Similarity in the bound carbohydrate groups of glycoproteins from cells of several vertebrate classes

Biochimica et Biophysica Acta, 719 (1982) 190-198

190

Elsevier Biomedical Press BBA 21256

SIMILARITY IN T H E BOUND CARBOHYDRATE G R O U P S OF G L Y C O P R O T E I N S FROM CELLS O F SEVERAL VERTEBRATE CLASSES D I A N A L. BLITHE *, H. FRED CLARK and LEONARD WARREN **

The Wistar Institute 36th Street at Spruce, Philadelphia, PA 19104 (U.S.A.) (Received April 7th, 1982)

Key words: Glycoprotein; Glycopeptide; (Species comparison)

The carbohydrate groups of the glycoproteins of human, hamster, chick, reptile and fish cells growing in culture have been fractionated in succession according to size (Sephadex G-50), affinity for concanavalin A, charge (DEAE-Sephadex) and by thin-layer chromatography. It was found that despite the complexity of the array of separable glycopeptides in each type of cell, most of these structures seemed to be common to all of the cells. This suggests that they have existed in a relatively stable state for several hundreds of millions of years throughout the evolution of the vertebrates.

Introduction

Materials and Methods

Our laboratory has been involved in characterizing and comparing the glycopeptides derived by exhaustive proteolytic digestion of glycoproteins from the surface and internal membranes of control and transformed cells [1,2]. By combining techniques which fractionate glycopeptides on the basis of size, lectin affinity and charge, followed by thin-layer chromatography it has been found that an extensive array of protein-bound carbohydrate groups is formed by the cell [3,4]. Although the patterns of glycopeptides are complex, they are reproducible [4]. In this study we provide evidence that despite quantitative and some qualitative differences, the complex patterns of glycopeptides from cells of vertebrates in culture bear a striking resemblance to one another.

As previously described [1,4] BHK21/C13 (hamster), W138 (human) and fathead minnow cells [27] in log phase of growth were cultured for 48 h in plastic T150 or roller flasks containing 50 ml medium in the presence of 25 #Ci D-[14C]glu cosamine or 50/aCi D-[ 3H]glucosamine. Cells were harvested by removing the medium, washing the cells three times with phosphate-buffered saline, and scraping them from the plastic surface with a rubber policeman. The cell pellet was suspended in Tris-HC1 buffer, pH 8.2, containing 1 mM CaC12, heated for 3 min at 100°C and exhaustively digested with pronase for 5 days at 37°C in the presence of toluene. The digest was applied to a column of Sephadex G-50 (fine) (3 × 120 cm) and developed with 0.01 M N H 4 acetate in 20% ethanol. The contents of appropriate tubes were pooled to form fractions A, B and C (Fig. 1, top). These were lyophilized. As previously described [4], each fraction was applied to a column of con A-Sepharose. Some glycopeptides did not adhere to the column and passed through to form concanavalin A ( - ) fractions. Those that adhered were eluted with

* Present address: The Institute for Child Health and Human Development, National Institute of Health, Bethesda, MD 20205, U.S.A. ** To whom correspondence should be addressed. 0304-4165/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

191

a-methyl-D-mannoside (40 mg/ml) to form concanavalin A ( + ) fractions. Concanavalin A ( + ) and concanavalin A ( - ) fractions were always checked by rechromatogrpahy on columns of Con A-Sepharose. Fractions were dialyzed and subjected to ion-exchange chromatography on DEAE-cellulose as described previously [4] and in the legend to Fig. 2. For thin layer chromatography, sheets of silica gel-60 (Precoated TLC plastic sheets, E. Merck, Darmstadt) were heated for 1 h at 100 ° and washed with 20% ethanol for 15h by ascending chromatography. The sheets were dried at room temperature. Radioactive (3000 cpm) and standard (25 /ag) glycopeptides were chromatographed in ascending fashion for 8 h in propanol/acetic acid/water (3 : 3 : 4) [8]. The sheets were dried and then subjected to a further 7 h of chromatography. Solvent reaching the upper edge of the plastic sheet was adsorbed by Whatman's 3MM paper. Vertical bands 1 cm in width containing the glycopeptides were cut into 0.5 cm pieces and each piece was assayed for radioactivity in 4 ml New England Nuclear Formula 963 scintillation fluid. Ovalbumin glycopeptides 1 and 4 [9], used as standard were located with an orcinol/sulfuric acid spray [8]. All chemicals were of reagent grade. Pronase (grade B) was purchased from Calbiochem, La Jolla, CA. Sephadex G-50 (fine), Con. A-Sepharose and DEAE-Sepharose were purchased from Pharmacia, Inc., Piscataway, NJ. Radioisotopes (250 m C i / m m o l D-[U- 14C]glucosamine hydrochloride; 20 C i / m m o l D-[6-N-3H]glucosamine hydrochloride were purchased from New England Nuclear, Boston, MA. Results

Cells in log phase of growth were cultured for 48 h in the presence of radioactive D-glucosamine. Since D-glucosamine is converted metabolically into N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and sialic acids, incorporation of radioactivity into essentially all protein-bound carbohydrate groups is effected. In Fig. 1 can be seen typical elution curves of material derived from cells in culture. They represents all of the protein-bound carbohydrate material of the cell. The initial peak (tubes 1-10)

1400

w z

1200-

i: ELUAIr E !

PRE A

A

I

B

I

C

I

D

I

I000II

800-

."i

I

/

"A

600-

4000 o

i~

/

iX

200-

.....~,

/

•....-...:~..~.....

IO

.

.... ~'

...,~.

20

30 FRACTION

"

..... "

40

.

50

.

.

.

....

60

NUMBER

Fig. 1. Sephadex G-50 chromatography. Elution patterns of radioactive glycopeptides derived from ( ) human (W138), ( - - - - - - ) hamster ( B H K 2 1 / C I 3 ) and ( . . . . . . ) fathead minnow cells growing in culture. The results of three separate runs are shown. Only fractions A, B and C were processed further.

is quite variable in size and consists largely of glycosaminoglycan [5]. Group D glycopeptides tubes (48-60) (mono to trisaccharides), highly variable in amount, were not examined further. Radioactive glycopeptide material in tubes 18-48 was divided into 3 parts; group A (M r 4200-5500), group B ( M 3000-4200) and group C (M r 15003000). 45-88% of the total radioactivity incorporated was found in the A, B, C fractions. Apparent molecular weights were determined primarily by analysis of materials and by chromatograTABLE I P E R C E N T O F T O T A L R A D I O A C T I V I T Y IN V A R I O U S FRACTIONS Fractions of z4C- or 3H-labeled glycopeptides obtained from a column of Sephadex G-50 were applied to a column of Con A-Sepharose (1 X 10 cm). Adherent ( + ) and non-adherent ( - ) fractions were obtained [4]. Radioactivity in the group D area (tubes 48-60) in Fig. 1 is not included in the calculations because of its great, almost random variability. Fraction

Eluate Pre A (A) concanavalin A (A) concanavalin A (B) concanavalin A (B) concanavalin A (C) concanavalin A (C) concanavalin A

(--) (+) (--) (+) (- ) (+)

Human

Hamster

Fish

(%)

(%)

(%)

35.7 15.8 11.1 0.5 13.4 5.5 10.6 7.4 100.0

30.3 7.5 16.5 0.3 15.2 10.9 9.2 10.1 100.0

46.1 10.4 7.1 0.6 4.7 5.6 16.0 9.5 100.0

192

Group A Con A ( - )

12

3

4

567

25O

250

2O0

200

150

150

100

100

50

50 /

0

0 Group B Con A 1+)

Group B Con A ( - ) I

2

3

',

254

4

II

I

5

6

I

I

7 I

I

E n o A

181

I I I

A:'AI

I 2 3 i ~ i

4 I

I

383

256

166

287

192

124

"1"1

I

127

191

95

41

E "1-

o

0 Group C Con A ( - ) I

150

I

2 I

3 I

I

4 I

113

I

5 t

.

J

6 I

I I

200

I 387-

150

280

75

.

.

.

.

0

.

Group C Con A (+1 2 I

3 I

4 t

I 374

280

194-

I

187

/Jl 38

0

83

97

0 80 100 120 140 160 180 ~

0

20

40

60

80 100 120 140 160 180

0

20

40

60

,

,

i

t

Fraction number Fig. 2. DEAE-Sephadex chromatography of glycopeptides. Glycopeptides from hamster cells metabolically labeled with D-[ 3 H]glucosamine that had been fractionated on columns of Sephadex G-50 and Con A Sepharose were mixed and homologous tac-labeled material from fish cells (fathead minnow, Pimephales promelas) [27]. Radioactive glycopeptides were applied to a column of DEAE-Sephadex [4]. The glycopeptides were eluted first with a linear gradient from 0 to 0.1 M sodium borate in 0.01 M pyridine acetate pH 4.5 (tubes 0-110). then with a second linear salt gradient from 0 to 0.1 sodium acetate in 0.1 M sodium borate and 0.01 M pyridine acetate pH 4.5: ( ) 14C-labeled glycopeptides from fish cells; ( - - - - - - ) 3H-labeled glycopeptides from hamster cells. Both fish and hamster cells were grown in Eagle's minimal essential medium containing 7.5% fetal calf serum. Fish cells were grown at 25°C, hamster cells at 39°C.

193 fraction which passed through the column and a concanavalin A ( + ) fraction which consisted of material that adhered to the column and could be eluted with ct-methyl-D-mannoside (40 m g / m l ) . The distribution of radioactivity in the various fractions is seen in Table I. It is clear that increase in size of glycopeptides is accompanied by a decreased ability to adhere to concanavalin A. This suggests that in smaller glycopeptides, the mannose residues with an affinity for concanavalin A are more exposed; as glycopeptides are enlarged the mannose residues are

phy of known standards on columns of Sephadex G-50 [4]. Glycopeptides derived from human, mouse, chick, reptile and fish cells in tissue culture closely resembled those of hamster in their behavior on columns of Sephadex G-50 (Fig. 1). Although fractions A, B and C were poorly resolved, upon rechromatography on columns of Sephadex G-50 they eluted at the same point as in the first elution [4]. Glycopeptides in groups A, B and C were then each fractionated into two parts on columns of Con. A-Sepharose [4,5], a concanavalin A ( - )

r

170

^

1281

Group A Con ~

I \I!I

8Sl-

/.

431

~ ~

}.,' ;I

I

4277

f I

4184

L /', \I;

E~

",/

s e r u m ,

-192

25C

Group B Con A

CL O

Fig. 3. DEAE-Sephadexchromatographyof glycopeptides.Procedure was essentially the same as that described in the legend of Fig. 2. Glycopeptides from BHK21/CI3 (hamster) cells labeled with 3H ( - - - - - - ) were compared with []4C]glycopeptides from VSW cells (Russel viper) ( ) grown at 3 0 ° in Eagle's-minimalessential medium containing 10% fetal calf

389

Group B Con A (+)

288

<

A

222 .~.

188

"1

I I 103

-

A

q

216

125

148

q)

E m "1"

I

I

I,-

'IA

oll

2

, ,,

o ~

orou,.ccon.,,-,"''" ,', ~

/I

45

30

iI ti I~

A

/ I

i I

~.,~.s .... 40 60

112

,,/ ', i

20

o ~

1

~,j~

is

0

r.,.,

I

~,

,5

37

3e ,/~

Fraction

3

o 262

I II

7s

';' / .... ~ 0 80 100 120 140 160 180

.

Group C Con A 1+1 350

iiii

113

',

,"

0

t/

0

20

number

q / ~ 40 60

87

80

0 100 120 140 160 180

194

171

181 135

Fig. 4. DEAE-Sephadex chromatography of glycopeptides. Procedure was essentially the same as that described in the legend of Fig. 2. Glycopeptides from BHK21/CI3 (hamster) cells labeled with 3H ( - - - - - - ) were compared with [14C]glyco-

90

grown in Eagle's minimal essential medium containing 7.5% fetal calf serum at 37°C.

Group A Con A (--) 128

A

86

43

'

0 v~,~. _ 175li P" |i ~

,

/AIt

/[IJ/

"

-

-0-7 20

GroupBConA(--)

i',

-,3,. ,~ I /JIr/ ~ ,

45

,, :',

/It

i i

't

200F--

/

]314 GroupBConA(+)/

{!

1~

150~ /

I~, I',

103

100

I~',

=

=

]235 3 / =

Ill', ~,

:=

51

, ,

0,,,,' ,-,, ..........

75~tI ~

-~- . . . .

/', /~

/

M~;~,-xr---T~,,,

0

20

40

60

/ \ Vi ~~ ,," ,,"

50

050

o~

125

2231

t

tf II

I ~,

78 ..~

o

331

,

,'"':'-

62

?-', , .( , ;~._~'~....~__, 80 100 120 140 160 180

112

//

0x_~

20

40

60

80

100 120

--

165

0 140 150 180

Fraction number more highly substituted [6,7]. Five fractions were then fractionated further on columns of DEAE-cellulose; group A ( - ) , group B ( + ) , group B (--), group C ( + ) and group C ( - ) . There was insufficient material in the group A ( + ) fraction for further analysis by ion-exchange c h r o m a t o g r a p h y (Table I). Radioactive glycopeptides from human, reptile, and fish cells in culture were co-chromatographed with glycopeptides from hamster on columns of DEAE-Sephadex. It can be seen in Figs. 2, 3 and 4 that the elution patterns are quite complex and that while

quantitative and some qualitative differences do exist in this internally controlled analysis, the great majority of peaks from the various species co-elute. Materials in these peaks were fractionated further by thin-layer c h r o m a t o g r a p h y on silica gel 60 [8]. The mobility of radioactive glycopeptides was c o m p a r e d to k n o w n glycopeptides derived from ovalbumin [9] and detected by orcinol spray [8]. Table II reveals that, to date 5 0 - 6 0 spots or peaks have been detected (presumably carbohydrate groups) arising from a single cell species. This does not include material in the eluate, pre A, group A

195

TABLE II T H I N LAYER C H R O M A T O G R A P H Y OF GLYCOPEPTIDES Values in the table were calculated by dividing the number of cm traveled from the origin by the unknown, by the distance traveled in cm by ovalbumin glycopeptide 4. Dashes indicate that radioactive glycopeptides were not found in areas where they were present in preparations from other species. Blank areas signify that glycopeptide fractions were not assayed. Numbering of fractions is based upon the pattern of elution of peaks seen in Fig. 2. Secondary chick mebryo fibroblasts were grown according to Vogt [27] C57/B1 mice were made radioactive and their glycopeptides isolated from kidney as previously described [28]. Glycopeptide fraction A (-)

Fish

1 2

3

4

0.82 0.97 0.87 1.06 0.44 0.82 0.89 0.75 -

5 6 7

13(-)

0.90 1.10

1

Viper

Chick

Mouse (kidney) 0.79 -

0.65 0.80 0.91 0.74 0.91 0.88 0.93 1.09

0.74 0.88 0.90 0.90 0.41 0.82 0.99 1.10 0.86 0.97

-

0.59 0.85

0.56 0.86

-

1.00

0.81 0.80 0.92

1.08

0.87

0.87

0.86

1.17 0.82

0.86

1.00

1.03

1.00

0.55 0.85

1.06

1.06

C(-)

0.44 0.85 0.44 0.82 0.77 0.88 0.73 0.90 1.130

Human

0.42 0.87 0.98 1.13 0.41 0.89 0.97

-

0.86 0.97

a(+)

Hamster

0.57 0.86

0.41 0.82 0.99 1.10 0.55 0.91

1.00

1.04

0.73 0.96 1.10 0.80 0.99 1.14 0.79

0.60 0.90

0.42 0.87 0.98 1.13 0.57 0.90

0.42 1.00 0.57 0.92

1.08

1.05

1.03

0.87

0.86

1.02

1.00

0.91 ! .02

0.92 1.13

0.95 1.06

0.79 0.98 1.13 0.65

0.99

196 TABLE II (continued) Glycopeptide fraction

Fish

0.93 1.17 1.02 1.14 1.10

4

C (+)

Viper

1 1.11

1.00 1.11 -

2

1.01 -

3

0.98 1.13 0.92 1.05

( + ) or group D populations. Of all the comparable spots from fractions of the various species, the great majority had mobilities that would suggest that they were the same (Table II). While correlation in size, affinity for concanavalin A, charge and mobility on sheets of silica gel 60, in sequence, do not establish identity of glycopeptides it strongly suggests that they are in fact very similar. Discussion

Our results suggest that the cell is capable of synthesizing a very complex array of carbohydrate groups attached to polypeptides and that most members of this array are common to cells in culture from fish, reptiles, bird, rodent and human. We have identified approx. 60 carbohydrate groups. It is probable that some, but not all, of these groups are homogeneous. Further fractionation by high performance liquid chromatography is now being explored. Identity of groups by actual chemical and structural analysis has not been carried out to date although the sugar components of several families of glycopeptides of the hamster have been determined [3,4,10]. In all there are probably at least 100 groups, the bulk of which appear to be linked to the amide-N of asparagine [4]. The cell also makes a large number of glycolipids so that the total number of carbohydrate groups is indeed impressive. It should be noted further that mouse liver and kidney tissues from the intact animal yield glyco-

Chick

1.06 1.16

Mouse (kidney)

Hamster

Human

0.85 1.06 1.03 1.11

1.07 1.21 1.12

1.05 1.18 1.01 1.15 0.93

0.92 1.11

1.05

peptides resembling those from primary chick fibroblasts and from estasblished lines of hamster and mouse (3T3) cells in culture (Table II; unpublished data). Further, our results suggest that the glycopeptides arising from the surface components of cells, released by treating intact cells with trypsin are quite similar to those derived from the cell pellet i.e. from the more inaccessible, interior part of the cell (data not shown). It may be that of all the theoretically possible protein-bound groups consisting of one to 30 sugar residues (A, B, C, D groups), perhaps 100 or more structures are actually formed in sialic acid-containing vertebrates [11] that we have examined. These groups would appear to constitute an alphabet whose letters are used repeatedly in various glycoproteins. They appear to have arisen with the sialic acid-containing vertebrates and have stabilized perhaps four to five hundred million years ago, suggesting a clear, selective advantage. Synthesis of these groups as lipid-linked saccharides and transfer to polypeptides is even more ancient since this pathway appears to be present in algae [12], plants [13,14], yeast [15] and insects [16]. However, sialic acids are not found generally in these groups [11] and glycopeptides derived from them should fractionate differently from those of vertebrates since fractionation depends to a large extent on the properties of sialic acids [3,4,10]. On the other hand, preliminary studies with glycopeptides from the sea urchin (Arbacea punctulata) indicate that despite the presence of

197 sialic acid [11], they are quite different from those of vertebrates. Hughes and Butters [18], in their discussion of glycosylation patterns as evolutionary markers distinguish between core glycosylation mediated b y a lipid-linked intermediate, and later processing and elongation of carbohydrate chains. While the former is c o m m o n to all eukaryotic organisms, later processing has undergone various changes during evolution related to function and survival. Results of the present study suggest that the second process took place early in the evolution of vertebrates and because of the remarkable stability of the complex array of products, must be highly advantageous. Since m a n y [19,20] if not most, glycoproteins display, a heterogeneity of their carbohydrate groups this would suggest that the various potential sites of glycosylation of a polypeptide chain, (O- or N-linked) m a y bear one of several groups or perhaps no carbohydrate group. However it is evident that various glycoprotein differ from one another in their carbohydrate groups in a characteristic m a n n e r so that there is a clear, but not absolute, preference for some of the 100 or so groups over others being present at specific sites on specific polypeptides. Recently it has been shown that the oligosaccharides of a-1-acid glycoprotein are different in rats and h u m a n s [21]. Finally, it should be noted that the array of protein-bound groups can be changed to a suprising extent b y changing the environment of the cell at the time of synthesis of the glycoprotein (see Refs. 2 2 - 2 4 for reviews). Shifts in the array of groups m a y follow from the fact that the biosynthesis of protein (and lipid) -bound carbohydrate groups is not constrained by a template but m a y depend on specificities of glycosyl transferases and glycosidases [25,26] and on several environmentally determined characteristics (availability of activated sugars, pH, availability of cations etc.). Perturbations in environment m a y lead to the preferred synthesis of some polypeptideb o u n d carbohydrate groups (letters of the alphabet) over others. The consequences of such structural changes in glycoproteins is u n k n o w n but is p r o b a b l y significant in the evolutionary process and perhaps in pathology.

Acknowledgements This work was presented to the G r a d u a t e Faculty of the University of Pennsylvania by D.L.B. in partial fulfillment of the requirements for the Degree of D o c t o r of Philosophy. This work was supported by grants from the U.S.P.H.S. CA 19130, C A 10815, Training G r a n t C A 09171 and A C S G r a n t BC 275. The expert technical assistance of E. Gilbert, C. Walz and R. Espiritu is gratefully acknowledged.

References 1 Buck, C.A., Glick, M.C. and Warren, L. (1970) Biochemistry 9, 4567-4576 2 Warren, L., Buck, C.A. and Tuszynski, G.P. (1978) Biochim. Biophys. Acta 516, 97-12 3 Glick, M.C. (1979) Biochemistry 18, 2525-2532 4 Blithe, D.L., Buck, C.A. and Warren, L. (1980) Biochemistry 19, 3386-3395 5 Baker, S.R., Blithe, D.L., Buck, C.A. and Warren, L. (1980) J. Biol. Chem. 255, 8719-8728 60gata, S., Muramatsu, T. and Kobata, A. (1976) Nature 259, 580-582 7 Muramatsu, T., Koide, N., Ceccarini, C. and Atkinson, P.H. (1976) J. Biol. Chem. 251, 4573-4679 8 Holmes, E.W. and O'Brien, J.S. (1979) Anal. Biochem. 93, 167(bl 170 9 Huang, C.-C., Mayer, H.E. Jr. and Montgomery, R. (1970) Carbohydrate Res. 13, 127-137 10 Santer, U.V. and Glick, M.C. (1979) Biochemistry 18, 2533-2540 11 Warren, L. (1963) Comp. Biochem. Physiol. 10, 153-171 12 Bause, E. and Jaenicke, L. (1979) FEBS Lett. 106, 321-324 13 Lis, H. and Sharon, N. (1978) J. Biol. Chem. 253, 3468-3476 14 Yoshima, H., Furthmayr, H. and Kobata, A. (1980) J. Biol. Chem. 255, 9713-9718 15 Parodi, A.J. (1979) J. Biol. Chem. 254, 10051-10060 16 Quesada-Aune, L.A. and Belocopitow, S. (1978) Eur. J. Biochem. 88, 529-541 17 Butters, T.D. and Hughes, R.C. (1980) Biochim. Biophys. Acta 640, 655-671 18 Hughes, R.C. and Butters, T.D. (1981) TIBS, pp. 228-230 19 Montgomery, R. (1972) in Glycoproteins (Gottschalk, A., ed.), Vol. 5, Part A, pp. 518-528, Elsevier/North-Holland Biomedical Press, Amsterdam 20 Spiro, R.G. (1973) Advances in Protein Chemistry, Vol. 27, p. 349 Academic Press, New York 21 Yoshima, H., Matsumoto, A., Mizuochi, T., Kawasaki, T. and Kobata, A. (1981) J. Biol. Chem. 256, 8476-8484 22 Megaw, J.M. and Johnson, L.D. (1979) Proc. Soc. Exp. Biol. Med. 161, 60-65

198 23 Warren, L. (1981) in Fundamental Mechanisms in Human Cancer Immunology, (Saunders, J.P., ed.), Vol. 5, pp. 99108, Elsevier/North-Holland Biomedical Press, Amsterdam 24 Warren, L., Baker, S.R., Blithe, D.L. and Buck, C.A. (1982) in Biomembranes (Nowotny, A., ed.), pp. 57-78, Plenum Press, New York 25 Struck, D.K. and Lennarz, W. (1980) in The Biochemistry of Glycoproteins and Proteoglycans, (Lennarz, W.J., ed.), pp. 35-83, Plenum Press, New York

26 Schachter, H. and Roseman, S. (1980) in The Biochemistry of Glycoproteins and Proteoglycans, (Lennarz, W.J., ed.), pp. 85-160, Plenum Press, New York 27 Gravell, M. and Malsberger, R.G. (1965) Ann. N.Y. Acad. Sci. 126, 55-565 28 Warren, L., Zeidman, 1. and Buck, C.A. (1975) Cancer Res. 35, 2186-2190 29 Clark, H.F., Cohen, M.M. and Lunger, P.D. (1973) J. Nat. Cancer Inst. 51,645-657