Protein degradation in canine tracheobronchial secretions

BIOCHEMIC’AI

MI:DI(‘INt

18.

6470 (19771

Protein Degradation Tracheobronchial MARK Department

J. REASOR’

oj’En~~irot7mentcrl Mrdirinr. (INS Public Health.

in Canine Secretions

AND ROBERT Tl7e Johns

Baltimore.

Hopkim Maryland

J. RUBIN Universit? 21205

School

ojHygienr

Received November 16, 1976

We have recently developed a novel method for the collection of tracheobronchial secretions (TBS) from anesthetized dogs using a plastic screen mesh to trap the secretions in situ in the trachea (1). TBS collected from humans and experimental animals by a variety of techniques have been shown to contain a number of proteins, many of which are important in defending the lungs against inhaled toxic agents (2). We were interested, therefore, in characterizing the protein composition of the secretions collected by our new method. During our intitial electrophoretic analysis, we observed profiles suggestive of degraded proteins (3). The present study deals with an examination of the cause of the apparent protein degradation and considers the implications relative to the study of respiratory secretions.

Collection

of Tracheobronchial

METHODS Secretions

Tracheobronchial secretions were collected on cylinders of plasticcoated fiberglass screen in the trachea of pentobarbital-anesthetized dogs as previously described (I). Following collection, TBS samples were processed at O-4” in one of the three ways outlined in Fig. I. (1) Unfractionated TBS were removed from the collecting screen by solubilization in phosphate-buffered urea (PBU) containing 0.1 M sodium phosphate, 6 M urea, and 0.02% sodium azide, pH 7.1. (2) The supernatant and pellet phases were obtained by centrifugation of the collecting screens as illus’ This research was presented in a thesis submitted by Dr. Reasor in partial fulfillment of the Ph.D. requirements of the Johns Hopkins University. Present address: Department of Pharmacology, West Virginia University Medical Center, Morgantown, W. Va. 26506. 64 Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.

ISSN OMlb2Y44

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FIG. I. Flow diagram describing the processing of canine tracheobronchial secretions (TBS). Following collection, samples were (1) solubilized in phosphate-buffered 6 M urea (PBU), (2) centrifuged, or (3) soaked in Sputolysin, as outlined.

trated. (3) Cells in the secretions were isolated by soaking the secretionladen screen in Sputolysin (buffered dithiothreitol, pH 7.0, Calbiochem, La Jolla, Calif.), followed by centrifugation at SO@ for 5 min. The cell lysate was prepared by solubilization of the cells in PBU in a manner identical to that used with the unfractionated and pellet-phase samples. Sodium Dodecyl Sulfate-Polyacrylamide

Gel Electrophoresis

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed on gel rods of 7.5% acrylamide and 0.2% iV,N’-methylenebisacrylamide, following the method of Weber and Osbom (4). Prior to electrophoresis, all samples were incubated in a buffer containing 0.01 M sodium phosphate, 0.1% SDS, and 0.1% 2-mercaptoethanol, pH 7.0, for 1 hr. Electrophoresis was performed for 5 hr at 8 mA/gel with 30 pg of protein/gel. Proteins were visualized by staining with 0.025% Coomassie brilliant blue according to the method of Fairbanks et al. (5). Myosin, bovine serum albumin, ovalbumin, pepsin, trypsin, ribonuclease, and cytochrome C were used as standards for molecular weight determinations (4). Immunoelectrophoresis

Immunoelectrophoresis was performed in 1% agar on microscope slides. Dog serum (prepared from the blood of mongrel dogs), dog serum albumin, and serum IgG (Pentex, Kankakee, Ill.) were used as reference standards. Samples containing 270 pg of protein were electrophoresed for

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2 hr at I50 V and 30 mA and then developed for 36 hr at 3” against rabbit anti-dog serum (Kallestad, Chaska, Minn.). Precipitin bands were visualized by indirect light and photographed.

Following incubation in the phosphate-SDS-mercaptoethanol buffer. aliquots of the supernatant phase were digested with the proteolytic enzymes, pronase (Calbiochem), trypsin (Worthington. Freehold, N.J.), or protease (Sigma Chemical Co., St. Louis, MO.). Each enzyme was prepared in the phosphate-SDS-mercaptoethanol buffer and incubated with samples in a ratio of 50 ,ug of supernatant-phase protein/l vg of enzyme protein for 15 min at 25’.

Aliquots of the supernatant and pellet phases were solubilized in the phosphate-SDS-mercaptoethanol buffer and mixed together to effectively reconstitute the unfractionated sample. They were mixed together in a ratio (3 mg of supernatant-phase protein/2 mg of pellet-phase protein) simulating the relative contribution of each phase to the whole sample (3). The mixture was then electrophoresed as previously described.

The protein content of samples was measured by the method of Lowry et crl. (6). using bovine serum albumin as the standard. RESULTS Unfractionated TBS gave an electrophoretic profile with nearly all of the protein migrating as a densely staining, low molecular weight region with virtually no resolution (Fig. 2, gel A). In contrast, the supernatant phase contained a wide range of peptides (gel B). The pellet phase gave a pattern similar to that of the unfractionated sample (gel C). Since the unfractionated sample would be expected to contain the peptides found in both phases of the secretions. it appeared that a degradation of proteins had occurred in the unfractionated sample. Samples of the supernatant and pellet phases were mixed together prior to electrophoresis in order to determine whether the profile of the unfractionated TBS or the sum of the two separate phases would be generated. Following electrophoresis of the mixture, a nonadditive profile resulted (gel D) which qualitatively resembled that of the unfractionated sample, suggesting that there was a factor present in the pellet phase responsible for the degradation. Degradation was seen in all preparations tested. The electrophoretic profiles represented for the secretions were observed in samples from 15

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C

UNFRAC TBS

PELLET

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Btc

E

S+ SOILED C

f

S+ CELL LVSATE

0

0+ PRONASE

Rc. 2. Electrophoretic profiles of various fractions and mixtures of tracheobronchial secretions. For each sample, 30 pg of protein was electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Protein was visualized by staining with 0.025% Coomassie brilliant blue. (A) Unfractionated TBS; (B) Supernatant phase; (C) Pellet phase; (D) Mixture of supematant and pellet phases; (E) Mixture of supematant phase and boiled pellet phase; (F) Mixture of supernatant phase and the TBS cell lysate; (Ci) Mixture of supernatant phase arid pronase. A scale of apparent molecular weights of rhe peptide bands is presented on the left.

dogs, while the effects of mixing were demonstrated using the supematant and pellet phases from 4 different dogs. When aliquots of the pellet phase were heated in a boiling-water bath for 15 min, then mixed with the supematant-phase sample and electrophoresed, there was retention of the peptides of the supernatant phase (gel E). The same result was found when the pellet phase proteins were precipitated with 15% (w/v) trichloroacetic acid (TCA) followed by mixing of the TCA-soluble or resolubilized TCA-insoluble pellet-phase fraction with the supematant phase prior to electrophoresis (gels not shown). Incubation of the supematant phase with the proteolytic enzymes pronase (gel G), trypsin, or protease prior to electrophoresis resulted in the same low molecular weight peptide profile observed for the unfractionated TBS. These results suggested that the degradation of proteins in TBS could be enzymic. It is well-established that serum proteins are normal constituents of respiratory secretions (2, 7). Immunoelectrophoresis of unftactionated

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TBS and the supernatant phase with the development against anti-dog serum revealed fewer precipitin arcs in the unfractionated TBS, reflecting the absence of certain proteins in this preparation (Fig. 3). In addition, the arcs corresponding to albumin and IgG appeared more diffuse than those in the supernatant phase, possibly due to size heterogeneity of the antigenic proteins resulting from proteolysis. Cells contained in the unfractionated TBS were found to sediment in the pellet phase of the secretions. These cells included alveolar macrophages and polymorphonuclear leukocytes (l), both of which contain rich complements of proteolytic enzymes (8). The cells were isolated from TBS and solubilized in PBU (cell lysate). When aliquots were mixed with the supernatant phase and electrophoresed, the resulting pattern consisted of degraded proteins (Fig. 2, gel F). When aliquots of the cell lysate were heated prior to mixing, no change was seen in the electrophoretic profile of the supernatant-phase proteins (gel not shown). DlSCUSSlON This study demonstrates the differences in electrophoretic profiles of TBS proteins which can occur depending upon whether or not cells are removed from the secretions prior to characterization. The results presented here suggest that the degradation of proteins in the unfractionated TBS results from the action of proteolytic enzymes released from cells in the secretions during urea-solubilization, a step we found necessary due to the adherence of the secretions to the collecting screens. Centrifugation of the screens following collection removed the cells and prevented the degradation. Prior to electrophoresis, all samples were incubated in a solution containing SDS and mercaptoethanol, denaturing and reducing agents, respectively. It might be expected that during the mixing experiments the catalytic activities of proteolytic enzymes would be lost under such seemingly unfavorable conditions. This did not seem to be the case in these

FIG. 3. Immunoelectrophoresis phase (TBS Sup.). Samples containing and developed against rabbit anti-dog to albumin and IgG are labeled.

of unfractionated TBS (Unf. TBS) and the supernatant 270 pg of protein were electrophoresed in 1% agar serum (A-Serum). The precipitin arcs corresponding

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experiments. In fact, Porter and Preston (9) reported that the proteolytic activities of trypsin and chymotrypsin were retained under such conditions. An example of the problem which can arise when cells are not removed from secretions is illustrated in a series of studies which have dealt with the characterization of proteolytic enzymes in purulent sputum from several human respiratory disorders (10-12). In these studies, the sputum was homogenized in distilled water before analysis. Since cells were not removed prior to homogenization, and due to the hypotonicity of the medium, it was unclear as to whether the enzymes were originally intraor extracellular. As a result, the actual significance of such enzymes in sputum is’unknown. The significance of proteolytic activity in tracheobronchial secretions in situ is unknown. It is conceivable that under some circumstances significant cell lysis could occur resulting in the liberation of proteolytic enzymes into the airways. Although emphysema is characterized by a destruction of lung tissue (13) and proteolytic mechanisms are suspect (14)) there is no indication that products from cell lysis may contribute to this disease. The human respiratory disorder, alveolar proteinosis, is characterized by the accumulation of proteinaceous material in the alveoli which appears to originate from degenerating cells (15). Whether this material contains proteolytic enzymes or is itself the result of degradation is yet to be determined. The results of the present study clearly indicate that it is imperative to consider the manner in which respiratory secretions are processed following collection. The arbitrary homogenization or solubilization as a first step in processing may lead to artifactual observations and conclusions concerning the properties of respiratory secretions, especially regarding the protein composition. SUMMARY

Major differences were observed in the electrophoretic profiles of the proteins of canine tracheobronchial secretions depending upon the method by which secretions were processed prior to characterization. Secretions solubilized in phosphate-buffered 6 M urea directly after collection gave only a diffuse, low molecular weight pattern suggestive of degraded proteins. In contrast, when secretions were first fractionated by centrifugation into supematant and cell-containing pellet phases, a wide range of higher molecular weight peptides was demonstrated in the supernatant phase. The protein degradation appeared to be due to the action of enzymes released from cells in the secretions during the initial solubilization of the samples in urea. These results emphasize the need to carefully consider the manner in which respiratory secretions are processed prior to characterization.

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ACKNOWLEDGMENTS The authors thank Mr. David Cohen and Mr. John Brooks for their skilled technical assistance and Dr. Henry Yeager, Jr. for his comments during the preparation of this manuscript. This work was supported by National Institute of Environmental Health Sciences Grants ES-00454. ES-00034 (M. J. R.) and Research Career Development Award ES-44887 (R. J. R.).

REFERENCES 1. Adams, G. K., Aharonson. E. F., Reasor. M. .I.. and Proctor, D. F., J. Appl. Physiol. 40, 247 (1976). 2.

3. 4. 5. 6.

Masson, P. L., and Heremans, J. F., in “Sputum: Fundamentals and Clinical Pathology” (M. Dulfano, Ed.), p. 404. Charles C Thomas, Springfield, III. (1973). Reasor. M. J., Ph.D. thesis. The Johns Hopkins University, Baltimore (1975). Weber, K., and Gsborn, M., J. Bioi. Chem. 244, 4406 (1969). Fairbanks, G., Steck, T. F.. and Wallach. D. F. H.. Biochemistry 10, 2606 (1969). Lowry, 0. H., Rosebrough, N. J.. Farr. A. L.. and Randall. R. J., .I. Biol. Chem. 193, 265 (1951).

7. Reynolds, H. Y., and Newball. H. H.. J. Lub. C/;rr. MeJ. 84, 559 (1974). 8. Harris, J. 0.. Olsen, G. N., Castle, J. R., and Maloney, A. S., Amer. Ret,. Respir. Dis. 111, 579 (1975).

9. IO. 11. 12.

Porter, W. H., and Preston, J. L., Anal. Biochem. 66, 69 (1975). Lieberman, J.. and Kurnick, N. B., Nature (London) 19, 988 (1962). Lieberman, J., and Kurnick, N. B., Pediatrics 31, 1028 (1963). Lieberman, J., Trimmer, B. M., and Kurnick. N. B., Lab. Zmvest. 14, 249 (196.5). 13. Anderson, A. E.. Jr., and Foraker. A. G., Amer. J. Med. 32, 218 (1962). 14. Kuhn, C., III, and Tavassoli, F., Lab. Invest. 34, 2 (1976). 15. Rosen, S. H.. Castleman, B., and Liebow. A. A., N. Eng. J. Med. 258, II23 (1958).