Physicochemical characterization of a minor mucin component from cystic fibrosis tracheobronchial secretions

Biol'hinJlca el Riophysim Acta, 1077 (1991) 332-338 1991 ElsevIer Sci"nce Publoshers B.V. 0167-4838/91/$03.50

332

ADONIS 0167483891001706

BBAPRO 33886

Physicochemical characterization of a minor mucin component from cystic fibrosis tracheobronchial secretions Nitin V. Padhye \ Bashoo Naziruddin 1, Vinay C. Desai 1, Santiago Reyes de la Rocha and Goverdhan P. Sachdev 1 I

2

Col/eKe of PharmuC}'. UniversitJ' of Ok/ahoma Health Sciences Center. Oklahoma Ci/.",. OK (US.A.) and ~ Cystic Fihrosis Center, Univenity of Oklahoma Health Sciences Center, Oklahoma City. OK (U.S.A.) (Received 17 August 1990) (Revised manuscript received 21 November 1990)

Key words: Respiratory mucus: Cystic fibrosis; Mucin glycoprotein

A minor mucin glycoprotein component (HTM-2) was purified from the tracheobronchial secretions of two cystic fibrosis patients using a protocol established in our laboratory. The secretions were solubilized in 0.1 M Tris-HCI buffer (pH 7.5) containing 0.22 M potassium thiocyanate and fractionated on a Bio-Gel A-5m column, followed by digestion with DNAase, rechromatography on the same column and chromatography on hydroxyapatite which resolved the major mucin (HTM-l) from the minor mucin component (HTM-2). The mucin component HTM-2 was further purified using Superose 6 chromatography. SDS-composite gel (2% polyacrylamide + 0.5% agarose) and 6% polyacrylamide gel electrophoresis showed that the purified HTM-2 was totally free of low-molecular-weight contaminants. Equilibrium density sedimentation centrifugation of purified HTM-2 usinjt CsCl gradients also showed the absence of proteoglycans and other low-molecular-weigbt proteins. Comparison of carbohydrate and amino acid compositions of the two mucin components indicated that HTM-2 was quite different from the major mucin, HTM-1, reported earlier from our laboratory (Biochemistry, 24, 7334,1985). This suggested that HTM-2 has a different polypeptide core and is perhaps a different gene product. The effects of 6 M guanidine-HCI and different concentrations of NaCi on the molecular size of HTM-2 and its ability to form aggregates was also investigated using the technique of static light scattering. In buffer containing 6 M guanidine-HCI, HTM-2 had a weight-average molecular weight of = 4.5 • 10". However, in the presence of buffer containing 0.03,0.10 or 0.15 M NaCl, the molecular weight of HTM-2 was estimated to be = 11 • 10". These data suggest aggregation of HTM-2 in the presence of a range of NaCl concentrations. In contrast to HTM-1, which is a more anionic glycoprotein, the apparent molecular size of HTM-2 did not decrease at the higher NaCl concentration.

Introduction Tracheobronchial mucus secretions play an important protective role in the normal functioning of airways. In chronic obstructive pulmonary diseases, such as cystic fibrosis (CF), there is hypersecretion of mucus with altered viscoelastic properties. Mucin glycoproteins present in the mucus secretions are responsible. to a large extent, for the viscoelastic properties of the secretions [1,2]. Any changes in the chemical and/or physical properties of the mucins may alter the viscoelastic properties of the mucus secretions which, in turn. may

Correspondence: G.P. Sachdev, College of Pharmacy. University of Oklahoma Health Sciences Center. P.O. Box 269m, Oklahoma City. OK 73190, U.S.A.

influence the clearance of the secretions by ciliated epithelium [3-5]. Thus. isolation and characlerization of the individual mucin components from the mucus secretions are required to understand the relationship between mucin structure and the physical properties of mucus. Our earlier studies have been focussed on biophysical and biochemical characterization of mucins purified from canine and human tracheobronchial secretions [6.7]. Human respiratory mucins isolated from tracheobronchial secretions of individuals with normal lungs and from palients with asthma or cystic fibrosis were found to vary in their amino acid composition, aggregation behavior and hydrophobic hinding properties [8,'l1. Our previously reported studies have been primarily directed toward the biochemistry of the major. high-molecular-weight mucin (HTM-l) isolated from

333 the tracheobronchial secretions of cystic fibrosis and non-cystic fibrosis individuals. However. recently. we have also isolated and purified a minor mucin glycoprotein component (HTM-2) from cystic fibrosis tracheobronchial secretions. The two mucin components (HTM-I and HTM-2) were isolated based on their varying degree of adsorption on hydroxylapatite. The major and the minor components of bovine, ovine and porcine submaxillary mucins have also been purified by hydroxyapatite treatment and characterized [10,11]. The purification of mucin components from the tracheobronchial secretions of cystic fibrosis and noncystic fibrosis individuals using hydroxyapatite chromatography has been reported earlier [12J. Although HTM-I from cystic fibrosis individuals has been very well characterized [7-9]. to date, no information is available regarding the physicochemical properties of HTM-2. The objectives of the present study were, therefore, to isolate and purify minor mucin component. HTM-2. from cystic fibrosis tracheobronchial secretions and to determine its physicochemical properties and compare with those observed for the major mucin, HTM-l. Materials and Methods Collection of respiratory mucus secretions Tracheobronchial mucus secretions (sputum specimens) were collected from two cystic fibrosis patients using a protocol approved by the Institutional Human Investigation Committee. Several specimens from the same patient were pooled for the purification of mucins. Initially the mucus specimens were treated with 0.1 M Tris-HCI buffer (pH 7.5) containing proteinase inhibitors, phenylmethylsUlfonyl fluoride (l mM) and Nethylmaleimide (I mM). Further processing of the mucus secretions has been described earlier [7]. Isolation and purification of HTM-2 The component HTM-2 was isolated and purified by introducing an additional step in the purification protocol described earlier [7J. In brief, the pooled respiratory mucus secretions from a cystic fibrosis patient were solubilized in 0.1 M Tris-HCl buffer (pH 7.5) containing 0.22 M potassium thiocyanate and 0.02% sodium azide. Following solubilization treatment. the suspension was centrifuged at 27000 X g for 4 h. The clear supernatant was applied to a Bio-Gel A-5m column (5 X 90 em) preequilibrated with the sample buffer followed by elution with the same buffer. The excluded fraction containing DNA and mucins was treated with bovine pancreatic DNAaseI (type IV. Sigma. St. Louis, MO) to digest DNA molecules. Subsequently, the mucins were separated from the DNA fragments by rechromatography on the Bio-Gel A-5m column. The excluded fraction, containing the DNA-free mucin

glycoproteins, was further fractionated on a hydroxyapatite column (2.5 X 30 em) equilibrated with 0.01 M potassium phosphate buffer (pH 6.8). The column was initially eluted with the equilibrating buffer and subsequently with a discontinuous gradient of 0.15,0.30 and 0.50 M potassium phosphate buffer (pH 6.8). The major component, HTM-I. did not hind to the column and was eluted with the equilibrating buffer. The minor mucin component. HTM-2 and some low-molecularweight proteins eluted with 0.15 M buffer while other contaminating impurities eluted later with the 0.3 and 0.5 M buffer solutions. Fractions corresponding to HTM-l and HTM-2 were pooled individually. dialyzed extensively and then lyophilized. Partially purified HTM-2 was further chromatographed on a Superose 6 (FPLC, Pharmacia-LKB) analytical column (10/30) equilibrated with 0.1 M Tris-HCI buffer (pH 7.5) containing 0.22 M potassium thiocyanate and 0.02% sodium azide. The column was eluted with the same huffer. The excluded fraction was collected. dialyzed and lyophilized. The included fraction was not further characterized. Cesium chloride density gradient centrifugation The purified mucin (HTM-2) sample (4 mg/l0 ml) was stirred overnight at 4°C in sodium phosphate buffer (16.7 mM) (pH 6.8) containing 4 M guanidine-HCl, 33 mM NaCl, 0.02% sodium azide and 42% (w/w) cesium chloride. The centrifugation was carried out in a T865.1 rotor in Sorvall OTD SOB ultracentrifuge at 42000 rpm for 72 h at 14°C. Subsequently, the gradients were fractionated into 0.5 ml fractions which were analyzed for protein. hexosamine [13) and density. Gel electrophoresis The purity of mucins was examined by sodium dodecyl sulfate (SDS) gel electrophoresis using composite gels (2% polyacrylamide + 0.5% agarose containing 0.1% SDS) and 6% polyacrylamide gels [6.14J. Duplicate gels were run for each sample, one of which was stained with the periodic acid-Schiff (PAS) reagent to stain for carbohydrates and the other with Coomassie brilliant blue [15) or silver stain method (16) for proteins. Analytical methodv The components HTM-l and HTM-2 were analyzed for carbohydrate and amino acid composition. Neutral hexoses were determined by the anthrone method [17), using a mixture of galactose and fucose (1 : 1 w/w) as the standard. Sialic acid was determined by the resorcinol method [18]. using N-acetylneuraminic acid as the standard. Fucose. galactose. N-acetylgalactosamine, and N-acetylglucosamine were determined by gas-liquid chromatography as described previously [6,19J. In addition, after hydrolysis (4 M HCl, 4 h at 100oq, the

334 amino sugars were also analyzed using an amino acid analyzer. Amino acid analyses were performed by the method of Spackman [20] using a Durrum D-500 ammo acid analyzer following hydrolysis of the samples WI th constant boiling HCl for 22 h at 110 0 C in evacuated sealed glass tubes. Sulfate was determined by the sodium rhodizonate method [6,21). Lipids were detected usmg an earlier described procedure [9]. Uronic acid(s) were determined by the carbazole method [22), using glucuronic acid as the standard. Light scattering studies . . Light scattering experiments were earned out usmg a state-of-the-art light scattering spectrophotometer, Photal DLS-700 (Otsuka, Japan), equipped with an He-Ne laser light source (632.8 nm). The DLS-700 instrument is provided with the Zimm plot. Berry plot, Debye plot and one concentration method software for the analyses of static light scattering data. For these experiments, mucin stock solutions were prepared by dissolving component HTM-2 (2 mg/ml) in 0.02 M Tris-HCI buffer (pH 7.4) containing 0.02% sodium azide and either 0.Q3 M NaCI, 0.10 M NaC!, 0.15 M NaCI or 6 M guanidine-He\. All buffer solutions used were filtered through a 0.22 /Lm filter to remove dust. Mucin solutions were centrifuged (5100 x g) for I h to sediment dust particles. The supernate was diluted to give the desired mucin concentrations, which ranged from 0.02 to 0.25 mg/m\. The intensity of light scattered by the solution was measured at various angles ranging from 30 0 to 120 0 • A minimum of four different mucin concentrations were used for each light scattering experiment. The refractive index increments (dn/dc) were obtained using a Waters R40l differential refractometer. Molecular weights of HTM-2 were obtained by extrapolation of the light scattering data to zero angle and to zero concentration, by the method of Zimm [23). Radii of gyration (R G ) of HTM-2 and second virial coefficients were obtained from the Zimm plots.

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Isolation and purification of mucin component HTM-2 The purification of the minor mucin component HTM-2, from cystic fibrosis tracheobronchial secretions, was achieved by modification of an earlier described protocol [7). The separation of HTM-l and HTM-2 was achieved on a hydroxyapatite column which showed different adsorbability for the two mucin components. The elution profile is shown in Fig. 1. Major mucin HTM-l (peak I) was not adsorbed on the column matrix and was hence eluted with the equilibrating buffer. The minor component HTM-2 (peak 11), adsorbed on the column, was eluted with 0.15 M phosphate buffer (pH 6.8). Subsequent elution with 0.3 and 0.5 M phosphate buffer resulted in the isolation of

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Fig. 1. Hydroxyapatite column (2.5 x:\o em) ~hromatography of DNA-free mucin fraction. The column was preequilihratcd with 0.01 M potassium pho'phate huffer lpH fi.K) at a now rale of IS ml/h and 2.5 ml fractions were collected. HTM-l (peak L fractIons 3-10) eluted with the equilihration huffer and HT\1-2 (peak II. fraction' 21-30) eluted with 0.15 M phosphate huffer. The respectiw fractions were pooled. dialy7.ed and lyophilized.

contaminating proteins. Peaks I (HTM -I) and II (HTM-2) represented about 60 and 30% of the total eluted material, respectively. PAGE analyses on 6% gels showed that peak 11 contained high-molecular-weight mucin component and contaminating low-molecularweight protein components. Further purification of the component HTM-2 was achieved on a Superose 6 (FPLC, Pharmacia-LKB) column. In brief, the fraction, peak n. dissolved in 0.1 M Tris-Hel huffer (pH 7.5), containing 0.22 M K S CN and 0.02% sodium azide, 1.0

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Results and Discussion

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Fig. 4. Analytical density gradient centrifugation of HTM-2. 4 mg of HTM·2 was dissolved in 10 ml of 16.7 mM sodium phosphate burrer (pH 6.8). containing 4 M guanidine·HCI. 33 mM NaCl. 0.02% sodium azide and 421 (w/w) cesium chloride by stirring overnight at 4°C and centrifuged for 72 h at 42000 rpm as described in the lext. Subse· quently. the gradients were fractionated (0.5 ml each) and analyzed for protein (e). hexosamines (0 I and density (D).

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Fill- 3. SDS gel electrophoresis of purified HTM-2. Composite gels (21 polyacrylamide + 0.5% agarose) containing 0.1 I SOS were used and stained with PAS reagent. Lanes I and 2 correspond to SO ,.g each of CF-5 and CF-6 mucins. respectively.

was applied onto the Superose 6 column and eluted With the same buffer. The elution profile is shown in Fig. 2. The mucin component HTM-2 eluted close to TABLE I Chemical composilions of purified compOllrnts HTM-J and HTM-l isolated from cyslic fibrosis trachrobronchial srcrrtion

Component

% Dry weight of mucin HTM-I

ProteinFucose b Galactose b N-Acetyl-galactosamine' N-Acetyl-glucosamine ' Sialic acid d Sulfate'

HTM·2

CF-5

CF·6

CF-5

CF-6

15.60

17.25

28.00

26.93

13.10

9.27

7.66

7.46

21.30 5.20 19.40 17.80 7.60

20.73 5.98 18.28 20.21 8.30

25.93

28.50 10.84 14.95

• Calculated from amino acid composition. b A....yed by GLC. : Assayed by amino acid analyzer. Assayed by the resorcinol reaction. • Assayed by the rhodizonate method.

10.96 14.43 8.09 4.93

7.26 4.03

the void volume while the contaminating protein components were included. Two purified cystic fibrosis mucin HTM-2 preparations were examined for purity by SDS-composite gel electrophoresis. The mucin components gave a single broad band when stained with the PAS reagent (Fig. 3). No low molecular components were detected when the gels were stained with Coomassie brilliant blue or by the silver stain method. thus indicating the absence of any low molecular weight proteinj glycoprotein in the purified preparations. furthermore. PAGE analyses on 6% gels showed the absence of low-molecular-weight protein contaminants in the purified preparations. Also. the purified component HTM-2 gave a single peak (close to void volume) when chromatographed on a Superose 6 FPLC column in buffer containing 6 M guanidine-HC!. The absence of other absorbance peak(s) in the elution profile. in strongly dissociative conditions. further supports the purity of the mucin preparations. The purity of component HTM-2 was also investigated by analytical equilibrium density sedimentation centrifugation using cesium chloride gradients. This technique is known to resolve large-molecular-weight molecules like mucins. proteoglycans. proteins and DNA [24]. The results are shown in Fig. 4. A single peak at buoyant density of 1.402 gjml was obtained. These data indicate the absence of any contaminating proteoglycans (buoyant density. .. 1.6 gjml) and also lowmolecular-weight glycoproteinj protein(s) (buoyant density < 1.3 gjml) in the purified HTM-2. Presence of major and minor mucin components has been reported earlier for bovine. ovine and porcine submaxillary mucins [10.11]. In all these cases. the differential adsorption properties on hydroxyapatite

336 were used for separation of the major and minor mucin components. The minor components were not extensively studied by the investigators.

TABLE II

Comparison of chemical compositions of mucin components HTM-J and HTM-2 The chemical compositions of purified cystic fibrosis mucins HTM-l and HTM-2 are shown in Table I. In both the mucins, carbohydrate analyses by gas-liquid chromatography revealed the presence of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. In addition, the purified mucins contained sialic acid and sulfate. The absence of mannose, deoxyribose and uronic acids suggested that the macromolecules were free of serum glycoprotein(s), DNA and proteoglycans, respectively. Mucin components HTM-l and HTM-2 had significantly different chemical compositions. The overall carbohydrate content of HTM-l was higher ('" 74 to 77%) than that of HTM-2 ('" 67 to 69%). In contrast. the protein content of HTM-2 ('" 26 to 28%) was higher than that observed for mucin HTM-l ('" 15 to 17%). Compared to HTM-I, HTM-2 had low amounts of sialic acid and sulfate. Both mucins HTM-l and HTM-2 showed the presence of less than I % non-covalently bound lipids with no detectable amounts of covalently bound lipids. While Mantle and Forstner [25] have reported less than 5% by weight of non-covalently bound lipids in the purified human intestinal mucin, purified bovine gall bladder mucin was found to have trace levels of lipids [26). On the other hand. Slomianv et al. [27), have reported the presence of large amo~nts of noneovalently and covalently bound lipids in the purified gastric mucins, as have Woodward et al. [28] in respiratory mucin. The reason for the presence of very low levels of lipids in the mucins we purified is perhaps due to the different purification protocol employed by us.

Amino at:iJ

Amino acid compositions of purified mucin components HTM·J and HTM-2 Amino acid compositions of HTM-l and HTM-2 are shown in Table II. Comparison of the data revealed significant differences in the amino acid compositions of the two components. For example. the purified mucin HTM-l had a higher combined content of the hydroxy amino acids ('" 43 to 45%) as compared to that observed for mucin component HTM-2 ('" 29 to 31%). Also, the proline content was higher in HTM-l when compared to that observed in mucin component HTM-2. For HTM-I, the five amino acids (i.e., threonine, serine, proline. glycine and alanine) comprised about 71 to 74% of the total amino acid residues. while the content of these five amino acids in the mucin component HTM-2 was about 56 to 59%. In addition, the mucin component HTM-2 contained higher levels of acidic amino acids

and ('ompo.\lflons of purified compOlle'lts JlTAf-J lind HTM-2 iW!Uled from (rSl1C fihrol'ls tracheobronchial secretions

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Residues/l000 residues HTM-l CF·5

Aspartic acid Threonine Serine Glutami<.:
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283 141 47

11K

HTM-2 CF·6

CF-S

CF-6

30 301

65 156 133 90 83 114 77 63

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59

9K

93 40 6 20 41

49

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5

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10 24 12 26

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24 39

19 24 24 42 37

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than that observed for HTM-l. For example. the combined content of aspartate and glutamate in HTM-2 component was '" 15% as compared to '" 6 to 7% observed for HTM-1. Also, HTM-2 had higher levels of lysine than HTM-l. These observed differences in the amino acid compositions of components HTM-l and

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SIN2 (0/2) + 10000 c Fig. 5. Zirnm plot of the light scattering data fm HTM~2 (CF-S) in 0.02 M Tris-HCI buffer (pH 74) containing 6 M guamdine-HC"1. The com:enlration range used was (1.02--0.25 mg/ml and Iht' angles used were. 30-120°. Molecular weight and Radius of gyratinn (R{;) were ?btamed ~rorn extrapolation to zero concentration and 7.Cro angle. K IS the ,optical. l.:onstant. (' is the mucin concentration, and R (0) is the Raylcl~h rallo of the mUl.:in solution [ = (r 2 i / '1))( 1 + cos 20 )]. where I IS Lh~ Jnte~sily or the sl.:attering light at the scattering angle 8. /0 is the mtcnslty of the' incident light, and r is the distance of the pholomulliplicr luhe from the scattering ~olulion.

337

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HTM-2 are consistent with those reported for the major and minor mucin components of bovine and ovine submaxillary mucins [10.11]. Tbe molecular size and concentration dependent aggregation properties of the purified mucin component HTM-2 were investigated using the technique of static light scattering. In addition, as studied earlier for major mucin HTM-l [8], the aggregation behavior of HTM-2 component at different concentrations of NaCI was also investigated. For these studies, scattering intensity was measured for native (non-reduced) purified HTM-2 at various concentrations ( 0.02 to 0.25 mg/ml) in buffer (0.02 M Tris-HCI, pH 7.4) containing 6 M guanidineHC! or containing 0.03, 0.10 and 0.15 M NaCI. The weight-average molecular weights and radii of gyration (RcJ, under above stated experimental conditions, were obtained from the Zimm Plots. These data are shown in TABLE III Welf{ht-averaKc molecular weIghts and rad" of gyra/lon of mucin component HTM-2 l.\'olaled from ()'stle /lbroSJS tracheobronchwl secretIOns·

Concentration

Buffer

range

additions

(X

Mol.Wt 10- 6 )

(nm)

0.02-0.25

6 M guan-HCI

0.02-0.25

0.03 M NaCI

0.02-0.25

0.10 M NaCi

0.02-0.25

0.15 M NaCi

eF-5: CF-6: CF-5: CF-6: CF-5: CF-6: CF-5: CF-6:

158 190 160 148 171 153 169 153

RG

(mg/ml) 4.76 4.25 11.1 10.4 13.4 11.7 12.4 11.4

• Data ohtained from Zimrn plots of light scattenng measurements (for details, see Materials and Methods).

Figs. 5, 6 and Table III. In the presence of 6 M guanidine-HCl, the molecular weight of component HTM-2 was estimated to be "" 4.5 X 10 6 and R G "" 174 nm. In this regard, the minor component HTM-2 showed close similarity to major mucin HTM-l which had a molecular weight of '" 5.0 x 10" under identical experimental conditions [8]. In the presence of buffer containing 0.03, 0.10 or 0.15 M NaC!, considerably higher molecular weights were observed. The observed increase in molecular weight of HTM-2 in buffer containing different NaCI concentrations as compared to buffer containing 6 M guanidine-HC!, is perhaps due to the aggregalion of mucin monomers under these experimental conditions. Interestingly, the R G values did not differ under various additions to the buffer. Recently, we investigated the effects of different NaCI concentrations on the molecular aggregation of mucin component HTM-l isolated from cystic fibrosis tracheobronchial secretions [8). In the presence of 0.03 and 0.1 M NaCl, HTM-l formed large aggregates but little aggregation was observed in the presence of 0.15 M NaCI. However, unlike HTM-l. HTM-2 had similar molecular weights in the presence of different NaC! concentrations (see above). Since major mucin HTM-l has higher sialic acid and sulfate content than the minor mucin component HTM-2, HTM-l is more anionic than the component HTM-2. The differences observed in the aggregation behavior of components HTM- 1 and HTM-2 in the presence of different NaC! concentrations, are perhaps due to relative charge shielding effects with increasing NaCI concentration. The observed differences in the amino acid compositions of HTM-l and HTM-2 suggest structural differences in the polypeptide cores of these two mucin components. It is possible that the two components are products of two different genes. Mucin macromolecules are secreted primarily by the goblet and submucosal gland cells in the tracheobronchial tree and the possibility remains that these cell types may synthesize and secrete mucins witb distinct polypeptide backbones. Each polypeptide chain may act as a substrate for glycosyl transferases during post-translational modi fication(s). Also. two immunochemically distinct mucins have been reported to be present in human colonic mucin [29]. In addition, Podolsky et al. [30] have reported the presence of six distinct mucin species in the purified normal colonic mucins that appear to differ in their amino acid compositions. In conclusion, in this report we have described the isolation, purification and physicochemical characterization of a new minor mucin component from cystic fibrosis tracheobronchial secretions. To the best of our knowledge, this is the first such report describing the characterization of the component HTM-2. Initial studies have indicated that tracheobronchial secretions of normal individuals and asthmatic patients also contain

338

the minor component HTM-2. However, it remains to be seen if there are differences in the composition of the mucin component HTM-2 isolated from the secretions of cystic fibrosis and non-cystic fibrosis individuals. Acknowledgement This work was supported in part, by a grant from the National Heart, Lung and Blood Institute (HL34012). References 1 Yeager. H. (1971) Am. J. Med. 50. 493-509. 2 Lilt, M., Khan, M.A., Chakrin. L.W., Wardell. J.R. and Christian. P. (1974) Biorheology 11, 111-117. 3 Chen, T.M. and Dulfano. MJ. (1978) J. Lab. Clin. Med 91. 423-431. 4 Gelman, R.A. and Meyer, F.A. (1979) Am. Rev. Respir. Dis. 120. 553-557. 5 Giordano, A.M., Holsclaw, D. and Lin, M. (1978) Am. Rev. Respir. Dis. 118, 245-250. 6 Sachdev, G.P., Fox, O.F.. Wen, G., Schroeder, T .. Elkins, R.C. and Carubelli, R. (1978) Biochim. Biophys. Acta. 536. 184-196. 7 Chace, K.V., Flux, M. and Saehdev, G.P. (1985) Biochemistry 24. 7334-7341. . 8 Chace, K.V .. ~aziruddin, B., Desai, V.c., Flux. M. and Saehdev. G.P. (1989) Exp. Lung Res. 15,721-737. 9 Shankar. V.• Naziruddin, B.• Santiago Reyes de 13 Rlx;ha and Saehdev, G.P. (1990) Biochemistry 29, 5856-5864. 10 Tellamanti, G. and Pigman, W. (1968) Arch. Bioehem. Biophys. 124,41-50. 11 Salegui, M.D. and Plonska, H. (1969) Arch. Biochem. Biophys. 129,49-56.

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