©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of Guamerin, a New Human Leukocyte Elastase Inhibitor from Hirudo nipponia(*)

Hyo Il Jung (1), Seung Il Kim (2), Kwon-Soo Ha (2), Cheol O Joe (1), Ke Won Kang (1)(§)

From the (1) Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea and the (2) Biomolecule Analysis Group, Korea Basic Science Center, Taejon 305-701, Korea

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A new human leukocyte elastase inhibitor was extracted and purified from a Korean native leech Hirudo nipponia. The inhibitor, called guamerin, has a molecular weight of 6,110 and shows inhibition constant (K ) of 8.1 10M. It is stable at a wide range of pH from 1 to 11 and heat-stable up to 90 °C. The complete amino acid sequence of guamerin reveals a cysteine-rich polypeptide of 57 amino acid residues that shows no similarity to any known elastase inhibitors but has 51% sequence homology with hirustasin. Guamerin has identical spacing of 10 cysteine residues as antistasin-type serine proteinase inhibitors, but the P1 reactive site residue is Met instead of Arg. The neighboring sequence of the reactive site consists primarily of hydrophobic amino acid residues. Based on examinations of the target proteinases and the reactive site specificity, guamerin is a new low molecular weight protein that inhibits elastases.


INTRODUCTION

Human leukocyte elastase is a serine proteinase capable of degrading important connective tissue proteins such as elastic fiber (1) and collagen (2) . It is stored in the cytoplasmic azurophil granules and released into tissues when the cell encounters necrotic tissues or microorganisms to be phagocytized (3) . After uncontrolled liberation at sites of inflammation, human leukocyte elastase may cause nonspecific proteolysis and trigger destructive processes associated with various chronic diseases including psoriasis (4) and emphysema (5). The functional activity of human leukocyte elastase is, in most cases, controlled by the presence of the inhibitors (6) . There has been growing interest in isolation and characterization of properties of leukocyte elastase inhibitors because the inhibitors may be used as therapeutic agents to prevent chronic inflammatory diseases (7) .

Elastase inhibitors have been purified from human skin (8, 9) , parotid secretions (10) , and bronchial mucus (11) , as well as from other sources such as soybean (12) , turkey ovomucoid (13) , and leech (14) . Eglin C is, in particular, a potent elastase inhibitor isolated from Hirudo medicinalis and characterized as a cysteine-free low molecular weight protein (14) . We hypothesized that Hirudo nipponia might also contain inhibitors that may have therapeutic application to control processes associated with elastases.

In this paper, we describe a new human leukocyte and porcine pancreatic elastase-specific inhibitor, named guamerin, purified from a Korean native leech H. nipponia. Its complete amino acid sequence was determined, and the specificities against other proteases were examined. We have found identical spacing of cysteine residues as antistasin type serine-proteinase inhibitors, but the reactive site P1 residue is a methionine instead of arginine that determined specificity of the inhibitor. To our knowledge, guamerin is the first cysteine-rich elastase inhibitor isolated from leeches.


EXPERIMENTAL PROCEDURES

Materials

Adult leeches were collected from a lake at Chonju, cultured in the laboratory for 6 months (15) , and starved for 2 weeks prior to being utilized. Human leukocyte elastase, porcine pancreatic elastase, bovine pancreatic trypsin, -chymotrypsin, subtilisin, pepsin, papain, bovine plasma factor Xa, bovine plasma thrombin, azocasein, 4-vinylpyridine, and all chromogenic substrates were purchased from Sigma. Endoproteinase Lys-C and trypsin (sequencing grade) were provided by Boehringer Mannheim. Sephadex G-75 and DEAE-Sepharose were purchased from Phamarcia Biotech Inc. Sep-Pak C cartridge and Delta-Pak C reversed phase HPLC() columns were obtained from Waters division of Millipore.

Purification of Guamerin

Starved leeches (40 g) were minced and ground on ice, and 200 ml of 80% (v/v) of cold aqueous acetone was added with stirring at 4 °C for 10 min. Then, sodium chloride and trichloroacetic acid were added to final concentrations of 0.3 and 0.4 M, respectively, with constant stirring for 60 min. The insoluble residue was removed by centrifugation at 6,000 rpm for 10 min. The resulting supernatant was mixed with 4 volumes of 100% ice-cold acetone to precipitate elastase inhibitory peptides and centrifuged at 12,000 rpm for 20 min. The pellet was suspended in 50 mM Tris-Cl, and about 5% of the pellet was solubilized in the buffer. No elastase inhibitory activity was found in the insoluble part, which was removed by centrifugation at 10,000 rpm for 20 min. The solution was applied to a Sephadex G-75 column (110 2 cm) equilibrated with 50 mM Tris-Cl buffer, pH 8.0 containing 200 mM NaCl and eluted with the same buffer at a flow rate of 0.5 ml/min. The inhibitor fractions were pooled, desalted, and then applied to a DEAE-Sepharose column (7 1.6 cm) equilibrated with the same buffer and eluted with a 60-ml linear gradient of NaCl from 0 to 400 mM. The inhibitor fractions were pooled, concentrated, and desalted with a Sep-Pak C cartridge. The desalted fraction was applied to a Delta-Pak C reversed phase HPLC column (3.9 300 mm) equilibrated with buffer A and eluted with a linear gradient composed of buffer B. Buffer A is 0.1% trifluoroacetic acid, and buffer B is 80% acetonitrile containing 0.1% trifluoroacetic acid. Four inhibitory peptides were separated at a range of 25-60% of buffer B. The inhibitor fractions were separately collected, dried, and stored at -20 °C until use.

Assay of Inhibitory Activity

The anti-elastase activity of the purified peptide was estimated from the residual activity of elastase in the mixture of peptide and the protease. The standard assay mixture contained 40 µl of 0.2 M Tris-Cl, pH 8.0, containing 20 µl of 0.37 mg/ml human leukocyte elastase and either 40 µl of guamerin (1.75 µM) or 40 µl of buffer. The reaction was initiated with the addition of 400 µl of succinyl-Ala-Ala-Ala-p-nitroanilide (from 0.2 to 1.6 mM) in the buffer. After incubation at 37 °C for 1 min the reaction was stopped by placing on ice. The appearance of p-nitroaniline was spectrophotometrically monitored at 410 nm (16) . The percentage of inhibition (%I) was calculated as %I = [(1 - A /A )] 100, where A and A are the absorbance with and without guamerin, respectively. The inhibition constant (K ) of guamerin for the hydrolysis of Suc-Ala-Ala-Ala-pNA was determined according to the method of Dixon (17).

Assay for inhibitory activity against thrombin and factor Xa was done essentially as described above. Tos-Gly-L-Pro-L-Arg-pNA and Bz-L-Val-Gly-L-Arg-pNA were used as substrates for thrombin and factor Xa, respectively. The buffers used were 10 mM HEPES, pH 8.0, for thrombin and 0.2 M Tris-Cl, pH 8.0, containing 2.5 mM CaCl to activate factor Xa.

The inhibitory activity of guamerin on the digestion of azocasein by the other proteases was measured using an assay mixture that included 150 µl of 50 mM Tris-Cl, pH 8.0, 50 µl of enzymes (from 0.05 to 0.2 mg/ml), 40 µl of guamerin (1.75 µM) or buffer (serving as control). In the case of papain and pepsin, 0.2 M Tris-Cl, pH 8.0, containing 1 mM cysteine, 20 mM EDTA, and 10 mM sodium acetate, pH 5.4, were used as reaction buffers, respectively. Upon the addition of 0.5 ml of 0.1% (w/v) azocasein in the same buffer, the tubes were incubated at 37 °C for 50 min. The reaction was terminated by adding 0.5 ml of 15% trichloroacetic acid, and the tubes were allowed to stand on ice for 15 min. The precipitate was removed by centrifugation, and the absorbance at 440 nm was measured (18) . The percentage of inhibition was calculated using the above equation.

Reduction and S-Pyridylethylation

Guamerin (80 µg) dissolved in 200 µl of 0.1 M Tris-Cl pH 8.0 containing 6 M guanidine hydrochloride was mixed with 10 µl of 2-mercaptoethanol. The mixture was flushed with nitrogen gas and then incubated at 50 °C for 7 h. The free sulfhydryl groups were exposed to 15 µl of 4-vinylpyridine, and the solution was stirred at room temperature in the dark for 2 h (19) . The reduced and S-pyridylated guamerin was then repurified on a Dalta-Pak C column (3.9 300 mm) by 0-50% gradient of acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min and used for subsequent analyses.

Enzymatic Cleavage of S-Pyridylethylated Guamerin

S-pyridylethylated guamerin (40 µg, 13 nmol) was digested with trypsin (2 µg) in 100 µl of 0.1 M Tris-Cl buffer, pH 8.0, at 37 °C for 20 h. In a separate reaction, this peptide (40 µg) was also digested with endoproteinase Lys-C (1.5 µg) in the same buffer at 37 °C for 24 h. The reaction was stopped by freezing on dry ice.

Isolation of the Peptides after Digestion

Peptide fragments resulting from enzymatic digestion were purified by a Waters HPLC system (510 HPLC pump, 717 plus automatic sampler, 996 photodiode array detector, and Millennium 2010 chromatography manager) equipped with a Delta-Pak C reversed phase column (3.9 300 mm) equilibrated with buffer A. Peptides were eluted from the column with a linear gradient of buffer B (0-50%, 0.42%/min) at a flow rate of 0.5 ml/min at 25 °C. Absorbance at 214 nm was measured, and peptides were manually collected for sequence analysis. Peptides were numbered according to the order of elution from the HPLC column. Peptides derived from trypsin and endoproteinase Lys-C were designated as T and L, respectively.

The amino acid and sequence analyses of native guamerin were done by the Pico-Tag method (Waters) after hydrolysis in constant boiling HCl (Sigma) at 110 °C for 24 h. In one sample, cysteine residues were oxidized to cysteic acid with a mixture of formic acid and hydrogen peroxide (19:1, v/v) (20) . To determine the tryptophan content, guamerin was directly digested with 20 µl of 4 N methanesulfonic acid (21) . Amino acid sequences of peptides were analyzed in an Applied Biosystems 476A protein sequencer. Prior to peptide digestion, the N terminus was investigated by automated Edman degradation.

Molecular Weight Determination by Matrix-assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)

One µl of guamerin (6.1 mg/ml) dissolved in aqueous 0.1% trifluoroacetic acid was mixed with 2 µl of sinapinic acid (70 g/liter). The mixture was loaded on a flat inert metal (e.g. silver, platinum) probe. The solvent was removed in a stream of warm air, and the sample was then transferred to the vacuum chamber. Thus the typical absolute sample amount used for analysis was about 10 pmol. Then the molecular weight of guamerin was detected by MALDI-MS (22) . Bovine pancreas insulin (M 5,734) and cytochrome c (M 12,328) were used as reference peptides to calibrate the molecular weight of guamerin exactly.


RESULTS

Purification of Guamerin

Crude extract from leeches was mixed with 50 mM Tris-Cl buffer, pH 8.0, and centrifuged to remove insoluble materials. Approximately 95% of nonactive proteins was removed as insoluble pellet. The supernatant enriched in anti-elastase activity was applied to a Sephadex G-75 column equilibrated with 50 mM Tris-Cl buffer, pH 8.0, containing 200 mM NaCl. Elastase inhibitory activity-containing fractions from the Sephadex G-75 column were pooled, desalted (Fig. 1A), and fractionated further by passage over a DEAE-Sepharose column preequilibrated with 50 mM Tris-Cl buffer, pH 8.0. The column was developed with a linear NaCl gradient from 0 to 400 mM, and inhibitory activity was eluted as a peak at around 150 mM NaCl (Fig. 1B). Following desalting and acidifying, the active fractions from DEAE-Sepharose were subjected to reversed phase C HPLC (Fig. 1C). The most activity was found as a major peak in Fig. 1C, designated as peak3. Its purity was confirmed by three different methods. 1) Tricine-SDS-polyacrylamide gel electrophoresis (23) showed a single band (data not shown); 2) MALDI mass spectrum provided a single sharp peak with a molecular weight of 6,110 (Fig. 2); and 3) gas phase protein sequencing has detected a single amino acid on the N terminus. The results of each purification of guamerin are shown in .


Figure 1: Purification steps of guamerin by sequential chromatography. A, Sephadex G-75 column profile. The inhibitory activities against human leukocyte elastase were found at fractions 30-40; B, DEAE-Sepharose column profile. The active fractions 16-20 were desalted and concentrated. The dottedline indicates a linear salt gradient from 0 to 400 mM; C, Dalta-Pak C HPLC column profile. Peptides were eluted with a linear gradient of acetonitrile. The active peaks were numbered based on the order of elution. The most active peak (peak3) was isolated in bulk and assayed subsequently. The remaining active peaks are mentioned under ``Discussion.''




Figure 2: MALDI mass spectra of guamerin and reference substances. The arrow shows the molecular weight (6,110) of guamerin. This value was confirmed to be correct by the result calculated from amino acid sequence analysis. The peak at mass/charge 12328.6 and 5734.5 indicates the molecular weight of cytochrome c and insulin, respectively.



Physicochemical Properties

The stability of guamerin at elevated temperatures was investigated. When guamerin was incubated in 0.1 M Tris-Cl buffer pH 8.0 from room temperature to 90 °C at 10 °C intervals for 15 min each no loss of the inhibitory activity was observed. Guamerin was also stable in a wide range of pH from 1 to 11 at 25 °C. Guamerin competitively inhibited the catalytic activity of elastase in a substrate-like manner in the presence of the low molecular weight synthetic peptide substrate (Suc-Ala-Ala-Ala-pNA) as well as a high molecular weight substrate (azocasein). This result was obtained by using human leukocyte elastase with a specific activity of 100 units/mg protein and 0.17 µM of guamerin according to the method of Dixon (17). The inhibition constant of guamerin was 8.1 10M, which is considerably lower than eglin C (10 10M) or elafin (6 10M) (9, 14) . In addition, when the enzyme and guamerin were preincubated for varying time intervals prior to adding the substrate to reaction vials, no differences in inhibitory activities were found, suggesting that guamerin is a fast binding and slow dissociating molecule.

Amino Acid Composition and Sequence Analysis

The amino acid composition of guamerin was determined by Pico Tag amino acid analysis, and the result is shown in . The number of residues/molecule analyzed by hydrolysis was based on a molecular weight of 6,110, which was identical to the result obtained from the complete sequence of the peptide. It is very interesting that guamerin consists of 10 cysteines and no tryptophan. The significance of a large number of cysteine residues is unclear except that they play a pivotal role in structural rigidity. The importance of disulfide bridges was confirmed by a subsequent experiment showing that reduction and S-pyridylethylation caused loss of inhibitory activity of guamerin (data not shown).

The complete amino acid sequence data are given in I. Following reduction and S-pyridylethylation of guamerin, direct sequencing gave 57 residues and a blocked N terminus was not found. The total sequence was confirmed by overlapping the sequences of fragments digested with trypsin or endoproteinase Lys-C. When S-pyridylethylated guamerin was digested with endoproteinase Lys-C followed by separation of the peptides with a Delta-Pak C reversed phase HPLC column, three peptide fragments (L1, L2, and L3) were obtained. Tryptic digestion produced four peptide peaks (T1, T2, T3, and T4) and a ghost peak containing no peptide under the same condition (data not shown). The alignment of all the tryptic and lysyl endoproteinase peptides is shown in Fig. 3.


Figure 3: Amino acid sequencing strategy of S-alkylated guamerin. The direct sequence was determined by automated Edman degradation without fragmentation. The bars represent the overlapping fragments derived from enzymatic cleavage of trypsin (T) and endoproteinase Lys-C (L). Peptides were numbered according to the order of elution from HPLC column after enzymatic digestion (data not shown).



The specificity of guamerin was studied with various kinds of proteases, which are known to have different active site residues. The molar ratio of guamerin to each enzyme was adjusted to 5. Guamerin inhibited human leukocyte and porcine pancreatic elastase by 86.4 and 83.2% on average, respectively (Fig. 4). However, guamerin had a very small effect on trypsin (serine protease), chymotrypsin (serine protease), and subtilisin (serine protease) and no effect on papain (thiol protease), pepsin (aspartyl protease), thrombin (serine protease), and factor Xa (serine protease). These results show that guamerin may be an elastase-specific inhibitor. It has been known that the reactive center() P1-P1` peptide bond of the inhibitor may be cleaved by its target protease and the peptide bond determines the specificity (24) . We attempted to examine which site(s) within guamerin are cleaved by human leukocyte elastase. When guamerin-human leukocyte elastase complex was subjected to automated protein sequencing after incubation for 48 h, we observed that the Met-Ile peptide bond within guamerin was hydrolyzed by elastase.


Figure 4: Inhibition of various proteinases by guamerin. Guamerin was effective for human leukocyte and porcine pancreatic elastase but had no significant effect on subtilisin, chymotrypsin, trypsin, papain, pepsin, factor Xa, and thrombin. The bar represents standard deviation of four separate experiments. The experimental conditions are described under ``Experimental Procedures.'' HL-Elastase, human leukocyte elastase; PP-Elastase, porcine pancreatic elastase.




DISCUSSION

We have purified an elastase-specific inhibitor, guamerin, from H. nipponia and determined its complete amino acid sequence. Purification of guamerin was done by stepwise gel filtration, anion exchange, and reversed phase HPLC. The inhibitory peptides from DEAE-anion exchange chromatography were separated with an HPLC gradient, producing four peptide peaks with anti-elastase activity (Fig. 1C). During the purification and sequencing of guamerin, three more active peaks were observed (in Fig. 1C, designated as peaks 1, 2, and 4) with a dual gradient of acetonitrile elution. Their molecular weights were 5734, 7800, and 6212, respectively. Analysis of the second most active peak (peak4) showed over 90% homologous amino acid sequence as well as identical cysteine spacing and the same P1 residue as guamerin. This suggests that it must be an isotype of guamerin. The rest of the active fractions will be further characterized to find sequences and variabilities.

When sequence similarity was studied, guamerin showed no homology to any known elastase inhibitors including eglin C and elafin but had 51% sequence identity to hirustasin, antistasin-type serine proteinase inhibitor from H. medicinalis and 39% sequence similarity to antistasin (domain-2), a factor Xa inhibitor, from the saliva of a Mexican leech Haementeria officinalis (Fig. 5). Neither hirustasin nor antistasin is, however, known for inhibitory activity against elastases (25, 26) . In addition to conservation of cysteine residues with identical spacing, it is a striking feature that octapeptide (TCSPAQVC) and nonapeptide (DENGCEYPC) are completely conserved in guamerin and hirustasin (Fig. 5). These homologies suggest that guamerin, hirustasin, and antistasin may have a common ancestor and that gene duplication and mutation events may be sources of diversity in these inhibitors (26, 27) . Guamerin is a good example of the case that even though it has a highly homologous sequence to hirustasin and antistasin, its target protease is different from them. The specificity of the inhibitor is generally determined by the P1 residue of the reactive site (24, 28) . In hirustasin and antistasin, Arg is identified as the reactive site residue (25, 26, 27, 29, 30) . The sequence alignment indicates that the P1 residue Met of guamerin corresponds to the Arg residue at the P1 position for hirustasin and to Arg, Arg for two domains of antistasin (Fig. 5). Thus, we can suggest that the mutational replacement of Met for Arg at the reactive site P1 of guamerin resulted in the change of its activity. The hydrophobic region surrounding the reactive site is known to be preferred for human leukocyte elastase (31) . From Kyte-Doolittle hydropathy profiles (32) we have evidence that the secondary structure of guamerin had a different environment around the reactive domain from hirustasin and antistasin domain-2. The reactive domain of guamerin lies in a hydrophobic region, whereas the reactive domain of the others are hydrophilic. In addition to the distinct reactive site, we suggest that hydrophobic groups around the reactive site play a role in the specificity of guamerin.


Figure 5: Amino acid sequence homology of guamerin with hirustasin and antistasin. Dark boxes show matches of cysteine residues, and the shadedboxes indicate homologous sequence. The arrow marks the scissile peptide bond (the reactive center) of the three inhibitors. The sequence of elafin, an elastase-specific inhibitor of human skin with 57 amino acid residues, has virtually no homology.



There are a few specific elastase inhibitors having a similar molecular mass. For example, elafin, a human leukocyte elastase-specific inhibitor from human skin, has 57 amino acid residues but has virtually no sequence homology (Fig. 5). The preliminary data from ingestates suggested that guamerin was not present in the saliva of leeches but in the body tissue, perhaps implying a primary role in regulation of tissue proteinases. Further studies including cloning of cDNA are necessary to ascertain structural and functional properties of this interesting protein with the long range goal of application as a therapeutic agent.

In conclusion, guamerin is similar in both size and cysteine content to hirustasin, an antistasin-type serine protease inhibitor isolated from leeches, but it has a different target proteinase with a specific reactive site. It appears that guamerin is an elastase-specific inhibitor and differs from previously characterized elastase inhibitors in primary structure and K value.

  
Table: Purification of guamerin


  
Table: Amino acid composition of guamerin


  
Table: Automated sequence analysis of S-alkylated guamerin

Approximate yields of phenylthiohydantoin (PTH)-derivatives were calculated from peak areas on HPLC.



FOOTNOTES

*
This work was supported by grants from the Ministry of Science and Technology and from the Korea Science and Engineering Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: KAIST, Taejon 305-701, Korea. Tel.: 82-42-869-2612; Fax: 82-42-869-2610.

The abbreviations used are: HPLC, high performance liquid chromatography; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry.

The notation of Schecter and Berger (33) is used to denote the relative positions of residues with respect to the reactive site (scissile) peptide bond of inhibitor.


ACKNOWLEDGEMENTS

We thank Dr. Jin Sung Kim for MALDI-MS analysis, Soo Hyun Kim and Jong Soon Choi for computer facilities, and Dr. In Jung Yoon and Jae Hie Shim for helpful discussions.


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