Identification of different molecular forms of human airway lysozyme

ANALYTICAL

BIOCHEMISTRY

Identification

160, 227-232 (1987)

of Different

JACKYJACQUOT,'

Molecular Forms of Human Airway Lysozyme

RACHIDBENALI,JEAN-MARIEZAHM,ANDEDITHPUCHELLE

Groupe d’Etude des Systemes Mucociliaires (Gemuc), INSERM, Faculte de Medecine, 51095 Reims Cedex France Received June 16, 1986 Human airway lysozyme (HAL) was separated into fractions of distinct molecular forms using a Mono S cation-exchange column on a fast-protein liquid chromatography system. This new and rapid (30 min) purification procedure of human lysozyme enabled the preparation of fractions, highly enriched in different isoenzymes of HAL. Purified HAL from (i) pathological purulent airway secretions, (ii) nonpurulent airway secretions, and (iii) normal tracheobronchial tissue culture medium was characterized by four, three, and only one enzymatically active molecular forms, respectively. All charge forms (separated or combined) recovered from either purulent or nonpurulent airway secretions or tracheobronchial culture medium exhibited the same apparent mOkCUhr weight of 15,000. 0 1987 Academic Pms, Inc. KEY WORDS: human airway lysozyme; purification; multiple forms.

Lysozyme found in human airway secretions derives from various sources. Konstan et al. (1) have shown that in normal human airways, the submucosal tracheal glands, the surface epithelial cells, and the pulmonary alveolar macrophages secrete lysozyme. In pathology, lysozyme can also originate from leucocytes (2,3) present in high concentration when airway secretions are infected by pathogen bacteria (4). Although the physiological roles of HAL2 are still incompletely identified (5), it has already been reported that this human enzyme (i) possesses antibacterial properties involved in pulmonary defense mechanisms (6,7) (ii) is capable of restructuring the gel structure and consequently the rheological and transport properties of airway mucus (8,9), and (iii) is a potent inhibitor of chemotaxis and of the production of toxic oxygen radicals by stimulated neutrophils (10). To study the functional and structural properties of HAL, we developed a new and rapid method for

purifying HAL by a fast-protein liquid chromatography (FPLC) system using a Mono S cation-exchange column. We describe the presence of at least four enzymatically active HAL forms depending on whether HAL was purified from purulent secretions, nonpurulent secretions, or serum-free culture medium of human tracheobronchial explants. MATERIALS

AND METHODS

Enzyme Assay Lysozyme lytic activity was evaluated spectrophotometrically by measuring the initial rate of lysis of a Micrococcus luteus cell wall suspension (Worthington Biochemical Corp.) according to Shugar’s method (11) modified as previously described ( 12). The specific activity was defined as units of enzyme activity per microgram of protein, determined by the Bio-Rad protein assay procedure (13) using hen egg-white lysozyme (HEWL, Worthington) as standard. Sources of HAL

’ To whom correspondence should be addressed. * Abbreviations used: HAL, human airway lysozyme; HEWL, hen egg-white lysozyme; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

Pathological airway secretions. Secretions collected by expectoration (sputum) were 227

0003-2697187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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obtained from patients with chronic bronchitis. Purulent airway secretions were defined as secretions containing active leukocyte elastase and cathepsin G in enzymatically detectable concentration. The proteolytic activity of the two enzymes was determined spectrophotometrically with the substrate succinyl- (Ala)3 -p- nitroanilide ( 14,15) and the substrate succinyl-(Ala)2Pro-Phe-nitroanilide ( 16). Nonpurulent airway secretions were characterized as containing no detectable proteolytic activities. Only 1% of the entire collected sputa was referred to as nonpurulent secretions. Tracheal tissue culture. Human tracheobronchial material obtained within 5 h of death at postmortem examination was collected from 5 patients (aged 59 to 81) without signs of respiratory tract infections or other respiratory diseases. Tracheal and bronchial strips (3-mm wide, IO-mm long) were maintained in culture by using the method of Konstan et al. (1). The tissue was cultured at 35°C in a water-saturated incubator containing 40% O2 and 5% C02. The culture medium (medium 199 with Earle’s salt, Biochrom KG.) supplemented with Lglutamine and antimicrobials was harvested daily for up to 3 days. Culture media were pooled, concentrated on an Amicon concentrator with a YM2 membrane (Amicon Corp, Lexington, MA), dialyzed, and stored at -20°C until needed.

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previously obtained from (i) purulent secretions after the Sephadex G-75 gel filtration step, (ii) nonpurulent secretions after the dialysis step, (iii) nonpurulent secretions after the Sephadex G-75 gel filtration step, and (iv) native tracheobronchial tissue culture medium were subjected to Mono S HR5/5 FPLC column (Pharmacia). The standard elution conditions at a constant flow rate of 1.0 ml/min (pressure 15 MPa) were as follows: O-5 min, wash with 50 mM phosphate, pH 7.2; 5-25 min, linear NaCl gradient O-l .O M in 50 mM phosphate, pH 7.2; 25-27 min, wash with 1.0 M NaCl in 50 mM phosphate, pH 7.2; 27-30 mitt, wash with 50 mM phosphate, pH 7.2. Protein recovery studies were performed using this gradient elution profile and the eluant was monitored at 280 nm. Tracheal explant culture medium containing HAL was directly applied onto the Mono S column after the sample had been centrifuged (2O,OOOg, 30 min) to remove macroscopic particles. The molecular weight of the different HAL forms was determined using a Superose 12 HR IO/30 column equilibrated with 50 IIIM phosphate 0.15 M NaCl, pH 7.2. The column was calibrated with Blue Dextran ( VO) and M, standard myoglobin (M, 17,800), HEWL (M, 14,400), and cytidine (M, 243) at a flow rate of 0.4 ml/min (pressure 10 MPa) with the FPLC system.

Gel Electrophoresis Isolation of HAL HAL was prepared from pathological purulent and nonpurulent secretions according to the method previously described ( 12,17). Briefly, the purification procedure involves NaCl extraction, trichloroacetic acid treatment, and dialysis followed by CM Sephadex C-25 and Sephadex G-75 chromatography (Pharmacia). The purified HAL showed a single protein band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using the method of Ring and Laemmli (18). For further evaluation, HAL preparations

Distinct molecular forms of HAL were analyzed by a cathodic PAGE system, performed on 15% acrylamide slab gel (180 X 200 X 1.5 mm) at pH 4.3 according to the method of Reisfeld et al. (19). Protein bands were visualized with PAGE blue 83. RESULTS

HAL was isolated from pathological airway secretions by a purification factor of 28fold and a recovery of more than 80% of the dialyzed secretion extract using a classical

PURIFICATION

OF HUMAN

purification procedure, as previously described (17). Purified HAL exhibited the same molecular weight (&& 15,000) and specific activity (three times higher than that of HEWL) as those previously reported for the lysozyme isolated from human tracheal explants (1) (data not shown). Figure 1 shows the comparison of Mono S FPLC patterns obtained with the HAL purified in our study. Multiforms of HAL were

obtained, depending on its source. Four charge forms of HAL from purulent secretions, three charge forms of HAL from nonpurulent secretions, and only one charge form of HAL from native tracheal tissue culture medium were isolated (Figs. la-d). Whatever the pathological secretion extracts or culture medium, each form of HAL referred to as A, B, C, and D was always eluted at NaCl concentrations of 0.24, 0.28, 0.30, /

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229

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FIG. 1. Comparison of elution profiles obtained on a Mono S column with (a) 50 pg of isolated HAL from purulent secretions obtained by CM Sephadex C-25 and Sephadex G-75 chromatography; (b) 50 fig of HAL from nonpurulent secretions obtained as in a; (c) 100 ~1 of nonpurulent secretion extract aRer dialysis; (d) 200 rl of concentrated supematant (X30) of tracheal culture medium; and (e) 30 pg of commercially available human milk lysozyme. The column was eluted with a ZO-min linear gradient of NaCl (O-l.0 M) in 50 mM phosphate buffer, pH 7.2. Forms A, B, C, and D of HAL were eluted at NaCl concentrations of 0.24, 0.28, 0.30 and 0.34 M, respectively.

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and 0.34 M, respectively. For purulent secretions (Fig. la), the most basic form of HAL (form D) represented 43%, form C 17%, form B 29%, and form A 11% of the total protein content. Specific activities of the charge forms were 3.4, 11.1, 11.6, and 11.4 for forms A to D, respectively. Aliquots from each peak (A-D) were desalted and rechromatographed on the same Mono S column. The elution positions of each protein and lytic activity peak remained identical and no redistribution of their lytic activity was observed. For comparison, commercially available human milk lysozyme (Calbiochem Brand Biochemicals) eluted under only one charge form (form D) and with a specific activity similar to the form D of HAL (Fig. le). Further, Fig. lc shows that the three charge forms of HAL isolated from nonpurulent secretions could be directly purified by applying samples of dialyzed nonpurulent secretion extract. The recovery of HAL from the Mono S column was greater than 95%. All four isolated charge forms of HAL have different electrophoretic mobilities in a cathodic PAGE system (pH 4.3). The electrophoretic mobility of the charge forms increased in a stepwise manner as the basic character of each form increased (Fig. 2). Commercially available HEWL was found to migrate slightly faster than the most basic form of HAL. All charge forms of HAL developed sharp precipitation arcs in an immunodiffusion test against specific polyclonal rabbit antiserum to human lysozyme (data not shown). All charge forms (separated or combined) recovered from purulent, nonpurulent secretions, and/or tracheal culture medium were analyzed by gel-filtration FPLC on a Superose 12 column. A single and sharp peak of protein was eluted with a retention volume of 16.5 ml, corresponding to an apparent molecular weight of 15,000 (Fig. 3). Under the same experimental conditions, commercially available human milk and placenta lysozyme were eluted with an identical retention volume. Commercially available HEWL

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FIG. 2. Cationic PAGE (pH 4.3) of the four forms of HAL obtained on a Mono S column. Direction of migration is from anode (top) to cathode (bottom). Forms A, B, C, and D of HAL correspond to lanes 1,2,3, and 4, respectively. Lane 5, HEWL (*) and cytochrome c (* *).

(&I, 14,400) was eluted with a retention volume of 17.5 ml. DISCUSSION

This paper reports a simple and reproducible one-step chromatographic procedure for the purification and separation of the isoenzymes of human airway lysozyme, using a Mono S FPLC system. This purification is more rapid (30 min) and more convenient than other conventional methods, such as affinity chromatography on a large number of adsorbents including heparine-Sepharose, C6 muropeptide-Affigel 202 column, immunoabsorbent to epoxy-activated Sepharose 6B and a variety of ion exchangers used for the purification of human lysozyme from different sources (5). Although it has been reported that isoenzymes of lysozyme are present in some animal material, such as bird egg white (20) and bovine stomach mucosa (2 1,22), the presence of isoenzymes of human lysozyme has not been clearly illus-

PURIFICATION

OF HUMAN

VOLUME

LYSOZYME

231

(ml)

FIG. 3. Gel filtration FPLC on a Superose 12 column of 50 pegof recombined forms of HAL from purulent and nonpurulent secretions and tracheal culture medium. The column was eluted with 50 mM phosphate buffer 0. IS M NaCl, pH 7.2, at a flow rate of 0.4 ml/min and calibrated with Blue Dextran ( VO) and Mistandard myoglobin (MT 17,800), HEWL (M, 14,400), and cytidine (M, 243).

trated. Mouton and Jolles (23) have indicated the presence of two forms of lysozyme in urine of patients suffering from acute myeloblastic leukemia. Charge heterogeneity in human lysozyme can result from differences in the peptide structure and/or in the content of amide groups on each charge form (23). Our gel filtration results suggest that the HAL isoenzymes consist of a polypeptide chain of identical molecular weight. Microheterogeneity in the polypeptide chain could account for these charge differences. The number of charge forms (A-D) of HAL appears to be related to the source and composition of airway secretions. Fresh explant culture medium contained only the most basic form (form D), while pathological nonpurulent and purulent expectorated secretions contained at least two or three supplementary charge forms (A-C), respectively. The additional forms seem to derive from the deamidation of the form D. The multiple forms of HAL might also be the result of limited proteolytic modification induced by proteases released from either leucocytes, bacteria, or other cells. Pahud and Widmer (2 1) have reported that proteinases might be responsible for the five molecu-

lar forms of lysozyme purified from cow stomach mucosa. Additional studies are under consideration to characterize the biophysical and physiological properties of isolated HAL subtypes as well as the in vitro interactions between the form D of HAL and purified human leukocyte elastase and cathepsin G. ACKNOWLEDGMENTS This work was supprted by Grants from CRE 855 019 and Contract INSERM-Synthelabo 83 022. The authors gratefully acknowledge the Laboratoire d’Histologie et de Cytologic (Professor J. J. Adnet) et d’AnatomoPathologie (Professor T. Caulet) C.H.R., Reims, France for their kind cooperation in providing normal human tracheobronchial tissue; J. M. Toumier for the collection of pathological purulent and nonpurulent airway secretions in the Service de Medecine orient6 vers I’Insuffisance Respiratoire (Professor P. Sadoul, CHU, Nancy, France); and A. Quiqueret and M. C. Rohrer for preparing the manuscript. REFERENCES 1. Konstan, M. W., Chen, P. W., Scherman, J. M., Thomassen, M. J., Wood, R. E., and Boat, T. F. (1981)Am. Rev. Respir. Dis. 123, 120-124. 2. Klockars, M., and Reitamo, S. (1975) J. Histochem. Cytochem.

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Bulletin No. 1069, Bio-Rad Laboratories, Richmond, CA. Bieth, J. G., Spiess, B., and Wermuth, G. C. (1974) Biochem. Med. I&350-357. Tournier, J. M., Jacquot, J., Puchelle, E., and Bieth, J. G. (1985) Am. Rev. Resp. Dis. 132, 524-528. Nakajima, K., Powers, J. C., Ashe, B. M., and Zimmerman, M. (1979) J. Biol. Chem. 254, 4027-4032. Marx, J., Jacquot, J., Berjot, M., Puchelle, E., and Alix, A. J. P. (1986) Biochim. Biophys. Acfa 870, 488-494. King, J., and Laemmli, U. K. (197 1) J. Mol. Biot. 62,465-473. Reisfeld, R. A., Lewis, U. J., and Williams, D. E. (1962) Nature (London) 338,281-283. Amheim, N. (1974) in Lysozyme (Osserman, E. F., Canfield, R. E., and Beychok, S., Eds.), pp. 153- 16 1, Academic Press, New York. Pahud, J. J., and Widmer, F. (1982) Biochem. J. 201,66

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22. Dobson, D. E., Prager, E. M., and Wilson, A. C. (1984) J. Biol. Chem. 259, 11607-l 1616. 23. Mouton, A., and Jollts, J. (1969) FEBS Lefr. 4, 337-340.