Introduction

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Species of Ambrosia are the major cause of pollinosis in North America, with Ambrosia artemisiifolia (common/ short ragweed) accounting for more cases of allergic rhinitis and its related diseases than all other plants combined. Because of the increased spreading of this plant, as well as the increased duration of pollination, there has been considerably more human exposure and the reported cases of ragweed hay fever have risen dramatically (1). Pollen grains contain a variety of proteins required for the fertilization of plants, and exposure to moisture immediately initiates their release. Thus, in allergic reactions, mucous liquid in the respiratory tract will cause swelling of the pollen, resulting in a rapid discharge of its contents, including many proteins that may be able to penetrate mucosal surfaces.

Ragweed pollen extracts are known to contain numerous proteins, some of which are antigenic and may have the potential of being allergenic in certain sensitized individuals (7, 8). In cases of allergic rhinitis, this results in the characteristic reactions of intense sneezing, watery eyes, nasal obstruction, itchy eyes and nose, coughing, and wheezing, all within minutes after exposure to the offending pollen (9). Such responses are examples of Type I immediate hypersensitivity, a reaction that is dependent on the activation of IgE-sensitized mast cells that release pharmacologic mediators (e.g., histamine) and cause inflammation (10, 11). This situation is further complicated by the imbalance, possibly through proteolytic inactivation, of regulatory neuropeptides, and in some individuals this may lead to an asthmatic response (12).

There are 52 antigens presently identified in aqueous extracts of ragweed pollen, at least 22 of which are allergens as defined by reactivity with human IgE and induction of a Type I hypersensitivity response (15). The primary focus of previous research has been on the major protein allergens responsible for IgE production, and it has been clearly demonstrated that each ultimately causes mast-cell degranulation (16, 17). Other studies have also identified and characterized numerous additional ragweed proteins, some of which have been found to possess enzymatic activities. Indeed, to date, at least 20 enzymes have been identified in ragweed pollen extracts (18, 19). However, it has only recently been shown that proteolytic activity exists in these plant gametes (20).

Our own investigations have focused on characterizing proteolytic enzymes produced and isolated from pollen extracts, the goal being to determine if any might play a role in exacerbating the conditions and complications associated with allergic disease. Previously, we identified, purified, and characterized a chymotrypsin-like serine proteinase with a specificity for cleavage after phenylalanine residues (20). This novel peptidase was capable of efficiently hydrolyzing neuropeptides (vasoactive intestinal peptide and substance P) required in the maintenance and recovery of bronchomotor tone, although it could not degrade protein substrates. In this report, we characterize an additional endopeptidase from ragweed pollen with specificity for cleavage after arginyl residues and that may also contribute to the symptoms associated with allergies and/ or asthmatic reactions.

	Materials and Methods

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Common/short ragweed (A. artemisiifolia) was obtained from Miles Allergy Products (Spokane, WA) and polyvinyl difluoride membranes from BioRad (Hercules, CA). Diisopropyl fluorophosphate (DFP), leupeptin, and 3,4-dichloroisocoumarin (3,4-DCIC), were purchased from Calbiochem (La Jolla, CA). Angiotensin 2 (ATII), angiotensin 1, antipain, aprotinin, atrial natriuretic peptide (ANP), idoacetamide, lima bean trypsin inhibitor, soybean trypsin inhibitor, N-Bz-DL-Arg-pNA (BAPNA), N-Suc-Ala-Ala-Pro-Phe-pNA, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), substance P, and vasoactive intestinal peptide (VIP) were obtained from Sigma Chemical Co. (St. Louis, MO). E-64 (trans-epoxysuccinyl-L-leucylamido[4-guanidino] butane), ethylenediaminetetraacetic acid (EDTA), and pepstatin were purchased from Boehringer (Indianapolis, IL), ovomucoid from Worthington Biochemicals (Freehold, NJ), and [1,3-³H]DFP from DuPont NEN (Wilmington, DE). All substrates and inhibitors not listed were a kind gift provided by Dr. James Powers (Georgia Institute of Technology, Atlanta, GA).

Enzyme Extraction and Enzyme Purification

Pollen extracts were prepared as previously described (20), and the ammonium sulfate fraction dialyzed at 4°C against 20 mM Bis-Tris-HCl, 5 mM CaCl₂ (pH 6.5) (buffer A), with three buffer changes over a 24-h period. The dialyzed solution was applied to a Cibacron Blue Sepharose CL-6B affinity column (2.5 × 30 cm, 147 ml), previously equilibrated with buffer A. After application, the column was washed with buffer A and fractions were collected until the A_{280 nm} baseline fell below 0.025. All fractions of the flowthrough were assayed for activities that either cleaved the synthetic peptide substrate Bz-Arg-pNA (trypsin-like) or N-Suc-Ala-Ala-Pro-Phe-pNA (chymotrypsin-like). In this step, it was found that all activity toward Bz-Arg-pNA passed unretarded through the CBS CL-6B column, while activity responsible for the cleavage of Suc-Ala-Ala-Pro-Phe-pNA bound tightly.

The active pooled, flowthrough fractions were applied to a DEAE-Sephacel ion-exchange column (2.5 × 50 cm, 246 ml) equilibrated with buffer A, and the column was washed with the same buffer until the A_{280 nm} baseline fell below 0.05. A linear gradient from 0 to 500 mM NaCl in buffer A was then applied in a total volume of 1,500 ml. Fractions were collected and those containing activity were pooled and dialyzed at 4°C against 30% ammonium sulfate in buffer A with three changes every 2 h. The dialyzed fraction was applied to a phenyl-Sepharose CL-4B hydrophobic interaction column (1.5 × 30 cm, 53 ml), previously equilibrated with 30% ammonium sulfate in buffer A and washed in the equilibration buffer until the A_{280 nm} baseline fell to zero. A gradient of 30% to 0% ammonium sulfate in buffer A was then applied in a total volume of 300 ml, the collected fractions were assayed against Bz-Arg-pNA, and those fractions possessing activity were pooled and concentrated by ultrafiltration, using an Amicon PM-10 membrane. This material was applied to a Sephacryl S-200 Superfine column (1.6 × 100 cm, 197 ml), equilibrated with 150 mM NaCl in buffer A, and the active fractions obtained after gel filtration again were pooled and dialyzed at 4°C against 50 mM Tris, 5 mM CaCl₂ (pH 7.4) (buffer B). The dialyzed sample was applied to a Mono Q HR 5/5 FPLC column (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) and equilibrated with buffer B, and the column washed at 1 ml/min until the baseline stabilized near zero. Using a flow rate of 1 ml/min, the bound, active enzyme was eluted in 15 min using a linear 0-125 mM NaCl gradient. Final purification to remove any minor contaminants was achieved by passing the active fractions through a TSK-GEL G300SW gel filtration column (TosoHaas Corp., Montgomeryville, PA) equilibrated with 200 mM NaCl in buffer A.

Enzyme Assays

Amidolytic activity was measured at 405 nm by following the hydrolysis of the substrate Bz-Arg-pNA (1 mM), in 0.2 M Tris-HCl, 5 mM CaCl₂ (pH 9.0). General proteolytic activity was measured using 2.0% (wt/vol) azocasein, as described by Barrett and Kirschke (21).

Electrophoresis and Autoradiography

All purification steps were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as described by Schagger and von Jagow (22), using a Tris-HCl/Tricine buffer system. [1,3-³H]DFP was used to radiolabel active enzyme, as previously described (20), in order to confirm molecular mass and purity of the desired protein.

Amino Acid Analysis

Analysis of peptide fragments from enzyme-treated polypeptide hormones was performed as described earlier (20).

Enzyme Specificity and Kinetics

For specificity studies, substrates were incubated at an enzyme:substrate molar ratio of 1:1,000 in 50 mM Tris-HCl, 5 mM CaCl₂ (pH 9.0) at 25°C for 30 min and the digestions stopped by acidification with 5% trifluoroacetic acid. Bioactive peptide degradation was measured using an enzyme:substrate ratio of 1:5,000, in 50 mM Tris-HCl, 5 mM CaCl₂ (pH 9.0) at 25°C. Aliquots were removed at various time periods and digestion was stopped by acidification with 5% trifluoroacetic acid. Peptide fragments were separated using high performance liquid chromatography, as previously described (20), and cleavage was determined through amino acid analysis. K_m and V_max values were measured using substrates at concentrations ranging from 10 to 50 µM with a final concentration of enzyme of 10 nM in 50 mM Tris-HCl (pH 9.0) at 25°C and calculated using Hyperbolic Regression Analysis (written by J. S. Easterby at the University of Liverpool, Liverpool, UK; obtained through shareware).

	Results

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Enzyme Purification

Simulating physiologic conditions (10 mM Tris [pH 7.3], 150 mM NaCl), it was found that extraction of pollen at room temperature yielded large amounts of both Bz-Arg-pNA and N-Suc-Ala-Ala-Pro-Phe-pNA activities in less than 1 min (data not shown). However, enzyme stability allowed for a 24-h extraction at 4°C in 10 mM ammonium bicarbonate (pH 8.0) in order to obtain its highest recovery. After centrifugation, no activities for either enzyme were found associated with the resuspended pollen precipitates. Gel filtration chromatography of the supernatant from the extract gave a highly reproducible profile showing two distinct (Bz-Arg-pNA and N-Suc-Ala-Ala-Pro-Phe-pNA) peaks of activities (data not shown). The use of a Cibacron Blue Sepharose CL-6B column chromatography step was a very effective way of isolating the two activities and resulted in their complete separation. Further fractionation resulted in the purification of a homogenous enzyme preparation with a molecular mass near 80 kD, which corresponded with the increasingly prominent protein band that was observed using Tricine SDS-PAGE (Figure 1). This result is in agreement with the estimated mass obtained during gel filtration of the crude extract. The isolation procedure developed here resulted in the purification of 409 µg of enzyme from 100 g dry weight pollen, with a 7.0% yield and a 2,600-fold purification (Table 1).

View larger version (84K): [in this window] [in a new window]   Figure 1.   SDS-PAGE of fractions from the purification of serine trypsin-like endopeptidase from ragweed (A. artemisiifolia) pollen. Lane 1: molecular mass markers (rabbit muscle phosphorylase b, 94 kD; bovine serum albumin, 67 kD; ovalbumin, 43 kD; bovine erythrocyte carbonic anhydrase, 30 kD; soybean trypsin inhibitor, 20.1 kD; bovine milk -lactalbumin, 14.4 kD). The following lanes contained boiled and reduced samples. Lane 2: extract of ragweed (A. artemisiifolia) pollen; lane 3: ammonium sulfate precipitate from extract; lane 4: Cibacron Blue Sepharose CL-6B affinity column; lane 5: DEAE-Sephacel ion-exchange eluate; lane 6: phenyl-Sepharose CL-4B hydrophobic interaction column eluate; lane 7: Sephacryl S-200 Superfine column eluate; lane 8: Mono Q HR 5/5 FPLC column eluate; lane 9: purified ragweed pollen serine endopeptidase from TSK-GEL G300SW gel filtration column.

SDS-PAGE Analysis

As noted above, after reduction and boiling in SDS, the enzyme migrated as a single band at 80 kD (Figure 1) in agreement with that obtained by gel filtration. Confirmation that this was indeed the desired proteinase was made by labeling studies with [1,3-³H]DFP before SDS treatment (Figure 2), using procedures as previously described (20). This resulted in the production of a radioactive band with apparent molecular mass of 80 kD.

View larger version (38K): [in this window] [in a new window]   Figure 2.   SDS-PAGE and autoradiography of purified serine endopeptidase from ragweed (A. artemisiifolia) pollen. Lane 1: molecular mass markers (rabbit muscle phosphorylase b, 94 kD; bovine serum albumin, 67 kD; ovalbumin, 43 kD; bovine erythrocyte carbonic anhydrase, 30 kD; soybean trypsin inhibitor, 20.1 kD; bovine milk -lactalbumin, 14.4 kD). Lane 2: purified enzyme radiolabeled with [1,3-3H]DFP (17 µCi/µg of protein) incubated for 30 min at room temperature, followed by 30 min incubation with 10 mM cold DFP. The sample was boiled under reducing conditions before electrophoresis was performed. Lane 3: autoradiograph of 3H-labeled enzyme exposed for 96 h to X-ray film.

NH₂-terminal Sequence Analysis

No amino terminal sequence could be detected utilizing either native or [1,3-³H]DFP-labeled enzyme, suggesting that it was blocked.

Stability

The enzyme was found to be stable over the pH range 4.5 to 10.0 for 24 h at room temperature with less than a 5% loss in activity during this time. It required Ca²⁺ for stability and lost < 2% of its activity after long-term storage (3 to 4 mo) at 80°C, either lyophilized or frozen in solution.

Inhibition Profile

Incubation of the enzyme with DFP or TLCK resulted in total loss of activity, supporting its classification as a trypsin-like serine peptidase. In addition, DCIC was also inhibitory; however, incubation with representatives of all other class-specific low molecular mass inhibitors resulted in no loss of enzyme activity (Table 2). Known protein inhibitors, including avian ovomucoids, Kunitz-type trypsin inhibitors and several human plasma serpins were also unable to inhibit the enzyme. In contrast, a variety of highly specific synthetic inhibitors of serine peptidases, including coumarins, peptidyl chloromethyl ketones, and organophosphates were found to be effective in inactivating inhibitors of the enzyme (Table 3). Significantly, the most active of these were those containing Arg in the P₁ position.

Protein and Peptide Degradation

The trypsin-like pollen endopeptidase did not digest azocasein or any other protein substrate but did cleave small synthetic substrates. K_m and V_max values for the hydrolysis of several of these compounds, performed at the pH optimum of 9.0, are given in Table 4. It should be noted that the amidolytic activity of the peptidase on synthetic peptide substrates agreed with the inhibitor studies described above. Specifically, it exhibited a preference for Arg in the P₁ position and Arg or Gly in the P₂ position. In addition, multiple substrates containing lysine in the P₁ position were not reactive, confirming specificity of the enzyme toward arginal residues. Attempts to digest proteins with a mixture of the trypsin-like and the previously described chymotrypsin-like ragweed endopeptidase (20) resulted in no additional detectable activity, indicating no synergy between the two endopeptidases on protein substrates.

Enzyme Specificity

The ragweed peptidase cleaved various biologically active peptides responsible for maintenance of bronchomotor tone (substance P and VIP) and inflammation (ATII and ANP). This is illustrated in Table 5, where cleavage was found to occur primarily after arginine residues. In contrast to the rapid hydrolysis of substance P and VIP by the chymotrypsin-like enzyme (20), at very low enzyme:substrate ratios (1:5,000), the trypsin-like peptidase hydrolyzed ATII and ANP far more efficiently (Figure 3). Further experiments to determine synergistic effects, where both enzymes were used in a 1:1 molar ratio, resulted in no additional cleavage of any tested protein substrates or serpins. Additionally, this combination did not expedite hydrolysis of any of the biologically active peptides tested nor result in additional fragmentation, supporting the previous results (20) and those presented here, regarding the exquisite specificity of each enzyme. This specificity may contribute to the biologic significance of the enzyme(s) since, with only limited natural substrates and no inhibitors, the unrestricted hydrolysis of specific regulatory neuropeptides could be of considerable consequence.

View larger version (15K): [in this window] [in a new window]   Figure 3.   Decrease in percentage of peak area of various bioactive peptides. Decrease in percentage of peak area of various biologically active peptides was measured using an enzyme:substrate ratio of 1:5,000, in 50 mM Tris-HCl, 5 mM CaCl2 (pH 9.0) at 25°C. Aliquots were removed at various time periods, and digestion was stopped by acidification with 5% trifluoroacetic acid. Peptide fragments were separated using high performance liquid chromatography, and cleavage was determined through amino acid analysis. Closed squares: angiotensin 2; closed triangles: atrial natriuretic peptide; open diamonds: vasoactive intestinal peptide; closed diamonds: substance P.

	Discussion

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In this report, we have described the isolation and properties of a novel endopeptidase from common/short ragweed pollen which is likely to be a member of the serine endopeptidase class, based on its inhibition by DFP, DCIC, and TLCK (Table ). It may be further classified as a trypsin-like endopeptidase because of its preference for Arg in the P₁ position, as demonstrated with peptidyl substrates and inhibitors (Tables and ). In contrast to the previously described chymotrypsin-like endopeptidase (20), this enzyme requires Ca²⁺ and can hydrolyze single and di-peptide synthetic substrates, as well as small peptides. From a physiologic point of view, this may be extremely important since, in vitro, the enzyme is highly active in the degradation of ATII and ANP, both of which are required to offset the activity of bradykinin (BK) and BK-related peptides.

After isolation of both endopeptidases, present in approximately equal concentrations and in milligram protein yields from 100 g of pollen, we were also interested in whether they might act synergistically to expedite the degradation of the same neuropeptide(s) or to facilitate the activation and/or degradation of proteins involved in the inflammatory response. However, animal models used to determine the production of factors that produced increased vascular permeability did not show this effect when the enzymes were injected individually or in combination (data not shown). Similarly, when both enzymes were utilized to degrade various neuropeptides, it was observed that there was only cleavage by one of the peptidases, not both, an indication of the exquisite sensitivity of these substrates to a specific ragweed pollen endopeptidase.

There are two autocrine systems that appear to be responsible for the generation of regulatory proteins and neuropeptides that would affect vasoconstriction and/or vasodilatation. These are the renin-angiotensin system, which results in the production of the potent vasoconstrictor ATII, and the kallikrein-kinin system, which yields BK and/or kallidin (Lys-BK) (23). Normally, these two systems are in balance; however, fragmentation of ATII would result in an equilibrium shift in favor of higher levels of kinins, leading to edema, inflammation, and hypersensitivity within the respiratory tract. In addition, it is also well known that ANP can modulate increased production of ATII (24). Therefore, proteolytic inactivation of this peptide would also cause a decrease in ATII production, again contributing to the increased kinin activity. Though it is difficult for us to speculate on peptidase concentrations in vivo, enzymatic disruption of these two systems is already known to be associated with allergies and the accompanying asthmatic complications (25).

It is well known that excessive enzymatic hydrolysis of regulatory proteins, peptides, and tissue proteins can result in the development of lung disease. This is particularly important in familial emphysema where uncontrolled host-proteinase activity (neutrophil elastase) results in the destruction of alveolar tissue because of the absence of -1-proteinase inhibitor (-1-PI) (26). However, in garden-variety emphysema and allergic bronchopulmonary aspergillosis, there is clear evidence that enzymatic inactivation of -1-PI can also result in excessive tissue damage by neutrophil elastase (27, 28). Previously, we demonstrated that a chymotrypsin-like enzyme isolated from ragweed pollen, which has no apparent regulating inhibitor in human tissues, had the ability to degrade lung neuropeptides required for normal lung function. Clearly, through unrestricted inactivation of ATII and ANP, the trypsin-like enzyme described here has the potential to interfere with normal bronchial function by increasing the kinin/ATII ratio at localized sites in the upper and lower respiratory tracts where enzyme concentrations in the microenvironment may be physiologically significant. The ragweed peptidase, though not as abundant as the cysteine proteinase Der P1, isolated from dust extracts, parallels the complications that are suggested in house dust allergies. It is believed that the dust mite major allergen activates the kallikrein/kinin system and raises kinin levels at affected sites (29).

A relationship between asthma and other disorders associated with allergies of the upper respiratory tract appears inevitable, considering the mucus membranes are continuous from the nose and paranasal sinuses to the alveoli, with secretions being transported in both directions. The obvious inference is that perturbation of either the upper or lower respiratory tract would have a general effect on the whole system (30). Indeed, respiratory allergies initially affect the mucus membrane of the upper respiratory tract, but it is the additional affect of the bronchi and lungs that manifest the clinical symptoms of asthma.

From the results presented here, we believe that it is reasonable to suggest a role for ragweed pollen endopeptidases in the degradation of neuropeptides involved in the maintenance of bronchial function. This would occur in two ways, first through the degradation of ATII and ANP in the upper respiratory tract, which would allow increased kinin activity and the production of edema and swelling of tissues. A second, more profound effect would be in the lower respiratory tract, where one would have unregulated kinin activity and the vasoconstriction and hyperresponsiveness characteristic of asthma.

Finally, it would be remiss not to mention the fact that pollen is a gamete. Therefore, it is likely that most of the ragweed allergens described to date do have some biologic activity, and this almost certainly applies to the two endopeptidases we have now characterized. We hypothesize that such enzymes, as well as others not yet characterized, may have significant importance in allergic rhinitis, asthma, and other respiratory complications associated with pollen exposure, not only through the production of IgE-type antibodies but also through a direct effect on the respiratory tract itself.