TIBS 17 - NOVEMBER 1992
REVIEWS THE GLYCOSYLINOSITOL PHOSPHOLIPID (GPI) anchors of proteins such as
Thy-1 of neurons and lymphocytes and the variant surface glycoprotein (VSG) of African trypanosomes are post-translationally linked to the carboxyl terminus of the corresponding proteins (Fig. 1) 1,2. The fact that many GPIanchored proteins can be released from cells by microbial phosphatidylinositol (PtdIns)-specific phospholipases implies that the proteins themselves do not traverse the lipid bilayer. Such proteins therefore cannot be subject t o cytoplasmic events such as direct linkage to the cytoskeleton and phosphorylation, which impact on transmembrane proteins (Fig. 2). For this reason it was anticipated both that the lateral diffusion rof GPI,anchored proteins would be high and that these proteins might be released by physiologically important phospholipases. The evidence in support of such conjectures with regard to function is, however, difficult to generalize. GPI-anchored class I histocompatibility antigens do appear to gain access to much of the cell surface3; however, GPI-anchored proteins are under-represented in coated pits 4. The lateral diffusion of GPI-anchored proteins is not uniformly higher than for transmembrane proteins. Furthermore, although there are a few experimental reports describing the release of GPIanchored proteins and although soluble forms of several GPI-anchored proteins are found in extracellular fluids, release appears to be the exception rather than the rule. Several GPI-anchored proteins are endocytosed very slowly4,5 and at least one GPI-anchored protein, Thy-1, has an unusually long half-life4 In neurons and some polarized epithelial cells, GPIanchored proteins are preferentially enriched at the apical plasma membrane, apparently because the GPI unit is an apical sorting signal and/or causes association with sphingolipids 6;. A few GPIanchored proteins have been identified in membranes of secretion granules. A. M1 Tartakoff and N. Singh are at the Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA.
470
Essentially all eukaryotic cells express proteins on their surface that are anchored by a glycoinositol phospholipid. This anchor moiety may endow such proteins with unusual properties. The definition of the biosynthetic path that constructs these anchors is now in its final stages. Mutations that interrupt this path are, remarkably, compatible with survival of cells in culture, but are associated with at least one human disease.
GPI anchors may also be important for allowing transport of certain proteins along the secretory" pathway. For example, mutant cells that do not add GPI anchors do not express the corresponding proteins at the cell surface 8-]3. The polypeptide precursors of GPIanchored proteins, which include a putative transmembrane segment that is normally destined to be replaced by the GPI unit (see below), do not reach to the cell surface. We therefore suggest that the cytoplasmic and transJ
0
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membrane domains of such proteins must be removed in order tO allow the GPI-anchored proteins to avoid retention along the secretory path and/or degradation. This may provide an unusual post-translational mechanism for regulating cell-surface expression of such proteins.
Biosynthesis Timing of addition of the GPI moiety has been studied in yeast, Mrican trypanosomes and in higher euka~-
0n
O"IPOCH2CH2NH2 1 o
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I °:
,
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Figure1 Structures of GPI anchors of membrane proteins. An identical core glycan is found in all cases, consisting of ethanolamine phosphate, three mannose'residues and non-acetylated glucosamine (arrow 1) linked to inositol; however, there are significant differences that distinguish the anchors of Thy-I,VSG and human erythrocyte acetylcholinesterase. Constituents that are found in some, but not all, anchors are outside the enclosed region. These can be additional ethanolamine phosphate, mannose and N-acetylgalactosamine (or galactose) residues, as well as an acyl chain linked to inositol C-2 or C-3 (arrow 2). In VSG, R1 and R2 are acyl chains in ester linkage. In animal cells the glycerol unit often bears an ether-linked chain on the first carbon (R1). The figure also indicates the site of anchor cleavage by nitrous acid (arrow 3); Ptdlns-phospholipase D (PLdlns-PLD)(arrow 4) and Ptdlns-phospholipase C (Ptdlns-PLC) (arrow 5). Ptdlns-PLC is not active if acylinositol (arrow 2) is present.
© 1992,ElsevierSciencePublishers,(UIO
TIBS 17 - NOVEMBER 1 9 9 2
otes 1'14-16. In all cases, it is added in the rough endoplasmic reticulum (RER) within a couple of minutes of chain termination. This is actually an event of anchor exchange, since the primary translation product includes a relatively orthodox putative membrane-spanning segment near its carboxyl terminus, which is removed before, or concomitant with, anchor addition L2,~7.The fate of this carboxy-terminal peptide and the enzyme(s) responsible for its removal have not been identified. A trans-amidation reaction may be involved, thus explaining why it is important to remove a carboxy-terminal peptide, rather than simply appending the GPI unit to the pre-existing carboxyl terminus. The peptide signal causing anchor addition consists of a hydrophobic domain preceded by a pair of small residues 18. Putative preassembled anchor units have been identified in both trypanosomes and murine T lymphoma cells 1,2,19-22. These GPI units closely resemble the anchor moieties found on the corresponding proteins expressed at the ceil surface. Ceil-free systems have been established that exhibit lowefficiency transfer of these GPI units to the corresponding acceptor proteins L17. A family of Thy-l-negative T-lymphoma mutants has been useful in defining the metabolic path of GPI anchor biosynthesis 8. It is ironic that these mutants were first studied before the existence of GPI-anchors was known. These recessive mutants have been assigned to complementation groups A-F and H, after introduction of appropriate drug-resistance markers and pair-wise fusion. In all cases, except for class D (where no Thy-1 is made), synthesis of an apparently normal Thy-1 peptide occurs, but GPI-anchored proteins are not expressed at the cell surface. This lack of surface expression is matched by the inability of each of the mutants to add a GPI anchor to Thy-19J°,12,13. Class B and E mutants secrete much of their Thy-1, while the others degrade the Thy-1 that they synthesize, probably in lysosomes 23. Each of these mutants is characterized by lesions which interrupt the synthesis of the preassembled anchor. Thus, in vivo, 3H-mannose labeling of wild-type T lymphomas detects mannolipids with characteristic sensitivity to microbial PtdIns-specific phospholipases and nitrous acid (which splits the amino sugar-inositol linkage; see Fig. 1). The most polar of these units - the
putative anchor precursor resists exo-mannosidase, indicating that its nonreducing terminus is blocked, presumably with ethanolamine phosphate. Exterior By contrast, parallel in vivo labeling shows that none of the mutants produces such putative Cytosol anchor precursor mannoGPI lipids. anchor In A, C and H mutants, there is no production of mannolipids sensitive to Figure 2 microbial phospholipases Cartoon illustrating the relation of GPI-anchored proand nitrous acid. In these teins (right) and a transmembra'ne protein (left) to the cases, taking into conplasma membrane. Clearly, the GPl-anchored protein cannot be directly linked to the cytoskeleton or to sideration data from in clathrin. It also cannot be phosphorylated by cytosolic vitro labeling of membrane protein kinases. preparations of lymphoma cells supplemented with UDP-GlcNAc and GDP-mannose21,24.there mediates and some mature GPIappears to be a lesion that interrupts linked proteins of animal cells (Ref. the biosynthetic path so early that even 25: M. E. Medof and T. Rosenberry, the N-acetylglucosamine residue is not unpublished). added (Fig. 3). Thus. the lesion may (4) The terminal (linker) ethanolamine affect synthesis or translocation of the phosphate residue is derived from proper variety of Ptdlns or the addition phosphatidyl ethanolamine (A. of N-acetylglucosamine to Ptdlns. Menon. unpublished). 3H-sugar labeling of the E mutant. (5) In a final step in trypanosomes, indiboth in vivo and in vitro, shows that GPI vidual glycerol-linked acyl chains of units are produced which are sensitive the PtdIns unit are removed and replaced 14. to microbial PtdIns-specific phospholipase D and nitrous acid. but have (6) Lateral galactose, N-acetylgalactoseither none or possibly one mannose amine and additional mannose (Fig. residue (step 4 or 5 in Fig. 3) ~92125. 1) may all be added after anchor addition 1,2. Comparable experiments indicate that the class B mutant is blocked in addition of the third mannose residue Several agents inhibit synthesis of (step 6, Fig. 3) while the class F mutant the mature precursor unit in vivo: manis blocked in terminal ethanolamine nosamine inhibits addition of the third phosphate addition (step 7. Fig. 3). mannose residue. PMSF inhibits adGiven the ability of azacytidine, amino- dition of ethanolamine phosphate, glycosides and sodium butyrate to par- fluoroglucose stops mannose addition tially correct class C, F and H mutants. and certain myristic acid analogues respectively, the corresponding struc- block myristate addition in trypanotural genes may be present but not tran- somes. scribed into active products 26. There are several further aspects of Role of dolichol-phosphoryl-mannose the biosynthetic path worth noting: Dolichol-phosphoryl-mannose contributes mannose residues to the anchor. (1) The biosynthetic origin of the PtdIns, Thus, mutant yeast, class E mutant lymwhich for animal ceils often has phoma ceils and B4-2-1 mutant Chinese a 1-alkyl, 2-acyl structure, is not hamster ovary (CHO) cells, none of known: which make normal amounts of doli(2) Addition of N-acetylglucosamine to chol-phosphoryl-mannose, do not proPtdIns (step 1) is followed by its duce GPI anchors uJ2,15,27. In one of the deacetylation (step 2) and sub- lymphoma mutants, this defect can be sequent inositol acylation (step 3) corrected by transfection with a plasmid before addition of successive man- that encodes yeast dolichol-phosphorylnose residues. mannose synthase H. Moreover, when (3) Lateral ethanolamine phosphate is the CHO cell mutant B4-2-1 is treated found on mannose-containing inter- with tunicamycin, which greatly boosts -
\
471
TIBS 17 - NOVEMBER 1 9 9 2
levels of dolichol-phosphoryl-mannose, the defect in anchor synthesis is corrected 27. This involvement of dolicholphosphoryl-mannose is important because, judging from studies of N-glycan synthesis, dolichol-phosphoryl-mannose is responsible for mannose addition at the cisternal (luminal) face of the RER membrane. Thus, at least some of the mannose residues of the GPI unit must be added at the cisternal face of the RER. Consistent with this genetic information on animal cells, biochemical studies of broken cell preparations of trypanosomes argue that dolichol-phosphoryl-mannose contributes the three core mannose residues to the anchor structureL Nevertheless, in vivo labeling studies of the class E lymphoma mutant show that it synthesizes a GPIFigure 3 Model of synthesis of the preassembled anchor precursor in animal cells. Judging from in vivo and in vitro studies of both animal cells and trypanosomes, the sequence of intermediates appears to grow one residue at a time. The bestdefined steps are numbered 1-7. Mannose-containing species may also include additional ethanolamine phosphate linked to mannose. Site(s) of interruption of the biosynthetic path in class A-H mutants are indicated by the corresponding upper case letters. After transfer of the mature unit to protein, additional lateral substituents may be added and the inositol may be deacylated (see Fig. 4).
related mannolipid even though dolichol-phosphoryl-mannose is absent ~9. Thus, both GDP-mannose and dolicholphosphoryl-mannose may contribute to GPI anchor synthesis in these cells and successive steps of synthesis of the preassembled GPI-Ptdlns unit may occur on different faces of the RER membrane.
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472
As mentioned above, higher eukaryotes and blood-stream trypanos0mes make anchor precursors which include acylinositol. One can therefore readily rationalize the observation that GPI anchors on many ceils (L929 fibroblasts, human red ceils, procyclic trypanosomes etc) also have acylinositoll~2,2°,23, which causes them to resist PtdIns-phospholipase C (Ptdlns-PLC) hydrolysis. Nevertheless, the anchors of surface proteins of many cells lack acylinositol (murine T lymphoma cells, CHO cells). Since fusion of L929 fibroblasts with such cells yields stable hybrids whose GPI-anchored proteins lack acylinositol, L929 may lack a GPI deacylase, which is present in the lymphoma cell (Fig. 4) 23. In principle, this deacylase might act just before or after anchor addition. The presence of inositol-linked acyl chains may be important for promoting the 'flip-flop' of key intermediates across the RER membrane or for securing the integration of GPI units into the plasma membrane. Implications
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The broader significance of these observations concerns the expression of an abundant class of surface molecules of diverse function. Many are enzymes, some have been described as receptors (.for example, the FclII receptor for immunoglobulin, folate receptor) and several can (surprisingly) transduce signals to ceils when they are liganded by appropriate antibodies. This activity may reflect association of GPI-anchored proteins with a tyrosine kinase 28. In addition, certain protozoa express complex surface, glycans, which are attached to similar anchors 1,2. Given the existence of mutant cells that do not express GPI-anchored proteins, synthesis and expression of such proteins is certainly not essential for survival of individual cells in culture. In the presence of additional cell types, expression of GPI-anchored proteins may govern the proliferation of the cells that bear them 29. Moreover, lesions similar to those of the Thy-l-negative lym-
TIBS 1 7 - N O V E M B E R
1992
phoma mutants may explain the lack of GPI-anchored proteins on red and white cells in the human hemolytic anemia, paroxysmal nocturnal hemoglobinuria 3°. Finally, lesions which stop GPI biosynthesis will eliminate surface expression of multiple GPl-anchored proteins and therefore may be important for allowing malignant cells to escape immune surveillance.
Acknowledgements Work from the authors' laboratory was supportedby DK38181 from the NIH.
(a)
(b)
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Thre GPI anchor
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(C) References Due to TIBS policy of short reference lists, the number of references cited in this article has been limited. Much work has therefore unfortunately been left unacknowledged. 1 Cross, G. A. M. (1990) Annu. Rev. Cell Biol. 6, 1-39 2 Ferguson, M. A. J. and Williams, A. (1988) Annu. Rev. Biochem. 57, 285-320 3 Edidin, M. and Stroynowski, I. (1991) J. Cell Biol. 112, 1143-1150 4 Lemansky, P. et al. (1990) J. Cell Biol. 110, 1525-1532 5 Lisanti, M. P. et al. (1990) Proc. NatlAcad. Sci. USA 87, 7419-7423 6 Brown, D. A. and Rose, J. K. (1992) Cell 68, 533-544 7 Dotti, C. G., Parton, R. G. and Simons, K. (1991) Nature, 349, 158-161 8 Hyman, R. (1988) Trends Genet. 4, 5-8 9 Conzelmann,A., Spiazzi, A., Hyman, R. and Bron, C. (1986) EMBO J. 5, 3291-3296 10 Conzelmann,A., Riezman, H., Desponds, C. and Bron, D. (1988) EMBO J. 7, 2233-2240 11 DeGasperi,R. et al. (1990) Science 250, 988-991 12 Fatemi, S. H. and Tartakoff, A. M. (1986) Cell 46, 653=657 13 Fatemi, S. H. and Tartakoff, A. M. (1988) J. Biol..Chem. 263, 1288-1294 14 Masterson, W. J. et al. (1990) Cell 62, 73-80 15 Orlean,P. (1990) Mol. Cell. BioL 10, 5796-5805 16 Takami, N. et al. (.1988) J. Biol. Chem. 263, 12716-12720 17 Kodukula, K. et al. (1992) Prec. Natl Acad. Sci. USA 89, 1350-1353 18 Moran, P. and Caras, I. (1991) J. Cell Biol. 115, 329-336 19 Lemansky, P. et al. (1991) Mol. Cell. Biol. 11, 3879-3885 20 Field, M. C., Menon, A. K. and Cross, G. A. M. (1991) J. Biol. Chem. 266, 1-9 21 Sugiyama, E. et al. (1991) J. Biol. Chem. 266, 12119-12122 22 Doering,T. L., Masterson, W. J., Hart, G. W. and Englund,P. T. (1990) J. BioL Chem. 265, 611~B14 23 Singh, N., Singleton, D. and Tartakoff, A. (1991) Mol. Cell. Biol. 11, 2362-2374 24 Stevens, V. L. and Raetz, C. R. H. (1991) J. Biol. Chem. 266, 10039-10042 25 Puoti, A., Desponds, C., Fankhauser,C. and Conzelmann,A. (1991) J. Biol. Chem. 266, 21051-21059 26 Tisdale, E., Schimenti, J. and Tartakoff, A. (1991) Som. Cell Mol. Genet. 17, 349-357 27 Singh, N. and Tartakoff, A. (1991) MoI. Cell. Biol. 11, 391-400 28 Stefanova, I. et al. (1991) Science 254, 1016-1019 29 Chen, S. et al. (1987) Cell 51, 7-19 30 Hirose, S., Ravi, L., Hazra, S. V. and Medof, M. E. (1991) Proc. Natl Acad. Sci. USA 88, 3762-3766
Figure 4 Cell fusion experiments show that Ptdlns-PLC sensitivity (the hallmark of the absence of acylinosito!) behaves as a dominant trait. When (a) a cell that synthesizes Ptdlns-PLCresistant anchored proteins is fused with (b), a cell that synthes!zes Ptdlns-PLC-sensitive anchored proteins, stable hybrids [(c)] express Ptdlns-PLC-sensitive proteins. The simplest explanation of this observation is that the GPI unit that resists Ptdlns~PLC because of the presence of an inositol-linked, third acyl chain, is deacylated in cells that express a deacylase (e.g. murine lymphomas, CHO cells) but not in other cells (L929 fibroblasts, red blood cell precursors). As illustrated, the deacylation might occur at the level of the anchor precursor or just after anchor transfer to protein. This result is not consistent with a model in which the second class of cell produces an anchor inositol acyl transferase which is absent from the first cell type. In each oval cell, an element of rough endoplasmic reticulure (ER) is illustrated. Within it are 'three-legged' and/or 'two-legged' GPI units and/or GPIanchored proteins. The 'three-legged' units are those which include an acyl chain linked to inositol, in addition to the glycerol acyl and/or alkyl substituents. In cell (a), the third chain is never removed, while in (b) and (c) it is removed just 5efore or after addition to protein.
473
REVIEWS THE GLYCOSYLINOSITOL PHOSPHOLIPID (GPI) anchors of proteins such as
Thy-1 of neurons and lymphocytes and the variant surface glycoprotein (VSG) of African trypanosomes are post-translationally linked to the carboxyl terminus of the corresponding proteins (Fig. 1) 1,2. The fact that many GPIanchored proteins can be released from cells by microbial phosphatidylinositol (PtdIns)-specific phospholipases implies that the proteins themselves do not traverse the lipid bilayer. Such proteins therefore cannot be subject t o cytoplasmic events such as direct linkage to the cytoskeleton and phosphorylation, which impact on transmembrane proteins (Fig. 2). For this reason it was anticipated both that the lateral diffusion rof GPI,anchored proteins would be high and that these proteins might be released by physiologically important phospholipases. The evidence in support of such conjectures with regard to function is, however, difficult to generalize. GPI-anchored class I histocompatibility antigens do appear to gain access to much of the cell surface3; however, GPI-anchored proteins are under-represented in coated pits 4. The lateral diffusion of GPI-anchored proteins is not uniformly higher than for transmembrane proteins. Furthermore, although there are a few experimental reports describing the release of GPIanchored proteins and although soluble forms of several GPI-anchored proteins are found in extracellular fluids, release appears to be the exception rather than the rule. Several GPI-anchored proteins are endocytosed very slowly4,5 and at least one GPI-anchored protein, Thy-1, has an unusually long half-life4 In neurons and some polarized epithelial cells, GPIanchored proteins are preferentially enriched at the apical plasma membrane, apparently because the GPI unit is an apical sorting signal and/or causes association with sphingolipids 6;. A few GPIanchored proteins have been identified in membranes of secretion granules. A. M1 Tartakoff and N. Singh are at the Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH 44106, USA.
470
Essentially all eukaryotic cells express proteins on their surface that are anchored by a glycoinositol phospholipid. This anchor moiety may endow such proteins with unusual properties. The definition of the biosynthetic path that constructs these anchors is now in its final stages. Mutations that interrupt this path are, remarkably, compatible with survival of cells in culture, but are associated with at least one human disease.
GPI anchors may also be important for allowing transport of certain proteins along the secretory" pathway. For example, mutant cells that do not add GPI anchors do not express the corresponding proteins at the cell surface 8-]3. The polypeptide precursors of GPIanchored proteins, which include a putative transmembrane segment that is normally destined to be replaced by the GPI unit (see below), do not reach to the cell surface. We therefore suggest that the cytoplasmic and transJ
0
O I
II I Pro,e.., -CNHC.2 CH20-.P;O I
membrane domains of such proteins must be removed in order tO allow the GPI-anchored proteins to avoid retention along the secretory path and/or degradation. This may provide an unusual post-translational mechanism for regulating cell-surface expression of such proteins.
Biosynthesis Timing of addition of the GPI moiety has been studied in yeast, Mrican trypanosomes and in higher euka~-
0n
O"IPOCH2CH2NH2 1 o
Manal 2 6n t-2Manal-6Manor 1-~GIcNH2e
I °:
,
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HO I OHl 61
Figure1 Structures of GPI anchors of membrane proteins. An identical core glycan is found in all cases, consisting of ethanolamine phosphate, three mannose'residues and non-acetylated glucosamine (arrow 1) linked to inositol; however, there are significant differences that distinguish the anchors of Thy-I,VSG and human erythrocyte acetylcholinesterase. Constituents that are found in some, but not all, anchors are outside the enclosed region. These can be additional ethanolamine phosphate, mannose and N-acetylgalactosamine (or galactose) residues, as well as an acyl chain linked to inositol C-2 or C-3 (arrow 2). In VSG, R1 and R2 are acyl chains in ester linkage. In animal cells the glycerol unit often bears an ether-linked chain on the first carbon (R1). The figure also indicates the site of anchor cleavage by nitrous acid (arrow 3); Ptdlns-phospholipase D (PLdlns-PLD)(arrow 4) and Ptdlns-phospholipase C (Ptdlns-PLC) (arrow 5). Ptdlns-PLC is not active if acylinositol (arrow 2) is present.
© 1992,ElsevierSciencePublishers,(UIO
TIBS 17 - NOVEMBER 1 9 9 2
otes 1'14-16. In all cases, it is added in the rough endoplasmic reticulum (RER) within a couple of minutes of chain termination. This is actually an event of anchor exchange, since the primary translation product includes a relatively orthodox putative membrane-spanning segment near its carboxyl terminus, which is removed before, or concomitant with, anchor addition L2,~7.The fate of this carboxy-terminal peptide and the enzyme(s) responsible for its removal have not been identified. A trans-amidation reaction may be involved, thus explaining why it is important to remove a carboxy-terminal peptide, rather than simply appending the GPI unit to the pre-existing carboxyl terminus. The peptide signal causing anchor addition consists of a hydrophobic domain preceded by a pair of small residues 18. Putative preassembled anchor units have been identified in both trypanosomes and murine T lymphoma cells 1,2,19-22. These GPI units closely resemble the anchor moieties found on the corresponding proteins expressed at the ceil surface. Ceil-free systems have been established that exhibit lowefficiency transfer of these GPI units to the corresponding acceptor proteins L17. A family of Thy-l-negative T-lymphoma mutants has been useful in defining the metabolic path of GPI anchor biosynthesis 8. It is ironic that these mutants were first studied before the existence of GPI-anchors was known. These recessive mutants have been assigned to complementation groups A-F and H, after introduction of appropriate drug-resistance markers and pair-wise fusion. In all cases, except for class D (where no Thy-1 is made), synthesis of an apparently normal Thy-1 peptide occurs, but GPI-anchored proteins are not expressed at the cell surface. This lack of surface expression is matched by the inability of each of the mutants to add a GPI anchor to Thy-19J°,12,13. Class B and E mutants secrete much of their Thy-1, while the others degrade the Thy-1 that they synthesize, probably in lysosomes 23. Each of these mutants is characterized by lesions which interrupt the synthesis of the preassembled anchor. Thus, in vivo, 3H-mannose labeling of wild-type T lymphomas detects mannolipids with characteristic sensitivity to microbial PtdIns-specific phospholipases and nitrous acid (which splits the amino sugar-inositol linkage; see Fig. 1). The most polar of these units - the
putative anchor precursor resists exo-mannosidase, indicating that its nonreducing terminus is blocked, presumably with ethanolamine phosphate. Exterior By contrast, parallel in vivo labeling shows that none of the mutants produces such putative Cytosol anchor precursor mannoGPI lipids. anchor In A, C and H mutants, there is no production of mannolipids sensitive to Figure 2 microbial phospholipases Cartoon illustrating the relation of GPI-anchored proand nitrous acid. In these teins (right) and a transmembra'ne protein (left) to the cases, taking into conplasma membrane. Clearly, the GPl-anchored protein cannot be directly linked to the cytoskeleton or to sideration data from in clathrin. It also cannot be phosphorylated by cytosolic vitro labeling of membrane protein kinases. preparations of lymphoma cells supplemented with UDP-GlcNAc and GDP-mannose21,24.there mediates and some mature GPIappears to be a lesion that interrupts linked proteins of animal cells (Ref. the biosynthetic path so early that even 25: M. E. Medof and T. Rosenberry, the N-acetylglucosamine residue is not unpublished). added (Fig. 3). Thus. the lesion may (4) The terminal (linker) ethanolamine affect synthesis or translocation of the phosphate residue is derived from proper variety of Ptdlns or the addition phosphatidyl ethanolamine (A. of N-acetylglucosamine to Ptdlns. Menon. unpublished). 3H-sugar labeling of the E mutant. (5) In a final step in trypanosomes, indiboth in vivo and in vitro, shows that GPI vidual glycerol-linked acyl chains of units are produced which are sensitive the PtdIns unit are removed and replaced 14. to microbial PtdIns-specific phospholipase D and nitrous acid. but have (6) Lateral galactose, N-acetylgalactoseither none or possibly one mannose amine and additional mannose (Fig. residue (step 4 or 5 in Fig. 3) ~92125. 1) may all be added after anchor addition 1,2. Comparable experiments indicate that the class B mutant is blocked in addition of the third mannose residue Several agents inhibit synthesis of (step 6, Fig. 3) while the class F mutant the mature precursor unit in vivo: manis blocked in terminal ethanolamine nosamine inhibits addition of the third phosphate addition (step 7. Fig. 3). mannose residue. PMSF inhibits adGiven the ability of azacytidine, amino- dition of ethanolamine phosphate, glycosides and sodium butyrate to par- fluoroglucose stops mannose addition tially correct class C, F and H mutants. and certain myristic acid analogues respectively, the corresponding struc- block myristate addition in trypanotural genes may be present but not tran- somes. scribed into active products 26. There are several further aspects of Role of dolichol-phosphoryl-mannose the biosynthetic path worth noting: Dolichol-phosphoryl-mannose contributes mannose residues to the anchor. (1) The biosynthetic origin of the PtdIns, Thus, mutant yeast, class E mutant lymwhich for animal ceils often has phoma ceils and B4-2-1 mutant Chinese a 1-alkyl, 2-acyl structure, is not hamster ovary (CHO) cells, none of known: which make normal amounts of doli(2) Addition of N-acetylglucosamine to chol-phosphoryl-mannose, do not proPtdIns (step 1) is followed by its duce GPI anchors uJ2,15,27. In one of the deacetylation (step 2) and sub- lymphoma mutants, this defect can be sequent inositol acylation (step 3) corrected by transfection with a plasmid before addition of successive man- that encodes yeast dolichol-phosphorylnose residues. mannose synthase H. Moreover, when (3) Lateral ethanolamine phosphate is the CHO cell mutant B4-2-1 is treated found on mannose-containing inter- with tunicamycin, which greatly boosts -
\
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TIBS 17 - NOVEMBER 1 9 9 2
levels of dolichol-phosphoryl-mannose, the defect in anchor synthesis is corrected 27. This involvement of dolicholphosphoryl-mannose is important because, judging from studies of N-glycan synthesis, dolichol-phosphoryl-mannose is responsible for mannose addition at the cisternal (luminal) face of the RER membrane. Thus, at least some of the mannose residues of the GPI unit must be added at the cisternal face of the RER. Consistent with this genetic information on animal cells, biochemical studies of broken cell preparations of trypanosomes argue that dolichol-phosphoryl-mannose contributes the three core mannose residues to the anchor structureL Nevertheless, in vivo labeling studies of the class E lymphoma mutant show that it synthesizes a GPIFigure 3 Model of synthesis of the preassembled anchor precursor in animal cells. Judging from in vivo and in vitro studies of both animal cells and trypanosomes, the sequence of intermediates appears to grow one residue at a time. The bestdefined steps are numbered 1-7. Mannose-containing species may also include additional ethanolamine phosphate linked to mannose. Site(s) of interruption of the biosynthetic path in class A-H mutants are indicated by the corresponding upper case letters. After transfer of the mature unit to protein, additional lateral substituents may be added and the inositol may be deacylated (see Fig. 4).
related mannolipid even though dolichol-phosphoryl-mannose is absent ~9. Thus, both GDP-mannose and dolicholphosphoryl-mannose may contribute to GPI anchor synthesis in these cells and successive steps of synthesis of the preassembled GPI-Ptdlns unit may occur on different faces of the RER membrane.
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As mentioned above, higher eukaryotes and blood-stream trypanos0mes make anchor precursors which include acylinositol. One can therefore readily rationalize the observation that GPI anchors on many ceils (L929 fibroblasts, human red ceils, procyclic trypanosomes etc) also have acylinositoll~2,2°,23, which causes them to resist PtdIns-phospholipase C (Ptdlns-PLC) hydrolysis. Nevertheless, the anchors of surface proteins of many cells lack acylinositol (murine T lymphoma cells, CHO cells). Since fusion of L929 fibroblasts with such cells yields stable hybrids whose GPI-anchored proteins lack acylinositol, L929 may lack a GPI deacylase, which is present in the lymphoma cell (Fig. 4) 23. In principle, this deacylase might act just before or after anchor addition. The presence of inositol-linked acyl chains may be important for promoting the 'flip-flop' of key intermediates across the RER membrane or for securing the integration of GPI units into the plasma membrane. Implications
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The broader significance of these observations concerns the expression of an abundant class of surface molecules of diverse function. Many are enzymes, some have been described as receptors (.for example, the FclII receptor for immunoglobulin, folate receptor) and several can (surprisingly) transduce signals to ceils when they are liganded by appropriate antibodies. This activity may reflect association of GPI-anchored proteins with a tyrosine kinase 28. In addition, certain protozoa express complex surface, glycans, which are attached to similar anchors 1,2. Given the existence of mutant cells that do not express GPI-anchored proteins, synthesis and expression of such proteins is certainly not essential for survival of individual cells in culture. In the presence of additional cell types, expression of GPI-anchored proteins may govern the proliferation of the cells that bear them 29. Moreover, lesions similar to those of the Thy-l-negative lym-
TIBS 1 7 - N O V E M B E R
1992
phoma mutants may explain the lack of GPI-anchored proteins on red and white cells in the human hemolytic anemia, paroxysmal nocturnal hemoglobinuria 3°. Finally, lesions which stop GPI biosynthesis will eliminate surface expression of multiple GPl-anchored proteins and therefore may be important for allowing malignant cells to escape immune surveillance.
Acknowledgements Work from the authors' laboratory was supportedby DK38181 from the NIH.
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(C) References Due to TIBS policy of short reference lists, the number of references cited in this article has been limited. Much work has therefore unfortunately been left unacknowledged. 1 Cross, G. A. M. (1990) Annu. Rev. Cell Biol. 6, 1-39 2 Ferguson, M. A. J. and Williams, A. (1988) Annu. Rev. Biochem. 57, 285-320 3 Edidin, M. and Stroynowski, I. (1991) J. Cell Biol. 112, 1143-1150 4 Lemansky, P. et al. (1990) J. Cell Biol. 110, 1525-1532 5 Lisanti, M. P. et al. (1990) Proc. NatlAcad. Sci. USA 87, 7419-7423 6 Brown, D. A. and Rose, J. K. (1992) Cell 68, 533-544 7 Dotti, C. G., Parton, R. G. and Simons, K. (1991) Nature, 349, 158-161 8 Hyman, R. (1988) Trends Genet. 4, 5-8 9 Conzelmann,A., Spiazzi, A., Hyman, R. and Bron, C. (1986) EMBO J. 5, 3291-3296 10 Conzelmann,A., Riezman, H., Desponds, C. and Bron, D. (1988) EMBO J. 7, 2233-2240 11 DeGasperi,R. et al. (1990) Science 250, 988-991 12 Fatemi, S. H. and Tartakoff, A. M. (1986) Cell 46, 653=657 13 Fatemi, S. H. and Tartakoff, A. M. (1988) J. Biol..Chem. 263, 1288-1294 14 Masterson, W. J. et al. (1990) Cell 62, 73-80 15 Orlean,P. (1990) Mol. Cell. BioL 10, 5796-5805 16 Takami, N. et al. (.1988) J. Biol. Chem. 263, 12716-12720 17 Kodukula, K. et al. (1992) Prec. Natl Acad. Sci. USA 89, 1350-1353 18 Moran, P. and Caras, I. (1991) J. Cell Biol. 115, 329-336 19 Lemansky, P. et al. (1991) Mol. Cell. Biol. 11, 3879-3885 20 Field, M. C., Menon, A. K. and Cross, G. A. M. (1991) J. Biol. Chem. 266, 1-9 21 Sugiyama, E. et al. (1991) J. Biol. Chem. 266, 12119-12122 22 Doering,T. L., Masterson, W. J., Hart, G. W. and Englund,P. T. (1990) J. BioL Chem. 265, 611~B14 23 Singh, N., Singleton, D. and Tartakoff, A. (1991) Mol. Cell. Biol. 11, 2362-2374 24 Stevens, V. L. and Raetz, C. R. H. (1991) J. Biol. Chem. 266, 10039-10042 25 Puoti, A., Desponds, C., Fankhauser,C. and Conzelmann,A. (1991) J. Biol. Chem. 266, 21051-21059 26 Tisdale, E., Schimenti, J. and Tartakoff, A. (1991) Som. Cell Mol. Genet. 17, 349-357 27 Singh, N. and Tartakoff, A. (1991) MoI. Cell. Biol. 11, 391-400 28 Stefanova, I. et al. (1991) Science 254, 1016-1019 29 Chen, S. et al. (1987) Cell 51, 7-19 30 Hirose, S., Ravi, L., Hazra, S. V. and Medof, M. E. (1991) Proc. Natl Acad. Sci. USA 88, 3762-3766
Figure 4 Cell fusion experiments show that Ptdlns-PLC sensitivity (the hallmark of the absence of acylinosito!) behaves as a dominant trait. When (a) a cell that synthesizes Ptdlns-PLCresistant anchored proteins is fused with (b), a cell that synthes!zes Ptdlns-PLC-sensitive anchored proteins, stable hybrids [(c)] express Ptdlns-PLC-sensitive proteins. The simplest explanation of this observation is that the GPI unit that resists Ptdlns~PLC because of the presence of an inositol-linked, third acyl chain, is deacylated in cells that express a deacylase (e.g. murine lymphomas, CHO cells) but not in other cells (L929 fibroblasts, red blood cell precursors). As illustrated, the deacylation might occur at the level of the anchor precursor or just after anchor transfer to protein. This result is not consistent with a model in which the second class of cell produces an anchor inositol acyl transferase which is absent from the first cell type. In each oval cell, an element of rough endoplasmic reticulure (ER) is illustrated. Within it are 'three-legged' and/or 'two-legged' GPI units and/or GPIanchored proteins. The 'three-legged' units are those which include an acyl chain linked to inositol, in addition to the glycerol acyl and/or alkyl substituents. In cell (a), the third chain is never removed, while in (b) and (c) it is removed just 5efore or after addition to protein.
473
