Structural Analysis of Purified Human Tracheobronchial Mucins R. GUPTA', N. JENTOFT*+,A. M. JAMIESON5, and J. BLACKWELL§

Departments of Pediatrics', Biochemistry*, and Macromolecular Science§, Case Western Reserve University, Cleveland, Ohio 44106

SY NOPSlS

Light scattering has been used to investigate the structure of human tracheobronchial mucin glycoproteins (HTBM) from the sputum of cystic fibrosis patients. The specimen was extracted using 6M guanidinium hydrochloride solution and fractionated by gel exclusion chromatography on Sephacryl S-1OOO. The fractionated HTBM was purified by density gradient ultracentrifugation. Purity of the resulting material was confirmed by SDS polyacrylamide gel electrophoresis and uv spectroscopy. Light scattering measurements on the fractionated mucins yield weight-average molecular weights M , , and z-average radii of gyration Rg,*. The native cystic fibrosis HTBM consisted of a high molecular weight fraction with M , = 9.3 x lo6 daltons and a lower molecular weight fraction containing partly degraded mucins. After reduction and carboxymethylation of the high molecular weight native fraction, the resulting material was separated into three pools with M , values of 5.1 X lofi, 1.6 X lo6, and 400,000.The derived molecular weights for the protein cores M,,,, and the experimental radii of gyration are found to be consistent with the M,, ,- R , relation established previously for submaxillary, cervical, and gastric mucins. These results imply that HTBM has the same extended-coil conformation reported for other mucins and has a molecular structure consisting of subunits, linked into linear chains via covalent (disulfide) bonds.

INTRO DUCT I 0N

Mucous glycoproteins (mucins) are a group of high molecular weight biopolymers responsible for the rheological properties of mucus secretions.'-3 The molecular architecture of mucins consists of a peptide core which is heavily substituted with oligosaccharides that are o-glycosidically linked to serine and threonine residues. Molecular weight estimates for mucins range from ca 2.0 x lo6 to over 40 x 10'. Mucins isolated from different biological loci are found to contain distinctly different side-chain structures. Mucins produced by the submaxillarv gland have on average the shortest side chains ( 1- 5 sugar residues). Cervical mucins have longer side chains (1-9 sugars). Gastrointestinal

and tracheobronchial mucins have the longest side chain lengths (1-20 sugars). Both physical4) and electron microscopy6 investigations have clearly established th a t the native macromolecular structure of mucins is a relatively stiff linear random coil of exceedingly high molecular weight, implying that mucin assembly must proceed via end-to-end attachment of subunit glycoproteins. Static and dynamic light scattering analysis further suggests that the conformational statistics of the polypeptide backbone is very similar for different families of mucins. Thus the radii of gyration of fractionated submaxillary mucins were found' to follow the same universal relationship as cervical mucins, when plotted as a function of Mp,w,the weight-average molecular weight of the protein backbone:

R g ,( A ) 1990 John Wiley & Sons, Inc. CCC C ~ ~ - . ~ 5 2 5 / 9 0 / 0 2 0 3 4 7 - 0 9$04.00 Biopolymers, Vol. 29, 347-355 (1990)

=

0.57M;T (daltons)

(

Likewise, the hydrodynamic radii, computed from sedimentation or translational diffusion coefficients 347

348

GUPTAETAL.

of submaxillary and cervical mucins, were found7 t o follow the relation

R, (A)= 0.27MiT

brosis. Cystic fibrosis (CF) patients produce excessive quantities of unusually viscous mucus secretions, compared to normal individuals. The increased viscosity of bronchial mucus makes it difficult for CF patients to clear airway passages, and the resulting pulmonary insufficiency and frequent pulmonary infections are the leading causes of death. Our studies utilized a purified fraction of high molecular weight HTBM from the void volume on a Sephacryl S-1000 column. Density gradient centrifugation was used to remove protein and DNA contaminantsz2 and lipids.23 These results are compared with those for reduced and alkylated HTBM, which were included on Sephacryl S-1000. Our observations suggest that the molecular architecture of HTBM has a subunit structure similar to that of other mucins.

Comparison of Eqs. (1) and (2) with the equivalent relations for nonglycosylated random protein coils indicates that the persistence length of the mucin than that for chain is 2.5 times larger (25 nonglycosylated chains ( 9.7 I t was also noted7 that literature R, values for porcine gastric mucin and human tracheobronchial mucins were quite consistent with Eq. (2)) suggesting that these mucin families exhibit common conformational characteristics with submaxillary and cervical mucins. These observations indicate that the expansion of the protein backbone of mucins is dependent only on the first few sugars in the oligosaccharide side chains. A recent study' of fractionated ovine subEXPERIMENTAL maxillary mucin, which has only two sugars on its side chain, concluded that two sugars are sufficient Materials to cause maximum extension of the polypeptide Guanidinium hydrochloride (GdnHCl) for preparabackbone. tive work was procured from Aldrich Biochemicals The extremely large molecular weights report(Milwaukee, WI), and was treated with activated ed for mucins suggest that they may be assemcharcoal and ultrafiltered before use. For mucin bled from smaller subunits. In fact, studies on purification and light scattering analysis, we utigastrointestinal," and cervical mulized GdnHCl (Schwartz-Mann Biotech, Cleveland, cins" do suggest that these mucins are made up of OH). Cesium chloride was from Bethesda Research subunits interconnected via disulfide linkages, perLaboratory (Bethesda, MD). Sephacryl S-1000 was haps through "linker proteins." The existence of a obtained from Pharmacia (Uppsala, Sweden). Desimilar subunit structure in tracheobronchial mucins, however, is a matter of c o n t r ~ v e r s y . ' ~ - ~ ~ oxyribonuclease I was from Worthington Biochemical Corp. (New Jersey) and Hyaluronidase was Although considerable direct and indirect evidence from Sigma Chemical Co. (St. Louis, MO). Benzahas been reported suggesting that tracheobronchial midine hydrochloride was from Aldrich. Other mucins also exist as disulfide-linked polymer^,'^. 2o reagents and inhibitors were either from Sigma recent studies by Chace et al.17 suggest that reChemicals or from Fisher Chemicals (Springfield, duced and alkylated tracheobronchial mucins have NJ). Polyacrylamide was purchased from Biorad. the same molecular weight as the parent materials Filters (0.22, 0.45, and 8.0 pm) were from Millipore. when measured by light scattering techniques but possess considerably smaller sizes as measured by gel filtration experiments carried out in the same Sample Preparation solvent. Houdret et al.'l have suggested that the disparity in results achieved by different groups Sputum from three different patients suffering from may be explained by the activation of sulfhydrylCF was collected over a period of two to three dependent proteases during the reduction step, months and immediately frozen. Extraction buffer since large decreases in molecular size were obconsisted of 6M GdnHC1,5 mm Na, EDTA, 0.25M served when reduction and alkylation were carried sodium acetate, pH 7.0. The protease inhibitors out in nondenaturing solvents but not in the presphenylmethyl sulfonyl fluoride (5 m M ) and benzaence of GuHC1. midene hydrochloride (5 m M ) were added to prevent proteolytic attack on the mucin glycoprotein. In this report, we present light scattering meaand M , of fractionated human Iodoacetamide (5 m M ) was added to block the free surements of Rg,= tracheobronchial mucin glycoproteins (HTBM) isosulfhydryl groups and N-ethylmaleimide was added lated from the sputum of patients with cystic fi(5 m M ) to prevent sulfhydryl exchange.24

-

A) A).

S T R U C T U R A L ANA1,YSIS OF H'THM

The sputum samples were pooled and stirred with the extraction buffer overnight, dialyzed against water, and centrifuged to remove insoluble material. The solution was then treated with DNase25and hyaluronidase26 to remove any DNA and proteoglycan contaminants. The pH of the solution was lowered to 5.0 to precipitate extraneous proteins and the solution was passed through CM cellulose to remove additional protein contaminants.

349

Sephacryl S-1000 as shown in Fig. 3. The resulting fractions were pooled as follows (Fig. 3 ) : pool I: 34-40, pool 11: 41-48.

Gel Electrophoresis

Native mucin was subjected to 7.5% SDS polyacrylamide gel electrophoresis (PAGE) using the method of Laemmli.,' Absence of any bands on staining with Coomassie and silver stains indicate the absence of protein contaminants.

Gel Filtration and Density Gradient Ultracentrifugation

After exhaustive dialysis against 5M GdnHCl, approximately 20-30 mL of the solution were loaded onto a (2.5 X 120) cm column of Sephacryl S-lo00 a t 4"C, and the column was eluted with 5M GdnHCl, pH 7.0. Fractions were assayed for carbohydrate by the periodic acid/SchitT assay.27 A-Phage DNA and adenosine monophosphate were used as void volume and total volume markers, respectively, in the calibration of the column. The peak eluting in the total volume had DNA fragments, low molecular weight glycoprotein fragments, as well as protein and other impurities. The peak eluting near the void volume contained HTBM of high molecular weight. The pooled fractions from this peak were concentrated a t 4°C by using an Amicon ultrafiltering unit (YM loo0 membrane), and then purified by density gradient ultracentrifugation. The solution of HTBM was adjusted to 4M GdnHC1,5 m M EDTA, 10 m M phosphate, pH 7.0, and solid CsCl was added to a density of 1.4 g/mL. The resulting solution was centrifuged a t 42,000 rpm for 48 h at 4°C in a Beckman Model L centrifuge, using a Type 60 fixed angle rotor. The resulting gradient was fractionated on a density gradient fractionator Model 185 from ISCO. Fractions were assayed by periodic acid Schiffs assay27 [optical density (OD) 555 nm], and by their absorbance at 220 and 280 nm. The top of the gradient contained protein contaminants and lipids,23 and was discarded. The density of each fraction was determined by weighing 25 pL aliquots. A mucin peak centered at a density of 1.4 g/mL, as shown in Fig. 2, was collected, dialyzed exhaustively against water, and lyophilized. The lack of any material at the bottom of the gradient indicates2" absence of any DNA contaminant (OD 260 nm). The purified high molecular weight HTBM was rechromatographed in 5M GdnHCl on

Reduction and Carboxymethylation

Mucin, corresponding to material eluting in pool I on Sephacryl S-1000 (Fig. 3), a t a concentration of 2 mg/mL, was dissolved in a buffer containing 6M GdnHC1,lO m M phosphate, 5 m M EDTA, pH 7.5. Dithiothreitol (15 mM) was added to the above solution and reduction was carried out under N, at room temperature for 6-8 h. The pH of the solution was then raised to 8.5 and iodoacetamide a t a concentration of 60 m M was added. The solution was stirred in the dark a t 4°C overnight. Finally, the solution was dialyzed exhaustively to remove any salt, concentrated, taken up in loading buffer, and loaded on to an S-lo00 column (2.5 X 120 cm) equilibrated in 5M GdnHCl, 10 m M phosphate, pH 7.0 (Fig. 4). Fractions were assayed by periodic acid Schiffs assay27and the peak was divided into three fractions as indicated by bars; pool A: 40-50; pool B: 51-57; pool C: 58-68.

Analytical Methods

Amino acid analysis of the purified HTBM and of the reduced and carboxymethylated HTBM was performed by the high performance liquid chromatography method of Heinrikson and Meredith29 using a Varian 5000 liquid chromatograph. The results are given in Table I and are similar to literature data for bronchial mucins. Quantitative amino acid analysis showed the presence of 22% protein, determined using a-aminobutyric acid as an internal standard. Carbohydrate analysis was carried out by the method of Jentoft3' and is summarized in Table 11. Again these data are consistent with literature values for HTBM. The absence of proteoglycan contaminants and DNA contaminants in the purified HTBM was confirmed by uronic acid assay3' and OD 260 nm, respectively.

350

GUPTA ET AI,.

Table I Amino Acid Analysis of HTBM Purified by Density Gradient Ultracentrifugation" Amino Acid ~

Residues/1000 Residues

Carboxymethyllated Material

44 74 146

26 43 154 86 13 252 108 42 127 7 40 2 18 43 17 16 6

according to:12

~~

Aspartic acid Glutamic acid Serine Glycine Histidine Threonine Alanine Arginine Proline Tyrosine Valine Methionine Isoleucine Leucine Phen ylalanine Lysine Cysteineh

91

22 226 86 39 99

6 54 5

21 47 21 19 nd"

"Quantitative amino acid analysis showed the presence of

- 22% protein determined by using a-aminobutyric acid as an internal standard. hCysteine was determined as S-Carboxymethyl cysteine. 'nd: not determined.

Table I1 Carbohydrate Analysis" of HTBM Purified by Density Gradient Ultracentrifugation Fucose Galactose GalNAC GlcNAC Sialic acid

0.91 1.88 1.o 1.52 .30

'Expressed as molar ratios reiative to GalNAC.

where (4) ii is the solution refractive index, A, = 6328 A is the wavelength of incident light, NA is Avogadro's number, and

4TTi

sin 8/2

4'-

(5)

A0

where 8 is the scattering angle, M , is the weightaverage molecular weight, R , * is the z-average radius of gyration, and A , is the second osmotic virial coefficient. The refractive index increment a t constant chemical potential was determined by exhaustive dialysis of HTBM solutions against 6M GdnHCl, using a Brice Phoenix Differential Refractometer. We determined ( d E / d ~ ) = , ~0.10 mL/g.

RESULTS Figure 1 shows the gel filtration pattern observed when the crude mucin extract from pooled sputum was chromatographed on Sephacryl S-1000. The relatively low amounts of high molecular weight material eluting near the void volume of the col-

Light Scattering Analysis

Light scattering measurements were performed using instrumentation described e l ~ e w h e r e . HTBM ~.~ solutions of specified concentrations in the range of 0.3-2.5 mg/mL in 6M GdnHC1, 10 m M phosphate, pH 7.0, were prepared by procedures given in detail in prior communications. These solutions were clarified by filtration through 8 pm Millipore filters. The amount of mucin lost was found to be negligible as estimated by periodic acid/Schiff determinationsz7 of the filtered and unfiltered solutions. Dilutions were performed using solvent filtered through 0.22 pm Millipore filters, and the solutions were centrifuged a t 8000 x g for 15 min immediately prior to light scattering analysis. Light scattering intensities a t scattering angles 0 = 30" + 90" were analyzed by Zimm plots

n 0

Fraction Number Figure 1. Chromatography of native HTBM on Sephacryl S-1ooc)in 5M GdnHCl, 10 m M phosphate, pH 7.0. Vertical arrows indicate the void volume y,, and the total volume V,. Horizontal arrow indicates the much material near the void volume, which was separated and subjected to further purification steps.

STRUCTURAL ANALYSIS OF HTBM

351

Table I11 Structural Parameters of HTBM

O

O

V

20

30

40

Native mucin Fraction I Carboxymethylated mucin Fraction A Fraction B Fraction C

9.3

1875

5.1 1.6 0.4

1358 653 -

Fraction Number Figure 2. Mucin peak isolated by density gradient centrifugation in 4M GdnHC1, 5 m M EDTA, 10 m M phosphate, pH 7.0, and CsCl ( p = 1.4 g/mL): (0) solution density, g/mL; (m) OD 550 nm/2.0; (0)OD 260 nm; (a)OD 280 nm; (0)OD 220 nm/lO.

umn presumably reflects degradation occurring in ciw due to proteases of host and bacterial origin,:3:J.14 Th e fractions indicated by the bars in Fig. 1 were pooled and further purified by density gradient centrifugation. The mucin was recovered as a single symmetrical peak in the density gradient run (Fig. 2j and rechromatographed on a Sephacrvl S-1000 column with the majority of the niucin chromatographing a t or near the void volume of the column (Fig. 3). This material was pooled for further analysis. I t was free of contamination by low molecular weight proteins as judged by the absence of bands on SDS PAGE. Proteoglycans and DNA were also absent as judged by uronic acid assays and uv absorbance a t 260 nm. Amino acid (Table I) and carbohydrate (Table 11) analyses were consistent with other studies on tracheobronchial mucins from patients with cystic fibrosis. The highest molecular weight fraction from this step was studied by static light scattering methods; the results of these experiments are summarized in Table 111 and by the Zimm plot shown

in Fig. 4. Its average molecular weight was 9.3 x

lo6.

After cleavage of disulfide bonds in the mucin sample by reduction with dithiothreitol and alkylation with iodoacetamide, it was again chromatographed on Sephacryl S-1000 (Fig. 5). Comparing these results with those from the intact mucin sample (Fig. 3 ) demonstrates that disulfide bond cleavage is accompanied by a considerable decrease in the hydrodynamic size of the mucin. The broad peak from Figure 5 was cut into three pools for static light scattering studies and the results of these experiments are summarized in Fig. 6, and Table 111. The obvious decrease in molecular weight accompanying disulfide bond cleavage suggests that the mucin consists of subunits linked by disulfide bonds. The width of the subunit peak on gel filtration (Fig. 5) and the different molecular weights for subfractions from this peak imply that these subunits are polydisperse with respect to molecular weight. The overall average molecular weight of the subunits can be calculated from the molecular weights of the subfractions (Table 111) and their mass fractions (pool A = 31%, pool B = 50%, and pool C = 19%of the total recovered material). This ~ calculation gives an average value of 2.5 x 1 0 for the molecular weight of the subunits. It is notewor-

Fraction Number Figure 3. Hechromatography of purified native HTBM on Sephacryl S-1000 in 5M GdnHCI, 10 m M phosphate, pH 7.0. Void volume fraction I was pooled and used for light scattering anaiysis.

352

GUPTA ET AL.

Fraction Number Figure 4. Chromatography of reduced and carboxymethylated HTBM on Sephacryl S-lo00 in 5M GdnHCl, pH 7.0. The mucin peak was divided into three pools as shown. and each pool was analyzed by light scattering.

8X

I

I

1.0

sin2e/2

I

1

2.0

30

+

3

1000~

Figure 5. Zimm plot of light scattered by native HTBM, pool I, in 6M GdnHCl, 10 m M phosphate, pH 7.0. Extrapolation to c = 0 and B = 0 gives M , = 9.28 x 106 and Rg, = 1875 A. ~

.. 13 12 I1

10

x

h

$5

9

8 7 6 5 4

3 2

I 1

I

I

I

.2

A

.6

8

I

1

1.0

sin2*/2

I

I

1.2 +

1.4

1.6

I

1.8

1

2.0

1

2.2

IOOOC

Figure 6. Zimm plot of light scattered by carboxymethylated HTBM, pools A and B, in 6M GdnHC1, 10 mM phosphate, pH 7.0. Extrapolation to c = 0 and 0 = 0 gives: pool A, M , = 5.1 X lo", Rg, = 1358 A; pool B, M,,, = 1.6 X lo6, R g , = 653 A.

STRUCTURAL ANALYSIS OF HTHM

353

-1

1

3.5-

/

-

3.0

2.5

5.0

1

5.5

I

6.0

I

6.5

I

7.0

Figure 7. Experimental values of radius of gyration for HTBM are plotted logarithmically against the log molecular weight of the protein core: ( x ) our data; (0) data of Chace et al."; (0)data of Carlstedt and Sheehan.15 Solid line is Eq. (1).

thy that the Zimm plots for both the intact mucin and the mucin subunits, shown in Figs. 4 and 6, demonstrate no sign of concentration dependent self-association since the osmotic second virial coefficients are quite large and positive. The average molecular weight of the subunits can also be calculated from the gel filtration data using a previously determined calibration relation for the behavior of mucins on columns of Sephacryl S-1000.8 Thus, from the elution profile of the carboxymethylated HTBM (V, = 53, Fig. 3) we can compute the distribution coefficient, K,, = ( V , - )/( V, - V,) = 0.42. In earlier studies, we established a relation8 between the hydrodynamic radius R ,, and K ,) for mucins eluting on Sephacryl s-1000,

v,

where A = 1.0 and B = (1/2090) A - I . From Eq. (6), we determine R , = 549.5 A, which leads, via Eq. (2), to MP,, = 513,000. Using the relation Mp, = cM,, where c = 0.22 is the protein fraction of HTBM (Table I), we deduce M , = 2.33 X lo6.This result is clearly numerically consistent with the overall value M , = 2.5 x lo6 computed from the average of the light scattering M , data on pools A-C. Thus, the light scattering and gel filtration experiments are each consistent in indicating that, upon cleavage of disulfide bonds by reduction and alkylation, the average molecular weight of the tracheobronchial mucin fraction used in this study decreased from M , = 9.3 X lo6 to M , = 2.5 X lo6.

The implication follows that the HTBM mucin consists of subunits attached via disulfide linkages.

DI SCUSSIO N This investigation is focused on the properties of the highest molecular weight fraction of human tracheobronchial mucins from CF patients. This fraction was chosen for study primarily because the lower molecular weight fractions almost certainly contain mucin fragments released by the high concentrations of proteases known to be present in the respiratory tract of these individu a l ~ . ~34 ' Not ~~~ surprisingly, the molecular weight of this sample (9.3 x lo6) is greater than values determined for mucins fractionated on Bio Gel A-5M17 simply because Sephacryl S-1000 has a considerably larger pore size. Other studies on the size of HTBM have used materials isolated from patients with chronic bronchitis or asthma. I t is not clear whether the mucins from these individuals have also been exposed to proteases but considerable variation in average molecular weights were observed. The most extensive studies are those of Creeth et al.,'" who used sedimentation velocity and viscosity measurements to determine the average molecular weights of mucins isolated from several individuals. Sizes ranged from 3 x lo6 to 7 x lo6 Da. Carlstedt and Sheehan, on the other hand, report an average molecular weight of 18 x 106for materials isolated in the presence of protease inhibitors and using

354

GUPTA ET AI,.

minimal shear forces3' while the sedimentation velocity studies of Feldhoff et al.37suggest a distribution of molecular weights ranging from 2 x 10' to 10 x 10'. Upon cleavage of disulfide bonds by reduction and alkylation, the molecular weight of the tracheobronchial mucin fraction used in this study decreased from approximately 9.3 x 10' to 2.5 x lo', with the concomitant release of a linker protein of 65,000 daltons as measured by SDS gel electrophoresis (R. K. Gupta and N. Jentoft, unpublished observations, 1988). A linker protein of 65 KDa has been previously described for normal HTBM by Ringler et al.38 The decrease in size of the mucin is readily apparent in both the light scattering studies and the gel filtration experiments. This observation indicates that the mucin consists of subunits that are attached via disulfide linkages, and it is consistent with the general model of mucin structure that has arisen from studies on gastrointestinal," cervical," and submaxillary m u c i n ~ . ~ , I" t is also pertinent to note that there are significant differences in the amino acid composition of the native and the reduced and carboxymethylated HTBM (Table I). In particular, we refer to a decrease in the aspartate and glutamate content. This difference would be consistent with loss of a linker protein containing comparatively high levels of these two amino acids.35 Similar findings have been reported by Creeth et al.," who demonstrated that tracheobronchial mucins isolated from several individuals had molecular weights ranging from 3.3 x 10' to 7 x lo', and that these released subunits of 0.6 X 10' to 2.5 x 10' with the higher molecular weight mucin samples giving rise to the largest subunits. These data are consistent with the subunit molecular weights calculated from both gel filtration (2.3 x lo6) and static light scattering (2.5 x 10') experiments in this study. The gel filtration data presented by Chace et aLI7are also consistent with these findings; materials of considerably smaller size are released upon reduction. Light scattering experiments by Chace et al.,17on the other hand, suggested that there was no decrease in molecular weight or radius of gyration upon reduction, and the difference between the results obtained by light scattering and those from the gel filtration experiments was ascribed to aggregation. Although mucins do aggregate, it is not clear that this can be the explanation for the discrepancy between the gel filtration and light scattering results since both experiments were carried out in the same solvent and at similar mucin concentrations. Under these

conditions, one would expect aggregation to affect the results from both systems in a qualitatively similar fashion. In addition, inspection of the published Zimm plots'7 does not reveal evidence for aggregation over the range of concentrations used in the light scattering experiments. Finally, again using the relation Mp,w = cMw, where c = 0.22, we can compare our experimental R g , values for HTBM with Eq. (1). As seen in Fig. 6, our experimental data for both the intact and the reduced and carboxymethylated HTBM are consistent with Eq. (1). We also plot in Fig. 6 the recent R , . values determined by Chace et al. for HTBM fractions isolated from CF and asthmatic patients, and a result of Carlstedt and Sheehan15 for HTBM from bronchitic patients. The former R , values are a little larger, while the latter is in good agreement with those predicted by Eq. (1). The discrepancy may be due to a higher polydispersity in the HTBM samples used by Chace et al.I7 Our mucin experiments have generally been performed on fractions isolated by preparative gel exclusion chromatography. In summary, the results reported here demonstrate that tracheobronchial mucins from CF patients share the disulfide-linked subunit structure established for other types of mucins. In addition, the data demonstrate that tracheobronchial mucins, with their relatively long oligosaccharide side chains, have the same relationships between hydrodynamic volume, radius of gyration, and the length of the peptide core that was previously established for submaxillary, cervical, and gastric m u c i n ~ . ~ , ~ , This provides further evidence that all mucins possess the same extended random coil conformation, and that this conformation is dependent on steric interactions between the core sugars of the attached oligosaccharides and neighboring amino acid residues. This work was supported by NIH grants DK 33365 and AM 27651, and by grants from the Rainbow Chapter of the Cystic Fibrosis Foundation.

REFERENCES 1. Carlstedt, I., Sheehan, J. K., Corfield, A. P., & Gallagher, J. T. (1985) Essays Bwchem. 20, 40-76. 2. Allen, A. (1983) Trends Biochem. Sci. 8, 169-173. 3. Bell, A. E., Allen, A., Morns, E. R., & Ross-Murphy, S. B. (1984) Znt. J . Biol. Macromol. 6, 309-315. 4. Meyer, F. A. (1983) Biochem. J . (1984) 701-704. 5. Sheehan, J. K., & Carlstedt, I. (1984) Biochem. J . 217,93-101.

STRUCTURAL ANALYSIS OF H T H M

6. Rose, M. C., Voter, W. A., Sage, H., Brown, C. F., & Kaufman, B. (1984) J . Biol. Chem. 259, 3167-3172. 7. Shogren, R. L., Jamieson, A. M., Blackwell, J. & Jentoft. N. (1986) Biopolymers 25, 1505-1517. 8. Shogren, R. L., Jentoft, N., Gerken, T. A., Jamieson, A. M., & Blackwell, J. (1987) Carbohydr. Res. 160, 317- 327. 9. Shogren, R. L., Jamieson, A. M., Blackwell, J. & Jentoft, N. (1984) J . Biol. Chem. 259, 14657-14662. 10. Shogren, R. L., Jamieson, A. M., Blackwell, J., Cheng, P. W., Dearborn, D. G., & Boat, T. F. (1983) Biopolyrners 22, 1657-1675. 11. Pearson,

Structural analysis of purified human tracheobronchial mucins.

Light scattering has been used to investigate the structure of human tracheobronchial mucin glycoproteins (HTBM) from the sputum of cystic fibrosis pa...
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