characterization of mucin isolated from rat tracheal transplants

357

Biochimica et Biophysica Acta, 588 (1979) 357--367 © Elsevier/North-Holland Biomedical Press

BBA 29119

CHARACTERIZATION OF MUCIN ISOLATED FROM RAT TRACHEAL TRANSPLANTS

JEFFREY N. CLARK and ANN C. MARCHOK

University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.)

(Received May 15th, 1979)

Key words: Mucin; Polysaccharide analysis; Amino sugar; (Rat trachea)

Summary

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4--6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepha- rose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation t reatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent ~-elimination in the presence of alkaline borohydride, demon- strating the presence of O-glycosidic linkages.

Introduct ion

The s tudy of tracheal mucins has been difficult because the major source of this material has been sputum, which also contains secretions from the lower respiratory tract and salivary glands. To overcome these problems, Wardell and coworkers [1] developed a tracheal pouch system in dogs which allowed repeated collection of small amounts of accumulated tracheal secretions. The system has been used in studies on the alterations in mucus secretion brought

358

about by neuroregulators [2--4] , and Sachdev and coworkers [ 5] have reported a detailed chemical characterization of a mucin fraction obtained from the tracheal pouch. A second approach to the study of tracheal mucins has been the use of in vitro organ culture systems of rat [6,7], rabbit [8], and canine [9,10] tracheas. Since the material produced in vitro is secreted only by the trachea, certain studies are possible on the synthesis and secretion of radioactively labeled material; however, the small amount of material obtained seriously limits chemical and rheological analyses of tracheal mucins.

The present s tudy was undertaken to determine if a rat tracheal transplant model developed by Kendrick and coworkers [11,12] could be used for studies of the biochemistry of mucous glycoproteins. The work of Kendrick and coworkers [11,12] and the present experiments show that tracheal transplants offer several advantages over previous systems since they: (a )became vascular- ized by the host and maintain nearly normal morphology of the mucosa for many months; (b) continue to synthesize and secrete mucous glycoproteins into the lumen for many weeks; (c) each produce several milligrams of mucus which is easily removed and solubilized and is suitable for chemical and rheological studies. Also, (d) the secretions are uncontaminated by mucins derived from other sources; (e) substances can be introduced into the transplant to test their effect on mucin metabolism, and (f) the model employs a relatively inexpensive animal already used extensively in studies of respiratory disease. Furthermore, the present report describes the isolation and characterization of a mucous glycoprotein fraction obtained from the lumenal contents of the tracheal transplant.

Materials and Methods

Materials. Specific pathogen-free female Fischer 344 rats 10--12 weeks old were used for the transplants. Column chromatography material was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). All sugar standards were obtained from Sigma Chemical Co. (St. Louis, MO) and Supelco, Inc. (Bellefonte, PA). Rat serum albumin was obtained from Miles Laboratories, Inc. (Elkhart, IN). Reagents for gas-liquid chromatography were obtained from Analabs, Inc. (New Haven, CI) and Supelco.

Establishment of the graft and collection of secretions. Tracheal transplants were established as previously described [11,12]. Briefly, tracheas were removed aseptically from donor rats, sutured to polyethylene tubing, tied closed at bo th ends, and placed in subcutaneous pockets on the backs of iso- genic recipient rats. After 4--6 weeks the transplants were aseptically removed and the lumenal contents of each were flushed out with sterile saline.

Initial solubilization of transplant secretions. The collected secretions were dispersed with a Pasteur pipette and solubilized by dialysis for 24 h against buffer A (0.01 M phosphate buffer (pH 7.6) containing 0.10 M NaC1, 5 mM 2-mer- captoethanol, and 0.59 mM EDTA). The solubilized secretions were then cen- trifuged at 3000 × g for 15 min and the supernatant further characterized as diagrammed in Fig. 1.

Fractionation of the transplant secretions. Transplant secretions solubilized by buffer A were chromatographed on a Sepharose CL-6B column (2.6 X 62.5

TRANSPLANTS

NATIVE SECRETIONS IN SALINE

Dialyze and solubilize with buffer A

Centrifuge at 3000 x g_

• "~'PELLET (discard)

SU PERNATANT

L SEPHAROSE CL-6B

CHROMATOGRAPHY

Voi d Volume

PEAK I

(unpurified mucln fraction)

included Volume

PEAKS 2-5 Rerun on Sepharose CL-6B Dialyze vs. distilled H20 Lyophl Ii ze

l SDS GEL ELECTROPHORESIS (6%)

359

Dialyze vs. distilled H20

SEPHAROSE CL-2B REDUCTIVE- CHROMATOGRAPHY ALKYLATION

SEPHAROSE CL-4B

CHEMICAL [ Void ANALYSIS q INCLUDED VOLUME t Volume

PEAKS B, C

SODIUM BOROHYDKIDE ~- PEAK A REDUCTION (purified mucin fraction) CHEMICAL ANALYSIS

SUGAR, SULFATE AND AMINO ACID ANALYSIS

~, SDS AGAROSE/ ACRYLAMIDE GEL ELECTROPHORES1S

Fig. 1. Scheme for i so lat ion and character izat ion of rat tracheal transplant secret ions .

cm) and eluted with buffer A at a constant f low rate of 18- -25 ml/h. The eluate of the column was constantly monitored by ultraviolet absorption at 206 and 254 nm and by hexose and protein determinations on an aliquot of each tube as described below. Following chromatography, the peaks were pooled, dialyzed against triple-distilled water, lyophilized, and stored at - -20°C under a desiccant until further analysis. A small amount (5 rag) of the void volume fraction from Sepharose CL-6B chromatography (peak 1) was chromatographed on a Sepharose CL-2B column (2.3 × 59.0 cm) packed in the

360

same buffer and eluted at a f low rate of 20 ml/h. Reduction of disulfide bonds and S-carboxymethylation of the mucin frac-

tion (peak 1). Reduct ion of the void volume fraction from Sepharose CL-6B chromatography (peak 1) was performed as described [13]. In a typical experiment 100 mg of lyophilized sample was treated and thereafter dialyzed against buffer A and finally fractionated on a Sepharose CL-4B column (2.25 X 53.5 cm) packed in buffer A in a similar manner to the Sepharose CL-6B column. The major fractions from this column were dialyzed against triple-distilled water, lyophilized, weighed and chemically characterized.

Gel electrophoresis. For sodium dodecyl sulfate polyacrylamide gel electrophoresis, 50--100-pg samples were applied to 6% slab gels by the method of Miner [14]. Electrophoresis with agarose/acrylamide composi te gels containing sodium dodecyl sulfate was conducted by the methods described by Holden and coworkers [15] as altered by Sachdev et al. [5]. The lyophilized sample (2--4 mg) was dissolved in 1 ml of the running buffer and kept at 37°C for 12 h. Electrophoresis was then carried out in glass tubes (90 X 5 mm inner dia- meter) as described by Miner [14]. The gels were fixed as described by Segrest and Jackson [16].

~-elimination reaction. This was carried out as described by Sachdev [5]. Lyophilized samples of 2--4 mg were treated and thereafter, subjected to amino acid analysis as described below. Changes in the total amounts of serine, threonine, and alanine and the appearance of ~-aminobutyric acid were studied.

Analytical procedures. Hexose and protein determinations were performed on individual Sepharose column fractions by the methods of Spiro [17] and Lowry et al. [18], respectively, with either glucose or rat serum albumin as standard. Amino acid analysis of 2--4-mg samples (dry weight) was performed as described [19] after a 20 h hydrolysis at 110°C in 6.0 M HC1 containing 18 mM dimethylsulfoxide. Carbohydrate analysis was performed on 1--2-mg samples after hydrolysis for 2 h at 100°C in 2.0 M trifluoroacetic acid as described by Albersheim and coworkers [20]. The monosaccharides were then deri- vatized to the corresponding alditol acetates with NaBH4 reduction and acetyl- ated by acetic anhydride by the method of Hellerqvist and Ruden [21]. Then gas-liquid chromatographic analysis was done on a 6 ft glass column of 3% SP- 2340 on 100/120 Supelcoport (Supelco, Inc.). Sialic acid was determined on 1 mg samples by the method of Warren [22], with N-acetylneuraminic acid as standard. Sulfate was determined by the rhodizonate method [23]. Deoxy- ribonucleic acid analysis was performed by the method of Kissane and Robins [24] and lipid analysis was performed as described by Snyder and coworkers [25].

Results and Discussion

Histology of the tracheal transplants Histological examination of several transplants demonstrated that the muco-

sal lining ranged from a columnar to a tall columnar hyperplastic mucociliary epithelium. This morphology is maintained for several months [11,12]. Secre- tions in the lumen of the transplants as well as many of the epithelial cells and submucosal glands were stained by the periodic acid-Schiff reagent.

3 6 1

Solubilization of tracheal transplant secretions Solubilization of the transplant secretions in buffer A prior to column chro-

matography was essential due to the high viscosity of the material. The treat- ment, although relatively mild, was sufficient to solubilize nearly 100% of the native material. Between 5 and 8 mg (dry weight of dialyzed lyophilized sample) o f secretions were usually obtained from each transplant.

Sepharose CL-6B chromatography The solubilized secretions were initially separated by molecular weight on

Sepharose CL-6B into five major components , designated peaks 1--5 (Fig. 2A). Peak 1, excluded from the gel and therefore of very high molecular weight, was tentatively identified as mucin material. This assumption was substantiated by the relatively high hexose to protein ratio (0 .225) of peak 1 and the tendency of this material to form a gel upon rehydration. The material in peaks 2--5 had lower hexose to protein ratios (0 .098, 0 .063, 0 .035, and 0.110, respectively) and lower molecular weights than peak 1 and are therefore thought to consist primarily of non-mucin glycoproteins and proteins, part of which were derived from serum transudation into the lumen of the transplant. This was further substantiated by electrophoresis on 6% gels (results not shown), which showed that peak 4 consisted almost entirely of a component which ran identically with rat serum albumin. Lieberman and coworkers [26] have shown that canine tracheal pouch secretions also contain immunoglobulins, particularly IgA. It is

A

tO-

i s ~) 0.5- -0.10 T N

-o.o6 E

- 0 . 0 2 ,N 0

o ib

12- i 4

i i i i

,.f~ , 3 , / ' , = T 'I~', , 2 , _ _ . , , u , L B :1 b . - " __ ' , i: , % /s % ~__ ,IX'. / \ ,

3 ' . . . . " ~o ~o ~o ~o v,~o

-6

I -4'_o

x

o -2 x

i-

OBO-

I ~ 0,60- 8

0~$0-

0,20-

0 0

~ ~ , . _ _ ~" 1.6- 1.2-

0.

E 0 4 -

, 6 I o

1 2 - x

4 ~ 0 Jo2'° ~o ~ 5'0 !,6'o

TUBE NO,

Fig. 2. ( A ) Sepharose CL-6B c o l u m n c h r o m a t o g r a p h y on a c o l u m n ( 2 . 6 X 6 2 . 5 c m ) in b u f f e r A o f tracheal transplant s e c r e t i o n so lubi l i zed w i t h b u f f e r A. 5 . 3 - m l f r a c t i o n s w e r e c o l l e c t e d at a f l o w rate o f 2 2 m l f n . A p p r o x i m a t e l y 1 5 0 m g o f sample (dry w e i g h t ) w a s appl ied . 9 5 % r e c o v e r y o f the mater ia l wa s a t ta ined . (B) Sepharose CL-2B c o l u m n c h r o m a t o g r a p h y o n a c o l u m n ( 2 . 3 X 59 c m ) in b u f f e r A o f p e a k 1 ob ta ined f r o m Sepharose CL-6B c o l u m n c h r o m a t o g r a p h y . 4 . 1 - m l f rac t ions w e r e c o l l e c t e d at a f l o w rate o f 21 mHh. A p p r o x i m a t e l y 3 0 m g o f s a m p l e (dry w e i g h t ) was appl ied. 90% r e c o v e r y o f the mater ia l wa s a t ta ined .

362

also possible, due to the prolonged period before collection of secretions, that autolysis o f exfoliated cells and subsequent release of hydrolases may result in some breakdown of the mucin fraction. This assumption was provided indirect proof by analysis of the five major peaks which showed that peaks 2 and 3 as well as 1 contain sulfate, whereas peaks 4 and 5 do not (data not shown). Since sulfate monoester groups have only been reported for mucins and mucopolysaccharides, it is probable that peaks 2 and 3 contain either smaller mucous glycoproteins or mucin breakdown products. This possibility was not investi- gated further.

Chemical analysis of peak 1 Amino acid analysis was performed on peak 1 to provide further evidence

that it consists o f tracheal mucins. Table I shows that peak 1 is approximately 40% protein, containing large amounts of threonine, serine, alanine, proline, and acidic amino acids and small amounts of aromatic and sulfur-containing amino acids. These characteristics have been described for several other tracheobronchial mucins [5 ,9 ,27- -30] .

Carbohydrate analysis of peak 1 (see Table I) showed it to contain large amounts of galactose, N-acetylglucosamine, and N-acetylgalactosamine, as is typical of tracheobronchial mucins [5 ,9 ,27- -30] . The absence of xylose and glucuronic acid suggests little or no contamination by mucopolysaccharides [30] . This mucin fraction contained a smaller amount of fucose, but an amount of sialic acid similar to that described by Ellis and Stahl for a canine tracheal mucin [9] . The relatively large amount of mannose (three times that

T A B L E I

C H E M I C A L C O M P O S I T I O N OF P E A K 1

Percent b y dry weight o f the total sample as determined b y the amino acid analysis results.

A m i n o acid m o l / 1 0 0 0 m o l Sugar total amino acids

t oo l /100 m o l total monosacchar ides

Histidi~ne 21 .39 Lysine 46 .21 Arginine 50 .19 Threonine 79 .28 Serine 81 .17 Aspartic acid 93 . 53 Glutamic acid 107 .42 Half-cysteine 18 .46 Meth ion ine 11 .19 Tyros ine 49 .13 Phenylalanine 33 .48 Proline 62 .71 Valine 69 .24 Iso leucine 35 .02 Leucine 79 .38 Glycine 70 .23 Alanine 92 .11 S - c a r b o x y m e t h y l c y s t e i n e < 1 . 0 Percent prote in 38.1

Fucose Xy lose Mannose Galactose N-Acety lg lucosamine N-Acety lga lac tosamine Sialic acid

Glucuronic acid

3.7 < 0 . 5

6.2 32.8 27.8 22.4

7.2

<0.5

363

found by Ellis and Stahl [9]) indicates that the material is probably contami- nated by serum-type glycoproteins.

Further chemical analyses determined that this mucin fraction contained (based on dry weight) less than 1% DNA, about 3% lipid (most of which was cholesterol), and about 2.6% sulfate. The sulfate is probably found in sulfated mucins described histochemically by McCarthy and Reid [31].

Initial attempts to further purify the mucin fraction Further purification of the mucin fraction, peak 1, was necessary. Two

methods were attempted to fractionate this peak. Sepharose CL-2B was initially used to purify peak 1 further, since it fractionates at a higher molecular weight than CL-6B. Fig. 2B shows that this column yielded a high molecular weight void volume fraction and a mixture of unresolved components of lower molecular weight. Agarose gel electrophoresis of peak 1 in sodium dodecyl sulfate (results not shown) gave rise to smaller fragments (mucin subunits or contamination material) in the 20 000--100 000 molecular weight range, but did not fractionate the very high molecular weight mucins into discrete bands. Moreover, most of the periodic acid-Schiff-positive material remained at the origin of the 0.5% agarose/2.0% acrylamide composite gels.

Fractionation of the mucin fraction following reductive alkylation Because of the poor resolution obtained with the two systems above, it was

felt that further fractionation could not be achieved by use of the native mucin fraction, but that prior reduction and alkylation of disulfide bonds under denaturing conditions should be performed.

As shown in the outline in Fig. 1, peak 1 was subjected to reductive alkylation. The reduced alkylated material was thereafter subjected to Sepharose CL- 4B column chromatography (Fig. 3). About 25% elutes as a high molecular weight fraction near the void volume (peak A), while the remaining material yields the lower molecular weight (peaks B and C). All three peaks contain

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1" -1ON

',~ 0 .8 -_8 .

,.u 0.6- ,,n -6 °

e= 0.4-_ 4 e,

,~ 0 ,2- -2 ~0

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2o 3; 4o ~,5o 8b TUBE NO.

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Fig. 3. Sepharose CL-4B c o l u m n c h r o m a t o g r a p h y o n a c o l u m n (2 .25 X 53 .5 cm) in bu f f e r A o f p e a k 1 f o l l o w i n g reduct ive a l k y l a t i o n in 8.0 M urea/Tris-HCl buf fe r (pH 8.0) con ta in ing 5 mM di th io th re i to l . 4 .0- ml f rac t ions w e r e c o l l e c t e d . A p p r o x i m a t e l y 3 0 m g o f sample (dry w e i g h t ) w a s appl ied. 9 5 % r e c o v e r y o f the mater ia l w as a t ta ined .

3 6 4

glycoprotein material as determined by the presence of both hexose and protein.

Chemical analysis of Sepharose CL-4B peaks Table II shows the amino acid composition of material from peaks A to C.

The three peaks contain large amounts of serine, threonine, proline, and acidic amino acids and small amounts of aromatic and sulfur-containing amino acids, as has been observed in mucous glycoproteins [30]. The formation of S-car. boxymethylcysteine as a result of the reductive alkylation procedure should be noted. Although the material does not contain as much hydroxy amino acids as has been reported by Sachdev et al. [5] for mucins isolated from the canine tracheal pouch, the amino acid composition is nearly identical to that reported by Ellis and Stahl [9] for a canine mucin isolated from tracheal explant organ culture. It was found that the peaks contain 16.5, 13.6, and 8.2% protein, respectively, which represents a considerable loss of protein from the original mucin fraction, peak 1 (see Table I). The lost protein probably consisted of small molecular weight peptides which were trapped in the fibrillar matrix of the native mucins and which were lost during the dialysis step after alkylation, as no peak of high protein content was found. Also, disulfide-linked peptides may also be present in the native mucin.

Table III shows the monosaccharide composition of peaks A--C. There were slight differences in the carbohydrate composition of peaks A and B, princi- pally in the galactose and N-acetylgalactosamine residues. Peak C contained

T A B L E I I

A M I N O A C I D C O M P O S I T I O N O F S E P H A R O S E CL-4B P E A K S O B T A I N E D F O L L O W I N G R E D U C T I V E A L K Y L A T I O N OF P E A K 1

Percent prote in by dry weight o f the tota l sample determined b y the amino acid analysis results, n .d. , no t determined .

m o l / 1 0 0 0 m o l tota l a m i n o acids

Peak A Peak B Peak C

Histidine 24.21 Lysine 56 .32 Arginine 38 .17 Threonine 85 .23 Serine 92 .71 Aspartic acid 104 .63 Glutamic acid 1 0 3 . 1 5 Half-cysteine < 1.0 Methionine n.d. * Tyros ine 25 .19 Phenylalanine 35 .77 ProHne 87 .39 Valine 56 . 04 Iso leucine 35 .16 Leucine 93 .52 Glycine 74.71 Alanine 70 . 60 S - c a r b o x y m e t h y l c y s t e i n e 17 . I 5 Percent prote in 16.5

22.41 23 .71 44 . 36 44 .11 44 .19 32.07 90 .93 102 .11 96 . 83 92 .07

104.67 119.01 104.13 99.75 <I.0 < 1.0

n.d. n.d. 24.69 36.10 42.22 39.41 83.12 61.02 66.47 57.28 43.62 36.18 87.37 87.91 70 .10 82 .08 64 .92 76 .49 14 .92 11 .18 13.6 8.2

3 6 5

T A B L E III

S U G A R C O M P O S I T I O N O F S E P H A R O S E C L - 4 B P E A K S F O L L O W I N G R E D U C T I V E A L K Y L A T I O N O F P E A K 1

S u g a r m o l / 1 0 0 m o l t o t a l monosacchar ides

Peak A P e a k B P e a k C

F u c o s e 3 .3 3 .8 < 0 . 5 X y l o s e < 0 . 5 < 0 . 5 < 0 . 5 M a n n o s e 3 .3 2 .5 11 .8 G a l a c t o s e 33 .1 2 7 . 0 4 9 . 0 N - A c e t y l g l u c o s a m i n e 29 .0 3 0 . 0 2 9 . 8 N - A c e t y l g a l a c t o s a m i n e 21 .3 2 8 . 5 7 .5 Sialic ac id 1 0 . 0 8 .0 1 .8 G l u c u r o n i c ac id < 0 . 5 < 0 . 5 < 0 . 5

considerably more galactose but much less N-acetylgalactosamine and sialic acid than peaks A and B. The mannose content was 50% lower for peaks A and B than for the original mucin fraction, peak 1 (Fig. 2), while the relative content of the other sugars was unchanged. Most of this lost mannose apparently is in peak C, which has a relatively higher amount of the monosaccharide than the original mucin fraction.

Sulfate analysis of peaks A--C revealed that peak 1 consists of 2% sulfate by dry weight, peak B contains 4.2% sulfate, and peak 3 contains no detectable sulfate.

Peak A was considered to be the major mucin fraction. This conclusion was based on its very high molecular weight, its formation of a gel upon rehydration, its relative insolubility, its low mannose content , and its amino acid and carbohydrate composit ion, which is similar to that of other tracheobronchial mucins [5,9,27--30]. Peak B, due to its carbohydrate and protein content and its molecular weight, is thought to consist of smalle~ mucin-type molecules which may or may not be breakdown products from the reductive alkylation of original peak 1 material. Peaks A and B appear to consist partly of sulfo- and sialo- mucins since both of these components are present. Peak C has a low molecular weight, a high mannose content and no sulfate monoester groups; it most likely consists of serum-type glycoproteins.

Sodium dodecyl sulfate agarose/acrylamide electrophoresis of peak A Since peak A (Fig. 3) was assumed to be the major mucin fraction its purity

had to be determined. It was found that peak A migrated as a single band on 0.5% agarose/2% acrylamide gels and stained with both protein and carbohydrate stains (results not shown}. No fast-moving bands, indicating the presence of serum-type glycoproteins, were detectable in the 0.5% agarose/2.0% acrylamide running gel or the 7.0% acrylamide support gel. Based on these data and those above, it was concluded that peak A consists of a homogenous mucous glycoprotein fraction free of serum glycoprotein contamination.

Determination of O-glycosidic linkages of peak A Since the O-glycosidic amino acids-sugar linkage between seryl or threonyl

3 6 6

T A B L E IV

C H A N G E S IN T H E A M I N O A C I D C O M P O S I T I O N OF P E A K A F O L L O W I N G A L K A L I N E B O R O H Y - D R I D E R E D U C T I O N

A m i n o acid Peak A u n t r e a t e d Peak A fo l lowing Loss and gain ( t oo l / 1000 tool) t r e a t m e n t o f a m i n o acids

( m o l / 1 0 0 0 m o l )

Serine 92 . 72 68 .38 - -24 .34 Alanine 70 . 60 9 2 . 5 2 +21 .92 Threon ine 85 .23 64 .12 - -2 1 .1 1 9 ' -Aminobu ty r i c acid < 1 . 0 18 .17 +18 .17

residues and N-acetylgalactosamine is a characteristic of mucins [30], it is im- perative that this be demonstrated in the suspected mucin fraction, peak A. A lyophilized sample was subjected to alkaline borohydride reduction. It can be seen in Table IV that both serine and threonine moieties are lost in the fi-elimination reaction. The loss of each was nearly equal to the gain of alanine and 7-aminobutyric acid, respectively, indicating that fi-elimination and subsequent reduction had taken place. About 26% of the total seryl and threonyl residues are involved. Because identical procedures were used, these data do show that only about half as many hydroxy amino acids are involved in the O-glycosidic linkage in the rat tracheal transplant mucin as in that of a purified mucin obtained from the canine tracheal pouch (reported as 60% [5]), but this amount is only slightly less than the number of residues involved in human tracheal bronchial mucous glycoprotein (reported as 32% [32]).

In conclusion, this report shows that the rat tracheal transplant provides a unique model for s tudy of mucous glycoprotein production in the rat. Normal morphology of the epithelium is maintained, and the accumulated secretions do consist of mucous glycoproteins and serum-type glycoproteins, both of which have been demonstrated by multiple parameters. The drawback of the system is that a long collection period (4--6 weeks) increases the probabili ty of breakdown of secretions by hydrolases released by the lysis of exfoliated cells; however, this can be minimized by use of a shorter collection period, since mucin can be collected after a week or less (unpublished data). The ver- satility of the transplant model system, as described in other reports [11,12, 33--35] , makes this system an ideal model for s tudy of purified tracheal mucins under normal and various pathological and experimental conditions.

Acknowledgments

We thank Katherine B. Stephenson for her excellent technical assistance. We are indebted to Drs. Fred Hartman and Tina Scott for the amino acid analyses and lipid-analysis, respectively. J.N.C. was supported by Training Grant No. CA 05296 from the National Cancer Institute and later by a Young Investigator Grant (HL21369-02) from the National Heart, Lung, and Blood Institute. This work was sponsored jointly by the National Cancer Institute under Interagency Agreement 40-5-63 and the Office of Health and Environmental Research, U.S.

367

Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.

References

1 Wardell, J.R., Jr., Chakrin, L.W. and Payne, A.J. (1970) Am. Rev. Respir. Dis. 101 ,741 - -754 2 Chakrin, L.W., Baker, A., DeSanctis, N., Wardell, J.R., Jr. and Spicer, S.S. (1970) Pharmacologist 12,

264 3 Chakrin, L.W., Baker, A.P., Spicer, S.S., Wardell, J.R., Jr., DeSanctis, N. and Dries, C. (1977) Am.

Rev. Respir. Dis. 105 ,368- -381 4 Chakrin, L.W., Baker, A.P., Christian, P. and Wardell, J.R., Jr. (1973) Am. Rev. ResPir. Dis. 108, 69--

76 5 Sachdev, G.P., Fox, O.F., Wen, G., Sehroeder, T., Elkins, R.C. and Carubelli, R. (1978) Biochim. Bin-

phys. Acta 536, 184--194 6 Clark, J.N. and Marehok, A.C. (1979) Differentiat ion, in the press 7 Marchok, A.C., Cone, M.V. and Nettesheim, P. (1975) Lab. Invest. 33, 451--460 8 Gallagher, J.T. and Kent, P.W. (1975) Biochem. J. 148, 187--196 9 Ellis, D.B. and Stahl, G.H. (1973) Biochem. J. 136, 837--844

10 Stahl, G.H. and Ellis, D.B. (1973) Biochem. J. 136 ,845 - -853 11 Kendrick, J., Nettesheim, P. and Hammons, A.S. (1974) J. Natl. Cancer Inst. 52, 1317--1325 12 Griesemer, R.A., Kendrick, J. and Nettesheim, P. (1974) in Exper imenta l Lung Cancer, Carcinogenesis

and Bioassays (Karbe, E. and Park, J.F., eds.), pp. 539--547, Springer-Veriag, New York 13 Hudson, B.G. and Spiro, R.G. (1972) J. Biol. Chem. 247, 4229---4238 14 Miner, G.D. (1973) Applicat ion Note AN32A, pp. 39--40, Ortec, Inc., Oak Ridge, TN 15 Holden, K.G., Yim, N.C.F., Griggs, L.J. and Weisbach, J.A. (1971) Biochemistry 10, 3105--3109 16 Segrest, J.P. and Jackson, R.L. (1973) Methods Enzymol. 28, 54---63 17 Spiro, R.G. (1966) Methods Enzymol. 8, 4--5 18 Lowry, O.H., Rosebrough, N.S., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 19 Beckman 121 M Amino Acid Analyzer, Applicat ion Note TB0028, Spinco Division, Beckman Instru-

ments , PaiD Alto, CA 20 Albersheim, P., Nevins, D.J., English, P.O. and Karr, A. (1967) Carbohydr. Res. 5, 340--345 21 Hellerqvist, C.G. and Ruden, U. (1972) Eur. J. Biochem. 25, 96--101 22 Warren, L. (1959) J. Biol. Chem. 234, 1971--1975 23 Terho, T.T. and Hartiala, K. (1971) Anal. Biochem. 41 ,47 1 - -476 24 Kissane, J.M. and Robins, E. (1958) J. Biol. Chem. 233, 184--191 25 Snydar, F., Malone, B. and Wyk]e, R.L. (1969) Biochem. Biophys. Res. Commun. 34, 40---47 26 Lieberman, P., Ricks, J., Chakrin, L.W., Wardeli, J.R., Jr. and Patterson, R. (1970) Proc. Soc. Exp.

Biol. Med. 135, 713--716 27 Lamblin, G., Lhermit te , M., Degand, P., Sergeant, Y.H. and Roussel, P. (1973) Biochim. Biophys.

Acta 322 ,372 - -382 28 Roberts, G.P. (1974) Eur. J. Biochem. 50, 265--280 29 Havez, R., Roussel, P., Degand, P. and Biserte, G. (1967) Clin. Chim. Acta 17 ,281- -286 30 Spiro, R.G. (1973) Adv. Protein Chem. 27 ,349- -467 31 McCarthy, C. and Reid, L. (1964) Q.J. Exp. Physiol. 49, 81--84 32 Boat, T.F., Cheng, P.W., Iyer, R.N., Carlson, D.M. and Polony, I. (1976) Arch. Biochem. Biophys.

177, 95--104 33 Griesemer, R.A., Nettesheim, P., Martin, D.H. and Caton, J.E., Jr. (1977) Cancer Res. 37, 1266--1271 34 Nettesheim, P., Gricscmer, R.A., Martin, D.H. and Caton, J.E., Jr. (1977) Cancer Res. 37, 1272--1278 35 Pal, B.C., Topping, D.C., Griesemer, R.A., Nelson, F.R. and Nettesheim, P. (1978) Cancer Res. 38,

1376--1378

characterization of mucin isolated from rat tracheal transplants

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