120 Cracow, Poland,² Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia 30602³

Received 27 June 2001/ Returned for modification 7 August 2001/ Accepted 15 October 2001

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A strain of the common mold Stachybotrys chartarum has been isolated from the lung of a child with pulmonary hemorrhage. We report the purification of stachyrase A, a new serine chymotrypsin-like proteinase from S. chartarum. This enzyme cleaves major protease inhibitors, several biologically active peptides, and collagen, all of which are found in the lung.

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Neutrophil degranulation and release of powerful proteolytic enzymes, including elastase and cathepsin G, comprise one of the major mechanisms of defense against air-carried pathogens in the lung (14). Since such enzymes can also be destructive for the lung itself (6), specific proteinase inhibitors, including ₁-proteinase inhibitor (₁-PI), are continuously present in fluids bathing the lung (17). However, invading pathogens releasing their own proteinases can cleave these inhibitors.

Stachybotrys chartarum is a common fungal organism that normally grows on decomposing organic material but has recently been found to colonize the lung (3). In the 1930s it was held responsible for the outbreak of a new disease in animals (4, 16). The first reports of stachybotryotoxicosis in humans appeared in 1940 in Russia where individuals afflicted with infection with S. chartarum have developed dermatitis, pain and inflammation of mucous membranes, cough, fever, headache, and fatigue (5). However, this organism attracted public attention as a result of acute idiopathic pulmonary hemorrhage-hemosiderosis and death occurring in infants from Cleveland, Ohio (2). The correlation between S. chartarum and acute idiopathic pulmonary hemorrhage-hemosiderosis has recently been questioned (1, 8), but simultaneously experimental lung mycotoxicosis was found in mice exposed to spores of S. chartarum (13). S. chartarum produces several mycotoxins, immunosuppressive agents, and hemolysin activity (9, 18). We hypothesized that this fungal pathogen might also produce proteinases that could lead to destruction of lung tissue.

S. chartarum strain JS5106 derived from the lung of an infant with pulmonary hemosiderosis (ATCC 201859) was grown in Luria-Bertani medium (15) containing 0.2% glucose and 0.2% elastin with moderate shaking for approximately 3 to 4 weeks. Cultures were assayed for amidolytic activity against 1 mM N-Suc-Ala-Ala-Pro-Phe-pNA in 50 mM Tris (pH 7.5) at 37°C. This analysis indicated that the medium contained proteinase, whereas the fungus itself contained limited amounts of the enzyme. To purify the enzyme 40% ammonium sulfate precipitation was applied, the pellet was redissolved in buffer A (50 mM Tris [pH 7.5] and 10 mM CaCl₂) and dialyzed, and ammonium sulfate was added to 4%. To remove dark pigment the sample was applied to a DE-52 column, the flowthrough was collected, ammonium sulfate was added to 15%, and the sample was applied to a phenyl-Sepharose column. The activity was eluted in a linear gradient from 10% to 0% ammonium sulfate in buffer A. Two separate peaks of activity were found that contained stachyrase A and another enzyme with different properties (unpublished data). Stachyrase A was further purified on a Mono-Q column (Amersham Pharmacia Biotech) equilibrated with buffer A, with elution in a linear gradient of 0 to 500 mM NaCl.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis (11) of the purified enzyme revealed a protein band with a molecular mass of 32 kDa (Fig. 1). The enzyme possessed a gelatinase activity and was easily radiolabeled with [1,3-³H]diisopropylfluorophosphate (7, 12), indicating that it belongs to the serine proteinase family (Fig. 1). Several attempts of amino-terminal sequence analysis yielded an N-terminal sequence of ATQTGA (Microchemical Facility, Emory University School of Medicine, Atlanta, Ga., and BioCentrum, Jagiellonian University, Cracow, Poland); however, a longer sequence could not be obtained. The activity of stachyrase A was detected over a broad pH range of 6.0 to 11.5 and the enzyme was stable for several weeks at 4°C.

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FIG. 1. SDS-PAGE of fractions from purification of stachyrase A and zymography of purified enzyme. Lane a, culture medium; lane b, 40% ammonium sulfate precipitate; lane c, DE-52 flowthrough; lane d, phenyl-Sepharose column eluate; lane e, purified stachyrase A eluted from MonoQ; lane f, gelatin zymography of purified stachyrase A; lane g, fluorography of purified stachyrase A labeled with [1,3-3H]diisopropylfluorophosphate. Samples were reduced and boiled (lanes a to e and g) or only boiled (lane f) before PAGE analysis.

Stachyrase A was inactivated by class-specific inhibitors of serine proteinases (Table 1). Tosyl-phenylalanine chloromethyl ketone was a strong inhibitor of stachyrase A, indicating that it is a chymotrypsin-like enzyme. The inhibition profile obtained with orthophosphonates suggested that the enzyme prefers phenylalanine at the P1 position and proline at the P2 position. Several chromogenic substrates were tested; however, only N-Suc-Ala-Ala-Pro-Phe-pNA was efficiently hydrolyzed (data not shown). Cleavage sites were further mapped using the insulin ß chain as a substrate (Fig. 2). It appears that stachyrase A can cleave after a variety of amino acids, including Phe, Leu, Val, Ala, Tyr, His, and Ser, indicating specificity that is broad but is primarily towards hydrophobic amino acid residues.

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TABLE 1. Inhibition profile of stachyrase A

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FIG. 2. Cleavage of insulin ß chain by stachyrase A. Carboxymethylated insulin ß chain and stachyrase A were incubated for 24 h at 37°C (substrate:enzyme ratio, 100:1). Degradation products were purified by reverse-phase high-pressure liquid chromatography and analyzed by mass spectroscopy (Electrospray) at the Chemical and Biological Sciences Mass Spectrometry Facility, University of Georgia.

Since stachyrase A may be secreted by fungus growing within the lung, the effect of this enzyme on substance P, bradykinin, neurotensin, and angiotensin I and II was analyzed. Peptides were incubated with stachyrase A (100:1) for 12 h in 50 mM Tris (pH 7.5) at 37°C and analyzed as described previously (7). The proteinase degraded all of the peptides, with multiple degradation products detected. In vivo, this may lead to local changes in blood pressure and permeability of blood vessels.

Both collagen and elastin are the major structural proteins of the lung, and the ability to degrade these proteins is most likely beneficial for successful growth within the lung. Therefore, elastin and several types of collagen were incubated with stachyrase A and samples were analyzed by SDS-PAGE. While stachyrase A was ineffective towards elastin it efficiently degraded all tested types of collagen (Fig. 3). In separate studies, we have found elastinolytic activity that is secreted by S. chartarum; however, this activity cannot be attributed to stachyrase A.

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FIG. 3. Stachyrase A possesses collagenase activity. Ten nanograms of stachyrase A was incubated with 10 µg of human placenta collagen type VI (Sigma) (a), bovine nasal septum collagen (Sigma) (b), human placenta collagen type X (Sigma) (c), or type I rat tail collagen (d) in 50 mM Tris (pH 7.6)-150 mM NaCl-1 mM CaCl2 for 12 h at 37°C. Samples were separated by SDS-PAGE.

Subsequently, we analyzed the effect of major plasma inhibitors (ART Inc., Athens, Ga.) on the activity of stachyrase A. Figure 4 demonstrates that both ₁-PI and ₁-antichymotrypsin (₁-ACT) were cleaved by stachyrase A, whereas ₂-macroglobulin (₂-M) was totally degraded. Surprisingly, sequencing of cleavage products indicated that the ₁-ACT cleavage site was located at the N terminus after Val residue 21 whereas ₁-PI was cleaved at the reactive loop. Figure 5 demonstrates the time-dependent degradation of ₂-M by stachyrase A, whereas incubation with trypsin, as expected, yielded one major band of cleaved ₂-M. In equimolar concentrations, neither ₁-ACT, ₁-PI, nor ₂-M had any substantial effect on enzyme activity (data not shown). At very high concentrations of inhibitors inhibition could be detected, suggesting that they act as competitive substrates. In comparison, Aspergillus-derived seaprose was found to degrade ₁-PI, form complexes, and inactivate ₁-ACT but was inhibited by ₂-M (10). In addition, we tested the effect of stachyrase A on secretory leukocyte protease inhibitor normally present in the lung (17). This protein was also cleaved by the fungus-derived enzyme (data not shown).

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FIG. 4. Effect of stachyrase A on plasma inhibitors. One microgram of 1-PI, 1-ACT, or 2-M was incubated with or without stachyrase A (200:1) for 24 h at 37°C. Samples were separated by SDS-PAGE (left panel) or by native PAGE (right panel).

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FIG. 5. Time-dependent degradation of 2-M by stachyrase A. One microgram of 2-M was incubated with or without stachyrase A or trypsin (200:1) for 1 or 6 h (as indicated) at 37°C. Samples were separated by SDS-PAGE.

Based on the presented results we postulate that the role of stachyrase A should now be considered together with mycotoxins and hemolysin when pathological effects of colonization by S. chartarum are discussed.

 
This work was supported by research grants from the National Institutes of Health (HL26148 and HL37090) (to J.T.).

We thank Hideaki Nagase for a gift of rat tail collagen and Hans Fritz for a gift of human SLPI.

 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, GA 30602. Phone: (706) 542-1713. Fax: (706) 542-1738. E-mail: JTravis{at}arches.uga.edu.

Editor: D. L. Burns

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Infection and Immunity, January 2002, p. 419-421, Vol. 70, No. 1
0019-9567/01/$04.00+0     DOI: 10.1128/IAI.70.1.419-421.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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