Glycoproteins and Glycolipids in Disease Processes - American

22 Fucosylation—A Role in Cell Function

Downloaded by CORNELL UNIV on October 5, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/bk-1978-0080.ch022

MARY CATHERINE GLICK Department of Pediatrics, University of Pennsylvania Medical School, Children's Hospital of Philadelphia, Philadelphia, PA 19104

The original observation that virus transformation is accompanied by an alteration of membrane glycoproteins (1) has been extended in a number of laboratories to many virus transformed and tumor cells, including human tumors (see ref. 2 and 3 for reviews). The appearance of specific glycopeptides has been directly correlated with tumorigenesis (4) and has been suggested as a diagnostic procedure for leukemia (5). The studies have progressed to the point of elucidating the partial structure of one of the major glycopeptides predominant on the surface of virus transformed cells (6). This glycopeptide is triantennary and appears to be more highly branched than the major fucose-containing glycopeptide isolated from the nontransformed counterpart. These initial results are compatible with the suggestion that the key to the alteration which leads to the formation of more highly branched oligosaccharides after virus transformation may reside around theβ-mannosecore. The partial structures were assembled from data obtained by enzyme degradation of the isolated glycopeptides with purified exoglycosidases and recovery of the released monosaccharides by gas liquid chromatography. Even though these glycopeptide differences were detected using radioactive L-fucose to mark the glycoproteins, no difference has been seen thus far between the transformed and nontransformed cell surfaces in fucose per se. In order to accent this point a variety of data from a number of cell types have been summarized (Table 1) and used to suggest that the fucosylation of membrane glycoproteins may have a special role in the functioning of the cell. The data which suggest a role for fucosylation fall into several categories: (a) more directly correlated to cell function; (b) the molecule of fucose; and (c) ancillary data. More Directly Correlated to Cell Function The first three observations listed in Table 1 fall into a category which may be more directly suggestive of a role for fucose in membrane glycoproteins. The first, the fact that most membrane glycoproteins contain fucose, has been shown with many 0-8412-0452-7/78/47-080-404$05.00/0 © 1978 American Chemical Society Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.


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Fucosylation—A Role in Cell Function

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c e l l types using double l a b e l i n g experiments with r a d i o a c t i v e L-fucose and D-glucosamine or D-glucose. S i m i l a r membrane g l y c o p r o t e i n s are l a b e l e d with e i t h e r r a d i o a c t i v e L-fucose or the more general l a b e l s , D-glucosamine or D-glucose when examined by polyacrylamide g e l e l e c t r o p h o r e s i s ( 7 ) . An example of t h i s i s shown i n Figure 1 which d e p i c t s the s u r f a c e membrane glycoprot e i n s of transformed baby hamster kidney f i b r o b l a s t s marked with r a d i o a c t i v e fucose (Figure l a ) , and the same clone and the normal counterpart marked with r a d i o a c t i v e glucosamine (Figure l b ) . The only r e p r o d u c i b l e d i f f e r e n c e between the precursors was i n the high molecular weight r e g i o n of the g e l ( F r a c t i o n s 5-15). Another example i s seen i n the p a r t i a l l y p u r i f i e d membrane g l y c o peptides from hamster transformed c e l l s or t h e i r normal counterp a r t s . These c e l l s were m e t a b o l i c a l l y l a b e l e d with both D - [ ^ C ] glucose and L - [ % ] fucose (6). Again the s i m i l a r i t y of the r a d i o a c t i v e patterns of the glycopeptides suggests that a l l of these membrane glycopeptides were f u c o s y l a t e d . The second p o i n t (Table 1), that fucose i s p o s i t i o n e d i n the core r e g i o n of a l l of the membrane g l y c o p r o t e i n s thus f a r examined, can be shown by the t o t a l recovery of the monosaccharide u n i t s a f t e r s e q u e n t i a l enzyme degradation of the r a d i o a c t i v e glycopeptides ( 6 ) . The r a d i o a c t i v i t y was recovered as fucose only a f t e r treatment with r a t t e s t i s a-L-fucosidase and was r e moved subsequent to the r e l e a s e of a l l the other monosaccharides with the exception of two residues of N-acetylglucosamine from the core r e g i o n . I f fucose was p o s i t i o n e d i n another l o c a t i o n , the enzymatic degradation of the o l i g o s a c c h a r i d e u n i t would not have been complete. The t h i r d p o i n t (Table 1) may be a phenomenon r e l a t e d to the g l y c o p r o t e i n s t r u c t u r e s observed. The a c t i v i t y of a-Lfucosidase was not e l e v a t e d a f t e r v i r u s transformation to the extent that the other g l y c o s i d a s e s were e l e v a t e d . The a c i d hydrolase a c t i v i t i e s of the v i r u s transformed c e l l s are expressed as percentage of the normal i n F i g u r e 2. A c i d phosphatase, which represented other lysosomal enzymes, was s i m i l a r i n both c e l l types, as were s e v e r a l exoglycosidases. The a c t i v i t i e s of $g a l a c t o s i d a s e , 3-N-acetylglucosaminidase, a-mannosidase, and a,3-N-acetylgalactosaminidase were a l l e l e v a t e d 150 to 300%. In c o n t r a s t , the a c t i v i t y of a-L-fucosidase was e l e v a t e d only 120%. In other experiments using 4-methylumbelliferyl-fucopyranoside as s u b s t r a t e , the a c t i v i t y of both c e l l types was even more s i m i l a r . Three of the four g l y c o s y l h y d r o l a s e s with e l e v a t e d a c t i v i t i e s are d i r e c t l y concerned with the degradation of the o l i g o s a c c h a r i d e u n i t s which compose the branches of the membrane g l y c o p r o t e i n s . The more branched g l y c o p r o t e i n s are those which are more predominant on the transformed c e l l s u r f a c e ( 6 ) . The f a c t that a-L-fucosidase a c t i v i t y i s s i m i l a r i n both transformed and nontransformed c e l l s suggests that even though the branching of the g l y c o p r o t e i n s i s a l t e r e d on transformation, the fucose residues and the core r e g i o n may remain unchanged.

Walborg; Glycoproteins and Glycolipids in Disease Processes ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE


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600

20

40

60

80

100

Fraction Number Figure 1. Polyacrylamide gel electrophoresis of surface membranes. Surface membranes were prepared from cells metabolically labeled with (a)-L-[ H] or -[ C] fucose and (b) d-[ H] or -[ C] glucosamine and examined by polyacrylamide gel electrophoresis. All details have been described (21). (a) C /B cells logarithmically growing (O—O) or plateu phase (•—•) and (b) C /B (O—O) and (BHK /C (•-•). 3

3

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TABLE 1 Observations which suggest a r o l e f o r f u c o s y l a t i o n i n membrane f u n c t i o n s of mammalian c e l l s . 1. 2. 3.


4. 5.

Most membrane g l y c o p r o t e i n s are f u c o s y l a t e d . Fucose i s p o s i t i o n e d near the glycopeptide core. a-L-Fucosidase a c t i v i t y i s not s i g n i f i c a n t l y elevated a f t e r v i r u s transformation. Fucose i s the only monosaccharide which occurs as an L-isomer and a deoxy sugar but never as the N-acetyl d e r i v a t i v e . Fucose i s not metabolized l i k e other monosaccharides.

100

200

300

Glycosidase Activity of C /B (% of BHK /C ) 13

4

21

13

Figure 2. Acid hydrolase activities of transformed (C /B,,) and nontransformed (BHK /C ) fibroblasts. The cells, harvested when growing exponentially, were homogenized in 0.1% Triton X100. The enzyme activities were determined on the appropriate p-nitrophenyl derivatives in 3mM citrate buffer, pH = 4.5, with the exception of sialidase where fetuin in 50mM acetate buffer, pH = 4.5, served as substrate. The enzyme activities (per mg protein) of C /B cells are expressed as the percentage of the particular activity obtained with BHK /C cells. The values represent the mean obtained from three different cultures of each cell type. Acid phosphatase, which is not a glycosidase, is included for comparison. 13

21

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13

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Uniqueness o f Fucose i n Mammalian C e l l s Point 4 i n Table 1 suggests that due to i t s s t r u c t u r e alone fucose may be i n a unique p o s i t i o n . Thus f a r , fucose has been described only as the L-isomer, i n c o n t r a s t to other monosacchar i d e s which occur as D-isomers. I t i s the only deoxy sugar present i n mammalian g l y c o p r o t e i n s and thus f a r has not been described as the N - a c e t y l d e r i v a t i v e . The f a c t that i t i s not subject to the normal metabolic pathways as the other monosaccharides i s a l s o suggestive o f a unique r o l e . This i s shown by the f a c t that r a d i o a c t i v e fucose i s recovered only as fucose not only i n c e l l s i n c u l t u r e (8), but a l s o i n animals (9). Subsequent s t u d i e s may not support a l l o f these p o i n t s , and i n f a c t , recent r e p o r t s suggest that, contrary to the current dogma, fucose i s not always found i n a t e r m i n a l p o s i t i o n (10,11). A n c i l l a r y Data A d d i t i o n a l data supporting the hypothesis o f the uniqueness of fucose i n mammalian c e l l membranes can be summarized from a number o f other sources. Perhaps the most compelling i s the presence of r e l a t i v e l y l a r g e amounts o f two unusual components a s s o c i a t e d with the s u r f a c e membrane c o n t a i n i n g fucose. One o f these i s the major high molecular weight g l y c o p r o t e i n exported by human f i b r o b l a s t s i n c u l t u r e (12) . Although t h i s g l y c o p r o t e i n has been shown by others to be a component of the f i b r o b l a s t matrix, and f o r t h i s reason c a l l e d F i b r o n e c t i n (13), l i t t l e a t t e n t i o n has been given to the f a c t that i t i s r e a d i l y l a b e l e d with r a d i o a c t i v e L-fucose. The second unusual f u c o s e - c o n t a i n i n g molecule i s a low molecular weight component o f the c e l l s u r f a c e (14). Chromatography on p r e c a l i b r a t e d F r a c t o g e l PGM 2000, as w e l l as other c r i t e r i a , show that t h i s f u c o s e - c o n t a i n i n g molecule i s a p p r o x i mately 500 daltons (Figure 3). This s u r f a c e component i s not the same as the low molecular weight component o f NRK c e l l s reported by S t e i n e r (10), s i n c e fucose was r e l e a s e d by p u r i f i e d a-L-fucosidase. This enzyme was f r e e o f other d e t e c t a b l e exoglycosidases using s y n t h e t i c and n a t u r a l substrates (15), so fucose had to be i n a t e r m i n a l p o s i t i o n . A d d i t i o n a l data concerning the presence o f fucose i n membrane g l y c o p r o t e i n s i s that i n many d i f f e r e n t c e l l types exami n e d , only 30-40% o f the fucose content o f the t o t a l c e l l was found i n the membrane (7>15). This was i n c o n t r a s t to the s i a l i c a c i d content o f the membrane, which was as h i g h as 70-80% o f the t o t a l c e l l content (16). In a d d i t i o n , fucose found i n the membrane g l y c o p r o t e i n s showed two d i f f e r e n t r a t e s o f a c i d h y d r o l y s i s and two d i f f e r e n t s p e c i f i c a c t i v i t i e s ( 8 ) . Only 40% of the fucose from the membrane g l y c o p r o t e i n s o f transformed hamster f i b r o b l a s t s ( C 1 3 / B 4 ) was removed a f t e r 2 hours with 0.1 N H2SO4 a t 100°. This materi a l showed a higher s p e c i f i c a c t i v i t y than the remaining fucose which was subsequently removed by a-L-fucosidase. The reasons f o r these d i f f e r e n c e s are not apparent a t t h i s time, but the two



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Role in Cell

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Fraction Number Figure 3. Low molecular weight fucose-containing components associated with the surface membrane (14). Radioactivity extracted from the surface of a human neuroblastoma cell line (IMR-32), metabolically labeled with L-[ H] fucose, was purified and chromographed on Fractogel PGM 2000. The column was precalibrated with BSA, bovine serum albumin; LNF, lacto-N-fucopentaose; GlcNAc-Asn, 2-acetamido-l-(\.-$-aspartamide)-l,2-di-deoxy-fi-D-glucose; Lac, lactose; Fuc, fucose. 3


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GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

specific activities suggest the presence of at least two fucosyltransferases. Role of Fucosylation All of the above facts can be summarized to suggest that fucose as it occurs in membrane glycoproteins has a number of unusual characteristics. It is hypothesized that because of these, fucose performs a special function for the cell membrane. It can be postulated that fucose may serve to bind proteins or polypeptides for reinsertion into membranes, or transport through the membrane, thus its position near the glycopeptide core would be an advantage. The exact role it may play, if any, remains to be shown. Cell surface variants of a number of cell types have been reported (17-20). Perhaps through use of these variants which are defective in membrane glycoproteins, the special role which fucose may have in the relationship of the surface membrane to the cell will be ascertained. SUMMARY The fucose-containing glycopeptides of the membranes of virus transformed and tumor cells are clearly different from those of their normal counterparts. This difference is that the virus transformed cells express on their surfaces glycopeptides more highly branched than those found in the controls. Although fucose served as a radioactive marker to detect these differences, no difference has, as yet, been found in fucose per se. The similarities of fucose among several cell types and some unique properties of fucose in mammalian glycoproteins have been summarized and used to suggest that fucose may play a special role in the relationship of the surface membrane to the cell. ACKNOWLEDGEMENTS Supported by U.S.P.H.S. Grants CA 14037, CA 14489, and HD 08536. Literature Cited 1. 2. 3. 4. 5. 6.

Buck, C.A., Glick, M.C., and Warren, L. (1970) Biochemistry 9, 4567-4576. Glick, M.C. (1976) In Fundamental Aspects of Metastasis, L. Weiss, ed. North Holland Publishing Co., Amsterdam, pp. 9-23. Poste, G., and Nicholson, G.L., eds. (1977) Cell Surface Reviews, Vol. 4. Elsevier-North Holland Inc., Amsterdam. Glick, M.C., Rabinowitz, Z., and Sachs, L. (1974) J . Virol. 13, 967-974. Van Beek, W.P., Emmelot, P., and Homburg, C. (1977) Br. J. Cancer 36, 157-165. Glick, M.C., and Santer, U.V. (1977) In Cell Surface Carbohydrate Chemistry, Adv. Chem. Series, R.E. Harmon, ed., Academic Press, New York, pp. 13-26.


22. GLICK 7. 8. 9. 10. 11. 12.


13. 14. 15. 16. 17. 18. 19. 20. 21.


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Scanlin, T.F., and Glick, M.C. VII International Cystic Fibrosis Conference. In press. Fischer, A., and Glick, M.C. Manuscript in preparation. Bocci, V., and Winzler, R.J. (1969) Am. J. Physiol. 216, 1337-1342. Steiner, S., in these proceedings. Hallgren, P., Lundblod, A., and Svensson, S. (1975) J. Biol. Chem. 250, 5312-5314. Scanlin, T.F., Voynow, J.A., and Glick, M.C. Unpublished observations. Vuento, M., Wrann, M., and Ruoslahti, E. (1977) FEBS Lett. 82, 227-231. Mihalik, G.M., and Glick, M.C. Unpublished observations. Santer, U.V., and Glick, M.C. Manuscript in preparation. Glick, M.C. (1976) In Mammalian Cell Membranes, Vol. 1, G. A. Jamieson and D.M. Robinson, eds. Butterworths, London, pp. 45-77. Stanley, P., Narasimhan, S., Siminovitch, L . , and Schachter, H. (1975) Proc. Natl. Acad. Sci. USA 72, 3323-3327. Meager, A., Ungkitchanukit, A., and Hughes, R.C. (1976) Biochem. J . 154, 113-124. Briles, E.B., L i , E . , and Kornfeld, S. (1977) J . Biol. Chem. 252, 1107-1116. Pouysségur, J., and Pastan, I. (1977) J. Biol. Chem. 252, 1639-1646. Greenberg, C.S., and Glick, M.C. (1972) Biochemistry 11, 3680-3685.

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April 17, 1978.


Glycoproteins and Glycolipids in Disease Processes - American

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