Glycoproteins and glycosaminoglycans in human pulmonary tuberculosis

255

Clinica Chimica Acta, 57 (1974) 255-261 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

CGA 6731

GLYCOPROTEINS AND GLYCOSAMINOGLYCANS PULMONARY TUBERCULOSIS

TAKAKICHI Department (Received

MAETA,

MASAHIKO

of Biochemistry,

Tohoku

END0

and ZENSAKU

IN HUMAN

YOSIZAWA

Universtiy School of Medicine, Sendai 980 (Japan)

July 11, 1974)

Summary The fractions containing glycoproteins and/or glycosaminoglycans were separated from normal, capsular and caseous tissues of human pulmonary tuberculosis by pronase digestion, followed by fractionation on Dowex 1 column chromatography. The experimental results indicate that all the fractions increased with the degree of pathological alteration of the tissues. Of these fractions, a dramatic increase in 0.3 M Fr (sialoglycopeptide) was observed. It is therefore suggested that sialoglycoprotein may play an important role in pathological change of lung tissue in this disease.

Introduction The presence of complex carbohydrates (glycoproteins and glycosaminoglycans) in lungs has been reported by many investigators [l-8]. However, little is known concerning change of these substances with pathological alteration of lung tissue in human pulmonary tuberculosis. We have been studying glycoproteins and glycosaminoglycans of human lung in order to obtain a clue to their role in pathological alteration of this tissue in pulmonary tuberculosis. This paper reports a comparative study on glycopeptides and glycosaminoglycans isolated from normal, capsular and caseous lung tissues of this disease. Experimental Materials

Lung tissues obtained monary tuberculosis were normal tissue without any caseous tissue; CAS, caseous

by surgical operation from 23 cases of human puldivided into the following three portions: NOR, macroscopic alteration; CAP, capsule surrounding tissue.

256

Chondroitin sulfate A was purchased from Seikagaku Kogyo Co. Ltd, Tokyo. Hyaluronic acid were prepared from human umbilical cord according to the procedure of Danishefsky and Bella [9]. Heparan sulfate was a generous gift from Dr M.B. Mathews, University of Chicago. Pronase P was purchased from Kaken Kagaku Co. Ltd, Tokyo. a-Amylase (Type II-A, 50--100 units/mg) was purchased from Sigma Chemical Co., St Louis. Cellulose acetate membrane, Separax, was purchased from Joko Sangyo Co., Tokyo. Other materials were from commercial sources. Preparation of ethanol-dried tissues The tissues were homogenized in a Waring Blender with the same volume of 99% ethanol, and then heated in a boiling water bath for 10 min. The mixture was cooled in an ice bath and then filtered with suction. The residues were treated again with ethanol by the same procedures as above, and the residues were washed twice with the same volume of ethanol and ether, in succession, and then dried over CaClz and paraffin in vacua. Separation of crude complex carbohydrate fraction by pronase digestion The ethanol-dried tissues were suspended in 20 vols of 0.1 M calcium acetate, and the pH of the suspension was adjusted to 8.0 with 1 M NaOH. To the suspension were added pronase (3 mg per g of the dried tissues) and small amounts of Kanamycin, benzoic acid and toluene. The mixture was incubated at 37” for 3 days. The same amount of pronase was added every 12 h, and the pH of the reaction mixture was maintained at 8.0 with 1 M NaOH during the incubation. The reaction mixture was subsequently dialyzed against running tap water for 1 day at room temperature. The non-di~yzable fraction was concentrated to approximately one-third volume and was subjected to pronase digestion by the same procedure as above. The incubation mixture was then cooled in an ice-bath. To the mixture was added chilled 50% trichloroacetic acid (TCA) to give a final concentration of 7%. After standing at 0” for 30 min, the mixture was centrifuged. The supernatant was dialyzed against running tap water for 1 day, The non-dialyzable fraction was concentrated to a small volume. To the concentrate were added 4 vols of 99% ethanol saturated with NaCl. The resulting precipitates were dissolved in an appropriate volume of water and then reprecipitated with ethanol as above. The precipitates were collected by centrifugation, washed with ethanol and ether, and then dried over CaClz in vacua. The dried powder was dissolved in 20 vols of water and then digested with pronase (30 mg per g of dried powder) as above. The incubation was terminated by the addition of TCA (final concentration, 7%). Subsequently, the supernatant was dialyzed, and the non-dialyzable fraction was concentrated to a small volume, To the concentrate were added 4 vols of 99% ethanol saturatd with NaCl. The precipitates were collected by centrifugation and then dissolved in 20 ml of water. The solution was dialyzed against 5 liters of distilled water for 2 days. The non-dialyzable fraction was then Xyophilized, yielding crude complex carbohydrate fraction.

257 mg/g 0.4

ethanol-dried 0.8

tissues

1.2

1.6

3,2

3,4

W 0.1 0.3 0.5 0 75 1.0 1.25 1.5 1.75

-

NOR

2.0 Fig. 1. Yields of the fractions obtained by Dowex 1 column chromatography of complex carbohydrate fractions. HzO, 0.1. 0.3 . . . 1.75, and 2.0 were Hz0 Fr, 0.1 M Fr. 0.3 M Fr ,.. 1.75 M Fr, and 2.0 M Fr, respectively.

Fractionation of g~y~opeptides and glycosaminog~ycans by Dowex 1 co~~rnn chromatography An aqueous solution of the above crude complex carbohydrate fraction (50 mg in 3 ml) was applied to a column (2 cm X 20 cm) of Dowex 1 X 2, 200-400 mesh, chloride form, which was subsequently washed with 350 ml of water. Stepwise elution was carried out with 300 ml each of NaCl solutions of increasing molarity from 0.1 M to 2.0 M, as shown in Fig. 1, at a flow rate of 50 ml/h. Fractions of 5 ml were collected. The content of hexose in each fraction eluted with H, 0, 0.1 M and 0.3 M NaCl, and that of uranic acid in each fraction eluted with NaCl solutions from 0.5 M to 2.0 M were determined. The fractions containing carbohydrate of each step were pooled and then dialyzed against 5 liters of distilled water for 3 days. The non-dialyzable fraction was concentrated and then lyophilized. The fractions obtained by the elution with HZO, 0.1 M, 0.3 M .., 1.75 M, and 2.0 M NaCl were designated as Hz 0 Fr, 0.1 M Fr, 0.3 M Fr . . . 1.75 M Fr and 2.0 M Fr, respectively. Amylase digestion A portion (1 mg) of a sample was dissolved in 0.1 ml of 0.02 M phosphate buffer (pH 6.0) containing 0.15 M NaCl. To the solution was added the same volume of 2% cll-amylase solution in the same buffer. The mixture was incubated at 37” for 1 h. The incubation was terminated by boiling for 3 min. The TCA (7%)-soluble fraction of the digest was dialyzed against 1 liter of distilled water. The non-di~yzable fraction was then lyophilized, Electrophoresis Electrophoresis was carried out on cellulose acetate membrane cm) in formic acid-pyridine buffer (pH 3.0) [lo], at 1 mA/cm staining with toluidine blue (0.05% in 70% ethanol). Determination of constituents Sugars were identified by paper chromato~aphy

(6 cm X 22 for 30 min,

of the hydrolysates

with

258

1 M HCl at 100” for 2 h or 4 M HCl at 100” for 4 h. The experiments were carried out on Toyo filter paper No. 51 with n-butanol-pyridine-water (6 : 4 : 3, v/v/v) by the descending technique at 16” for 24 h. Alkaline silver reagent was used for staining. Constituents were determined by the following methods: hexosamine by the method of Garde11 [ll] ; uranic acid by the modified carbazole-H, SO4 method [ 121 ; hexose by the phenol-Hz SO, method [ 131; L-fucose by the cysteine-H, SO4 method [ 141 ; sialic acid in the hydrolysate with 0.05 M Hz SO4 at 80” for 1 h by the method of Warren [ 151; protein by the method of Lowry et al. [16], using crystalline bovine albumin as a standard. Results and Discussion Crude complex

carbohydrate

fraction

The yields of crude complex carbohydrate fractions from normal tissues (NOR), capsular tissues (CAP) and caseous tissues (CAS) are shown in Table I. As can be seen in this table, the relative yield (mg/g of ethanol-dried tissues) of crude complex carbohydrate fraction increased with the degree of pathological change. Fractions

obtained

by Dowex

1 column

chromatography

The relative yields (mg/g of ethanol-dried tissues) of the fractions obtained by Dowex 1 column chromatography of crude complex carbohydrate fraction are shown in Fig. 1. The data show that the relative yields of all the fractions, except H2 0 Fr, increased with the degree of pathological change. Of these fractions, the changes of 0.3 M Fr, 0.1 M Fr and 0.5 M Fr were remarkable; especially a dramatic increase of 0.3 M Fr was observed. Characterization

of

the fractions

obtained

by Dowex

1 column

chromato-

graphy

The results of the paper chromatography showed that the corresponding fractions obtained from NOR, CAP, and CAS contained substantially identical sugars. A representative paper chromatogram is shown in Fig. 2. A large amount of glucose was found in Hz 0 Fr. Hz 0 Fr, 0.1 M Fr and 0.3 M Fr contained the constituent sugars of glycoproteins, while 0.5 M Fr2.0 M Fr contained those of glycosaminoglycans. Of the latter fractions, 0.5 M Fr, TABLE

I

YIELDS

OF CRUDE

(CAP),

Wet

AND

tissues

complex

Relative

(CAS)

CARBOHYDRATE

tissues

FRACTIONS

NORMAL

NOR

CAP

307

110

58.5

(9)

carbohydrate

FROM

(NOR),

CAPSULAR

TISSUES

(g)

Ethanol-dried Crude

COMPLEX

CASEOUS

fractions

(mg)

218

16.5 127

CAS

50 8.0 105

yield

(mg/g

ethanol-dried

(ratio

to NOR)

tissues)

3.7

7.7

13.1

1.0

2.1

3.5

259

GalNGlcN Gal Glc Man Fuc

”

\, n

40

”n

1 0.3 ”n \I n

0

dUAGlcUA ;alN GlcN ja, Glc 0.5

0.75

1.0

1.25

1.5

1.75 2.0

Fig. 2. Tracing of paper chromatograms of the fractions separated from normal tissues. Paper chromatography was carried out on Toyo filter paper No. 51 with n-butanol-pyridine-water (6 : 4 : 3. v/v/v) by the descending technique at 16’ for 24 h. Alkaline silver reagent was used for staining. HzO, 0.1.0.3 . .. 1.75. and 2.0 were denoted the same as in Fig. 1. 1, mannose (Man); 2. galactose (Gal); 3, galactosamine (GalN); 4, L-fucose (Fuc); 5, glucose (Glc); 6. glucosamine (GlcN); 7, L-iduronic acid (IdUA); 6, glucuronic acid (GlcUA).

0.75 M Fr, 1.0 M Fr, and 1.25 M Fr contained glucosamine and glucuronic acid. In addition, galactosamine and galactose were found in 0.75 M Fr. On the other hand, 1.5 M Fr, 1.75 M Fr, and 2.0 M Fr contained galactosamine, glucuronic acid and L-iduronic acid. Additional glucosamine was found in 2.0 M Fr. Electrophoretograms on cellulose acetate membrane of 0.5 M Fr2.9 M Fr showed similar profiles between the corresponding fractions from NOR, CAP and CAS, except those of 1.75 M Fr and 2.0 M Fr from CAP. A representative one is shown in Fig. 3. 0.5 M Fr and one component of 0.75 M Fr migrated similar to hyaluronic acid. Another component of 0.75 M Fr migrated between hyaluronic acid and heparan sulfate, which resembled sulfated glycopeptides reported previously (cf. ref. 17). 1.0 M Fr migrated slightly more slowly than heparan sulfate. The mobility of 1.25 M Fr was similar to that of heparan sulfate. The profiles of the two bands of 1.5 M Fr from NOR, CAP and CAS, and of 1.75 M Fr and 2.0 M Fr from NOR and CAS resembled those of chondroitin sulfates A and B. However, 1.75 M Fr and 2.0 M Fr from CAP gave a broad band each migrating more slowly than heparan sulfate, although 2.0 M Fr migrated slightly faster than 1.75 M Fr. A band corresponding to heparin was found in all 2.0 M Fr from the three different tissues. Amylase digested a large portion of Hz 0 Fr and small portions of 0.1 M Fr and 0.3 M Fr, indicating the presence of glycogen in these fractions. Molar ratios of hexose, L-fucose and sialic acid to hexosamine and peptide contents of Hz 0 Fr, 0.1 M Fr and 0.3 M Fr are listed in Table II. Large

260

I

HA

I

HS Chs-E

I

3

I

Chs-P

I

HEP

1 ID

0.5 0 75

I

10

1

1.25 1 5

II

1 75

II II I

20

_

-_LI

2.0

0

40

cm

+

Fig. 3. Tracing of electrophoretograms on ceIIulose acetate membrane of the fractions separated from normal tissues. Electrophoresis was carried out on cellulose acetate membrane (6 cm X 22 cm) in formic acid-pyridine buffer (pH 3.0) at 1 mA/cm for 30 min. staining with toluidine blue. 0.5, 0.75 . . . 1.75, and 2.0 were denoted the same as in Fig. 1. HA, hyaluronic acid; HS, heparan sulfate; Chs-B, chondroitin sulfate B. Chs-A, chondroitin sulfate A; HEP, heparin.

amounts of hexose in Hz 0 Fr, specifically that from NOR, might be due to the presence of glycogen. The presence of sialic acid in 0.1 M Fr and 0.3 M Fr indicate that these fractions contained sialoglycopeptides. The fractions from pathological tissues contained much more sialic acid than those from normal tissues. The above observations indicate that these fractions contained the following substances: Hz 0 Fr, glycogen plus a small amount of neutral glycopeptide; 0.1 M and 0.3 M Fr, sialoglycopeptide plus a small amount of glycogen; 0.5 M Fr, hyaluronic acid; 0.75 M Fr, hyaluronic acid plus sulfated glycopeptide;

TABLE

II

MOLAR RATIOS OF HEXOSE. L-FUCOSE AND CONTENTS OF Hz0 Fr. 0.1 M Fr, AND 0.3 M Fr

SIALIC

NOR

Hexosamine (as glucosamine) Hexose (as glucose) L-Fucose SiaIic acid (as N-acetylneuraminic acid) Peptide (%)

ACID

TO HEXOSAMINE

CAP

AND

PEPTIDE

CAS

H20 Fr

0.1 M 0.3M Fr Fr

Hz0 Fr

0.1 M 0.3 M Fr Fr

Hz0 Fr

0.1 M 0.3 M Fr Fr

1.0 59.3 0.8

1.0 2.0 0.2

1.0 2.3 0.4

1.0 12.6 0.4

1.0 2.6 0.3

1.0 2.7 0.5

1.0 13.7 0.2

1.0 2.2 0.2

1.0 2.1 0.4

_

0.04 8.1

0.09 5.2

-

0.13 6.9

0.23 4.4

_

0.07 5.2

0.19 6.9

5.8

5.6

8.1

261

1.0 M Fr and 1.25 M Fr, heparan sulfate; 1.5 M Fr, chondroitin sulfates B and A and/or C; 1.75 M Fr from NOR and GAS, chondroitin sulfates B and A and/or C; 2.0 M Fr from NOR and CAS, chondroitin sulfates B and A and/or C plus heparin. Chondroitin sulfates eluted with 1.75 M and 2.0 M NaCl might be oversulfated. However, 1.75 M Fr and the major component of 2.0 M Fr from CAP were not characterized because of the small amounts of the samples. The presence of neutral glycoprotein, sialoglycoprotein, hyaluronic acid, chondroitin sulfates A and B, and heparin in lungs of some animals and human being were previously reported [l--6]. Also, the presence of heparan sulfate in lungs ]7,8] and sulfated glycoproteins in respiratory tract (cf. ref. 17) has been reported. As mentioned already, although glycoproteins and glycosaminoglycans of lungs have been studied by many investigators, little is known concerning the change of these substances with pathological alteration in human pulmonary tuberculosis. The present results show that the relative amounts of all the fractions containing glycopeptides and/or glycosaminoglycans increased with the degree of pathoiogi~~ alteration. Of these complex c~bohydrates, sialoglycopeptide (0.3 M Fr) increased dramatically with the degree of pathological change of the tissues. Moreover, the proportions of sialic acid in sialoglycopeptides from the pathological tissues were higher than those from the normal tissues. It is therefore suggested that sialoglycoproteins may play an important role in pathological change of lung tissue in human pulmonary tuberculosis. Acknowledgements We thank Professor Emeritus T. Maki, Department of Surgery, for sending T. M. to the Department of Biochemistry and for his interest throughout this work. Thanks are extended to Dr M.B. Mathews for a gift of heparan sulfate. This work was supported by a grant from the Ministry of Education of Japan. References 1 2 3 4 5 6

H. Masamune. Z. Yosizawa and K. Tokita, Tohoku 3. Exp. Med. 66 (1957) 51-60 K. Tokita. Tohoku J. Exp. Med. 66 (1957) 351-361 C. Itoh, Tohoku J. Exp. Med. 72 (1960) 150-162 C. Itoh. Tohoku J. Exp. Med. 72 (1960) 385-397 J.S. Brimacombe, and J.M. Webber, Mucopolysaccharides, Elsevier, Amsterdam, 1964 J.C. HiIborn and P.A. Anastassiadis, Biochim. Biophys. Acta 215 (1970) 57-69

7 J.A. CifonelIi, in E.A. Balazs (ed.), Chemistry and Molecular Biology of the Intercellular Matrix Academic Press, London, pp. 961-967 8 C.P. Dietrich, H.B. Nader, L.R. Britto and M.E. S&a, Biochim. Biophys. Acta 237 (1971) 430-441 9 I. Danishefsky and A. Bella, Jr, J. Biol. Chem. 241 (1966) 143-146 10 M.B. Mathews, Biochim. Biophys. Acta 48 (1961) 402-403 11 S. Gardell, Acta Chem. Stand.. 7 (1953) 207-215 12 T. Bitter and H.M. Muir, Anal. Biochem. 4 (1962) 330-334 I.3 M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and F. Smith, Anal. Chem., 28 (1956) 350-356 14 2. Dische and L.B. Shettles, J. Biol. Chem., 176 (1948) 595-603 15 L. Warren. J. Biol. Chem., 234 (1959) 1971-1975 16 O.H. Lowry, N.J. Rosebrough, A.L. Farr and RJ. Randall, J. Biol. Chem., 193 (1951) 266276 17 2. Yosizawa, in A. Gottschalk led.), Glycoproteins, Elsevier, Amsterdam, 1972, pp. 1000-1018