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Addition of a Glycophosphatidylinositol to Acetylcholinesterase

2001; Elsevier BV; Volume: 276; Issue: 30 Linguagem: Inglês

10.1074/jbc.m010817200

1083-351X

Françoise Coussen, Annick Ayon, Anne Le Goff, Jacqueline Leroy, Jean Massoulié, Suzanne Bon,

Computational Drug Discovery Methods

We introduced various mutations and modifications in the GPI anchoring signal of rat acetylcholinesterase (AChE). 1) The resulting mutants, expressed in transiently transfected COS cells, were initially produced at the same rate, in an active form, but the fraction of GPI-anchored AChE and the steady state level of AChE activity varied over a wide range. 2) Productive interaction with the GPI addition machinery led to GPI anchoring, secretion of uncleaved protein, and secretion of a cleaved protein, in variable proportions. Unproductive interaction led to degradation; poorly processed molecules were degraded rather than retained intracellularly or secreted. 3) An efficient glypiation appeared necessary but not sufficient for a high level of secretion; the cleaved, secreted protein was possibly generated as a by-product of transamidation. 4) Glypiation was influenced by a wider context than the triplet ω/ω + 1/ω + 2, particularly ω − 1. 5) Glypiation was not affected by the closeness of the ω site to the α10 helix of the catalytic domain. 6) A cysteine could simultaneously form a disulfide bond and serve as an ω site; however, there was a mutual interference between glypiation and the formation of an intercatenary disulfide bond, at a short distance upstream of ω. 7) Glypiation was not affected by the presence of an N-glycosylation site at ω or in its vicinity or by the addition of a short hydrophilic, highly charged peptide (FLAG; DYKDDDDK) at the C terminus of the hydrophobic region. We introduced various mutations and modifications in the GPI anchoring signal of rat acetylcholinesterase (AChE). 1) The resulting mutants, expressed in transiently transfected COS cells, were initially produced at the same rate, in an active form, but the fraction of GPI-anchored AChE and the steady state level of AChE activity varied over a wide range. 2) Productive interaction with the GPI addition machinery led to GPI anchoring, secretion of uncleaved protein, and secretion of a cleaved protein, in variable proportions. Unproductive interaction led to degradation; poorly processed molecules were degraded rather than retained intracellularly or secreted. 3) An efficient glypiation appeared necessary but not sufficient for a high level of secretion; the cleaved, secreted protein was possibly generated as a by-product of transamidation. 4) Glypiation was influenced by a wider context than the triplet ω/ω + 1/ω + 2, particularly ω − 1. 5) Glypiation was not affected by the closeness of the ω site to the α10 helix of the catalytic domain. 6) A cysteine could simultaneously form a disulfide bond and serve as an ω site; however, there was a mutual interference between glypiation and the formation of an intercatenary disulfide bond, at a short distance upstream of ω. 7) Glypiation was not affected by the presence of an N-glycosylation site at ω or in its vicinity or by the addition of a short hydrophilic, highly charged peptide (FLAG; DYKDDDDK) at the C terminus of the hydrophobic region. glycophosphatidylinositol acetylcholinesterase phosphatidylinositol-specific phospholipase C Many proteins are anchored at the cell surface through a glycophosphatidylinositol (GPI)1 that is covalently attached to their C terminus (1Low M.G. Biochem. J. 1987; 244: 1-13Crossref PubMed Scopus (408) Google Scholar, 2Ferguson M.A.J. Williams A.F. Annu. Rev. Biochem. 1988; 57: 285-320Crossref PubMed Scopus (952) Google Scholar, 3Ferguson M.A. J. Cell Sci. 1999; 112: 2799-2809Crossref PubMed Google Scholar). GPI-anchored proteins are recruited to glycosphingolipid/cholesterol-rich membrane microdomains (4Friedrichson T. Kurzchalia T.V. Nature. 1998; 394: 802-805Crossref PubMed Scopus (479) Google Scholar, 5Kenworthy A.K. Petranova N. Edidin M. Mol. Biol. Cell. 2000; 11: 1645-1655Crossref PubMed Scopus (391) Google Scholar, 6Pralle A. Keller P. Florin E.L. Simons K. Horber J.K. J. Cell Biol. 2000; 148: 997-1008Crossref PubMed Scopus (845) Google Scholar), where they may interact functionally with molecules involved in intracellular signal transduction; this is, for example, the case of the APP protein, the precursor of the β-amyloid peptide that forms amyloid deposits in Alzheimer's disease (7Mouillet-Richard S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (678) Google Scholar). GPI anchoring also seems to be directly related with the pathological misfolding of the PrP prion protein (8Lehmann S. Harris D.A. J. Biol. Chem. 1995; 270: 24589-24597Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Glypiation, the process of GPI addition, implies the cleavage of a C-terminal peptide and the concerted linkage of a preassembled GPI anchor, forming an amide bond between an ethanolamine moiety and the carboxylic group of the ω residue, at the C terminus of the mature protein. The structural requirements of the C-terminal signal peptides that induce GPI addition have been investigated extensively by the groups of Caras and Udenfriend, by mutagenesis of the "decay-accelerating factor" (9Caras I.W. J. Cell Biol. 1991; 113: 77-85Crossref PubMed Scopus (34) Google Scholar, 10Moran P. Caras I.W. J. Cell Biol. 1991; 115: 1595-1600Crossref PubMed Scopus (53) Google Scholar, 11Moran P. Caras I.W. J. Cell Biol. 1992; 119: 763-772Crossref PubMed Scopus (50) Google Scholar, 12Moran P. Caras I.W. J. Cell Biol. 1994; 125: 333-343Crossref PubMed Scopus (104) Google Scholar) and of placental alkaline phosphatase (13Micanovic R. Gerber L.D. Berger J. Kodukula K. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 157-161Crossref PubMed Scopus (78) Google Scholar, 14Kodukula K. Gerber L.D. Amthauer R. Brink L. Udenfriend S. J. Cell Biol. 1993; 120: 657-664Crossref PubMed Scopus (101) Google Scholar, 15Maxwell S.E. Ramalingam S. Gerber L.D. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1550-1554Crossref PubMed Scopus (40) Google Scholar, 16Udenfriend S. Kodukula K. Annu. Rev. Biochem. 1995; 64: 563-591Crossref PubMed Scopus (435) Google Scholar, 17Ramalingam S. Maxwell S.E. Medof M.E. Chen R. Gerber L.D. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7528-7533Crossref PubMed Scopus (31) Google Scholar). They showed that glypiation requires a C-terminal hydrophobic sequence and an upstream cleavage/addition site (2Ferguson M.A.J. Williams A.F. Annu. Rev. Biochem. 1988; 57: 285-320Crossref PubMed Scopus (952) Google Scholar, 13Micanovic R. Gerber L.D. Berger J. Kodukula K. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 157-161Crossref PubMed Scopus (78) Google Scholar). The two groups found that positions ω and ω + 1 (according to the group of Caras) or ω and ω + 2 (according to the group of Udenfriend) must be occupied by residues with small side chains. In addition, Caras and colleagues found that, for optimal processing, the ω site should be located between 10 and 12 residues upstream of the C-terminal hydrophobic sequence (10Moran P. Caras I.W. J. Cell Biol. 1991; 115: 1595-1600Crossref PubMed Scopus (53) Google Scholar). A systematic analysis of all reported GPI-anchored proteins and of the effects of mutations in their C-terminal region has led Eisenhaber et al. (18Eisenhaber B. Bork P. Eisenhaber F. Protein Eng. 1998; 11: 1155-1161Crossref PubMed Google Scholar, 19Eisenhaber B. Bork P. Eisenhaber F. J. Mol. Biol. 1999; 292: 741-758Crossref PubMed Scopus (363) Google Scholar) to formulate a prediction algorithm, which confirmed that the volumes of the side chains located near the cleavage site exert a major influence, probably because they must be accommodated within the catalytic pocket of a transamidase (15Maxwell S.E. Ramalingam S. Gerber L.D. Udenfriend S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1550-1554Crossref PubMed Scopus (40) Google Scholar). Several components of the transamidase complex have recently been cloned (20Riezman H. Conzelmann A. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 756-759Google Scholar, 21Ohishi K. Inoue N. Maeda Y. Takeda J. Riezman H. Kinoshita T. Mol. Biol. Cell. 2000; 11: 1523-1533Crossref PubMed Scopus (107) Google Scholar). In mammals, acetylcholinesterase (AChE; EC 3.1.1.7) subunits containing the alternative C-terminal peptide H (AChEH) produce GPI-anchored dimers (22Massoulié J. Pezzementi L. Bon S. Krejci E. Vallette F.M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1050) Google Scholar, 23Massoulié J. Anselmet A. Bon S. Krejci E. Legay C. Mayat E. Morel N. Simon S. Doctor B.P. Quinn D.M. Rotundo R.L. Taylor P. Structure and Function of Cholinesterases and Related Proteins. Plenum Press, New York1998: 3-24Crossref Google Scholar, 24Futerman A.H. Low M.G. Silman I. Neurosci. Lett. 1983; 40: 85-89Crossref PubMed Scopus (73) Google Scholar, 25Futerman A.H. Low M.G. Ackermann K.E. Sherman W.R. Silman I. Biochem. Biophys. Res. Commun. 1985; 129: 312-317Crossref PubMed Scopus (106) Google Scholar); this peptide is sufficient to induce the addition of a GPI anchor when added to a foreign protein (26Duval N. Krejci E. Grassi J. Coussen F. Massoulié J. Bon S. EMBO J. 1992; 11: 3255-3261Crossref PubMed Scopus (39) Google Scholar). The sequences encoded by the alternative H exons of Torpedo and rat AChEs contain one or two cysteines, which form intersubunit disulfide bonds in AChE dimers, and hydrophobic C-terminal regions of 15 or 19 residues. Otherwise, the sequences ofTorpedo and mammalian H peptides do not appear homologous and may have arisen independently in the AChE genes (23Massoulié J. Anselmet A. Bon S. Krejci E. Legay C. Mayat E. Morel N. Simon S. Doctor B.P. Quinn D.M. Rotundo R.L. Taylor P. Structure and Function of Cholinesterases and Related Proteins. Plenum Press, New York1998: 3-24Crossref Google Scholar). By introducing threonines at different positions in TorpedoAChE, Bucht and Hjalmarsson found that the last two of a group of three consecutive serines (Ser7–Ser9) could function as ω sites (27Bucht G. Hjalmarsson K. Biochim. Biophys. Acta. 1996; 1292: 223-232Crossref PubMed Scopus (15) Google Scholar). In the case of mammalian AChE, biochemical analyses of C-terminal peptides from the human erythrocyte enzyme showed that the GPI anchor is linked to glycine 14 in the H peptide (28Roberts W.L. Rosenberry T.L. Biochemistry. 1986; 25: 3091-3098Crossref PubMed Scopus (34) Google Scholar, 29Haas R. Brandt P.T. Knight J. Rosenberry T.L. Biochemistry. 1986; 25: 3098-3105Crossref PubMed Scopus (57) Google Scholar, 30Haas R. Jackson B.C. Reinhold B. Foster J.D. Rosenberry T.L. Biochem. J. 1996; 314: 817-825Crossref PubMed Scopus (24) Google Scholar). In the present work, the H peptide of rat AChE was mutated or replaced by Torpedo or composite C-terminal regions, and we report the effects of these modifications on the production of GPI-anchored AChE, on the level of active AChE in cells, and on its secretion in the culture medium. The cDNA encoding the rat AChE subunit was inserted in the pEF-BOS vector, under the control of the human EF-10c promotor (31Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1502) Google Scholar). Site directed mutagenesis was performed as described previously (32Bon S. Massoulié J. J. Biol. Chem. 1997; 272: 3007-3015Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In the case of rat AChE with chimericTorpedo/rat C-terminal peptides, we removed the noncoding regions, so that all constructs were identical, except for the 3′ sequence, encoding the C-terminal peptides. For transfections, DNA was purified on Nucleobond AX columns (Macherey-Nagel). COS-7 cells were transfected by the diethylaminoethyl-dextran method, as described previously (33Selden R.F. Howie K.B. Rowe M.E. Goodman H.M. Moore D.D. Mol. Cell. Biol. 1986; 6: 3173-3179Crossref PubMed Scopus (471) Google Scholar). For cultures of transfected cells, the fetal calf serum and the Nu-serum were treated with soman (10−6m) to block irreversibly any cholinesterase activity; this treatment was performed at least 1 week before use, so that excess soman was hydrolyzed during storage at 4 °C. The cells were usually extracted 2 or 3 days after transfection. The cells were extracted with TMg buffer (1% Triton X-100, 50 mm Tris-HCl, pH 7.5, 10 mmMgCl2) at 20 °C, because the sphingolipid/cholesterol microdomains are partially insoluble in Triton X-100 in the cold. For PI-PLC digestion, detergent extracts were incubated in TMg buffer for 1 h at 30 °C with 3 units/ml of PI-PLC from Bacillus thuringiensis (Glyko Europe, Upper Heyford, United Kingdom). To ensure that digestion was complete, some samples were incubated a second time, after the addition of the same quantity of fresh PI-PLC. Control extracts were incubated in the same conditions without PI-PLC. Solubilization of cell surface GPI-anchored AChE was performed by treating intact cells with the same concentration of PI-PLC for 20 min at 37 °C; the released activity was assayed in the medium after centrifugation at 17,000 × g for 15 min to remove cell debris. The AChE activity was assayed by the colorimetric method of Ellmanet al. (34Ellman G.L. Courtney K.D. Andres V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21500) Google Scholar). Enzyme samples (10 µl) were added to 0.2 ml of Ellman assay medium, and the reaction kinetics was monitored at 414 nm, at 15-s intervals for 3 min, using a Multiskan RC microplate reader (Labsystems, Helsinki, Finland). All mutants were expressed at least five times and most were expressed more than 10 times in independent transfections that included different sets of mutants used for various comparisons; experimental variations in the relative levels of activity and the fraction of PI-PLC-sensitive AChE did not exceed 15%. The indicated values were obtained from a representative experiment that included all mutants shown in a given table or figure. Aliquots of extracts were equilibrated with 1% Brij-96, loaded on 5–20% sucrose gradients in 1% Brij-96, 10 mm MgCl2, 25 mm Tris-HCl, pH 7.Escherichia coli β-galactosidase (16 S) and alkaline phosphatase (6.1 S) were included as internal sedimentation standards. The gradients were centrifuged for 18 h at 36,000 rpm in a SW-41 rotor, at 5 °C. Fractions of 300 µl were collected and assayed for AChE, β-galactosidase and alkaline phosphatase activities. Electrophoresis of active AChE was performed in nondenaturing conditions, in 7.5% horizontal polyacrylamide gels, in the presence of detergent, as described by Bonet al. (35Bon S. Toutant J.P. Méflah K. Massoulié J. J. Neurochem. 1988; 51: 786-794Crossref PubMed Scopus (57) Google Scholar). AChE activity was revealed after electrophoresis by the method of Karnovsky and Roots (36Karnovsky M.J. Roots L. J. Histochem. Cytochem. 1964; 12: 219-222Crossref PubMed Scopus (2996) Google Scholar). The gels were scanned and quantified with the TINA software (version 2.07d, Raytest Isotopenmessgeräte GmbH) to determine the relative intensities of each band. The percentage of lytic nonamphiphilic component preexisting before PI-PLC treatment (L) and of PI-PLC-resistant amphiphilic component (R) were determined from the profiles obtained for control and PI-PLC-treated samples, respectively; the fraction of GPI-anchored AChE was obtained as 100% − (L + R). Two days after transfection, COS cells were preincubated for 45 min in Dulbecco's modified Eagle's medium without cysteine and methionine and labeled for the indicated time with 150 mCi/100-mm dish of [35S]methionine/cysteine (Amersham Pharmacia Biotech). After labeling, the cells were rinsed with PBS and chased in medium containing Nu-serum. AChE from cell extracts or medium was immunoadsorbed on protein G immobilized on Sepharose 4B Fast Flow beads (Sigma). The beads were first washed and saturated with 5% bovine serum albumin in a buffer containing 150 mm NaCl, 5 mm EDTA, 50 mm Tris-HCl, pH 7.4, 0.05% Nonidet P-40. Samples (90 µl) of cell extracts or media were incubated with 40 µl of a 10% suspension of beads for 3 h to eliminate nonspecific adsorption and the beads were discarded. The samples were then incubated with 1:500 A63 anti-AChE antiserum (37Marsh D. Grassi J. Vigny M. Massoulié J. J. Neurochem. 1984; 43: 204-213Crossref PubMed Scopus (113) Google Scholar) or with 1:250 anti-FLAG M2 monoclonal antibodies (Eastman Kodak Co.), overnight at 8 °C, with gentle agitation on a rotating wheel; 80 µl of a 10% suspension of bovine serum albumin-saturated washed beads was then added and incubated for 1 h. After immunoadsorption, the beads were washed three times with 1 ml of buffer containing 1% Triton X-100, with centrifugations at 17,000 × g for 5 min. All incubations were performed at 8 °C under mild rotatory agitation. For polyacrylamide electrophoresis under denaturing conditions, samples of the washed beads were resuspended in 30 µl of 0.125 mTris-HCl buffer, pH 6.8, containing 1% SDS, 0.002% bromphenol blue, 5% mercaptoethanol, heated at 98 °C for 5 min, and centrifuged at 17,000 × g for 5 min at room temperature. Aliquots (10 µl) of the supernatant were submitted to electrophoresis in SDS-polyacrylamide gels, and the resulting bands were analyzed in a Fuji image analyzer (BAS 1000) or by autoradiography. The predictor is accessible on the World Wide Web (19Eisenhaber B. Bork P. Eisenhaber F. J. Mol. Biol. 1999; 292: 741-758Crossref PubMed Scopus (363) Google Scholar). The H peptides correspond to amino acids 536–577 of rat AChE and 536–566 of TorpedoAChE (numbering of Torpedo AChE), but for simplicity we use numbering from their first residue (TablesIandII). The rat H peptide contains two cysteines (Cys6 and Cys8), each of which is sufficient for the formation of disulfide-linked GPI-anchored dimers. 2S. Bon, unpublished result. The structure of the mutants is shown in Tables I, II, andIII. Mutations that were restricted to the C-terminal peptide did not modify the catalytic activity of rat AChE. Mutants containing a GPI signal derived from the rat H peptide are designated rm2 to rm44 (for "rat mutant," rm1 being the wild type). Mutants rm2–rm40 contain mutations around the ω site and sometimes minor insertions or deletions. Mutants rm41–rm43 were deleted to place the ω site at the position of Cys6. The rm44 mutant contains an internal FLAG peptidic epitope. The FLAG peptide was also added at the C terminus of some mutants (rm1-f, rm5-f, rm13-f, and rm18-f). The Torpedo H peptide is totally different from that of rat; mutagenesis showed that each of its first three consecutive serines (residues 542–544) can serve as an ω site. The catalytic domain of rat AChE was associated with Torpedoand with chimeric rat/Torpedo GPI addition signals (rr, rt, tr, and tt), which were made in long (L) and short (S) versions, by insertion or deletion of five residues (Table II, top). Composite constructs (cc) contained modified TorpedoGPI-addition signals (Table II, bottom). Finally, we analyzed mutants in which a few residues of the catalytic domain were deleted, to reduce the distance between the α10 helix and the ω site (Table III). Throughout, we indicate the immediate peptidic environment of the putative ω site (ω − 1/ω + 3), underlining the ω site itself or underlining its position when it is inactivated.Table IStructure of the modified rat GPI addition signalsNameω − 1/ω + 3MutationsCommentsCell. act.Glyp.Secr. act.Score%%%rm1HGEAAWild typeProline at 40, as in mouse100751006.7rm1′HGEAAC8SWild type with only the first cysteine100751006.2rm1"HGEAAS25PSuppression of a potential ω site at Ser25100751005.2rm2HPEAAG14PSuppression of the natural ω site Gly14312111−3rm3HPEAAG14S/S25PSuppression of the two preceding sites242211−4.7rm4HGEPAA16PProline at ω + 219327−3.9rm5HGEAAA12TThreonine at ω − 29661536.7rm6HPEAAT7P/G14P/S25PProlines at 7, 14, and 2520911−4.3rm7HPEPAG14P/A16P/S25PProlines at 14, 16, and 251527−5.2rm8HGNASE15N/A17SPotentialN-glycosylation site at ω + 1917011311.6rm9HGNAAE15NNon glycosylable control of rm81096511114rm10HGEASA17SNon glycosylable control of rm886621277.3rm11HNEAAG14NReplacement of Gly by Asn at the ω site112681046.7rm12HNESGNESGT at 14–18, S25PPotentialN-glycosylation at ω site127661736.8Nameω − 1/ω + 3CommentsCell. act.Glyp.Secr. act.Scorerm13PSPTRMutant showing essentially no glypiation250.411−3.4rm14ESGSRSGS at ω/ω + 1/ω + 27565226.5rm15ESGTRSGT at ω/ω + 1/ω + 25654206.9rm16ESEGRSEG at ω/ω + 1/ω + 2 (breaks the ω + 1 rule)3021164.6rm17ESGERSGE at ω/ω + 1/ω + 2 (breaks the ω + 2 rule)302916−4.2rm18EGGTRGGT at ω/ω + 1/ω + 23125164.1rm19EGEARGEA at ω/ω + 1/ω + 2 (as in the wild type)197.591.6rm20ECEARCEA at ω/ω + 1/ω + 2250.992.4rm21PSGTRLike rm15, but with proline at ω − 15354183.6rm22ESGTPEffect of a proline at ω + 2 (compare with rm15)115752227.3rm23HNGGREffect of an histidine at ω − 1 (compare with rm28)137671025.8rm24HCGGREffect of a cysteine at ω (compare with rm41)2512166.0rm25ESGSSSGS at ω/ω + 1/ω + 2 (nonglycosylable control of rm26)12375228.1rm26ENGSRPotentialN-glycosylation at the engineered ω site8762224.9rm27EGGSRNonglycosylable control of rm263030184.4rm28ENGGRNonglycosylable control of rm268762227.0rm29ETGGRSuppression of the preceding ω site by a threonine111691.2rm30HGGSREffect of an histidine at ω − 1 (compare with rm27)7656424.4rm31HSGTREffect of an histidine at ω − 1 (compare with rm15)142721165.0rm32HGEAREffect of an histidine at ω − 1 (compare with rm19)404036−0.4rm33CGEAREffect of a cysteine at ω − 1 (compare with rm19)363418−2.0rm34KGEAREffect of a lysine at ω − 1 (compare with rm19)8856491.3Nameω − 1/ω + 3Sequence of ω regionCommentsCell. act.Glyp.Secr. act.Scorerm35HNGGRPCTCPSPAH––NGGRPGPALPResidues preceding ω as in wild type112801076.7rm36HGEAAPCTCPSPAHGEAAPRPGPA––Reduced spacer after ω84661110.5rm37HGSSPPCTCPSPAH–––GSSPGPALPω and upstream as in wild type5666716.2rm38HGNAPPCTCPSPAH–––GNAPGPALPω and upstream as in wild type93651133.4rm39ENGGRPCTCPSPAHGPENGGRPGPALPWild type sequence upstream of ω6766367.2rm40ETGGRPCTCPSPAHGPETGGRPGPALPSuppression of preceding ω site3732181.4rm41HCGGR––––––––––HCGGRPGPALPDisulfide bond by ω site cysteine?2575406.5rm42HNGGR––––––––––HNGGRPGPALPControl of preceding: no cysteine97901116.3rm43HTGGR––––––––––HTGGRPGPALPControl of rm41, suppression of ω3165430.6rm44HGEAAPCTCPSPAHGEAAPRPGPInsertion of FLAG peptide upstream of the346028−19.5DYKDDDDKALShydrophobic regionThe C-terminal peptides of mutants rm1 to rm44 are derived from the rat H peptide, except that the third residue before the C terminus is a proline rather than an arginine, as in mice (48Li Y. Camp S. Rachinsky T.L. Getman D. Taylor P. J. Biol. Chem. 1991; 266: 23083-23090Abstract Full Text PDF PubMed Google Scholar). The sequence of the wild type H peptide of rat AChE (the cysteines are doubly underlined, the ω site is underlined, and the hydrophobic region is shown in boldface type) is as follows.Numbering of the mature AChE is indicated on the line (Torpedo numbering), and numbering of the H peptide is indicated below. In the second column, the predicted ω site position is underlined. In the top of Table I, mutants rm1 to rm12 contain point mutations in the wild type GPI-addition signal. The columns on the right indicate the level of cellular activity (cell act.), as a percentage of the wild type, the proportion of GPI-anchored AChE in the cell extracts (Glyp.), the level of secreted activity as a percentage of the wild type (Secr. act.), and the score given by the big-PI algorithm. In the middle part, mutants rm13 to rm34 differ only in the 15–19 interval, the rest of the C-terminal peptide being identical: ATEVPCTCPSPAHP [.....] PGPALPLSLLFFLFLLHSGLPWL. The bottom of the table shows mutants rm35 to rm44 which contain deletions (dashes) or insertions (doubly underlined). All of these mutants share the same N- and C-terminal segments: ATEV [.....]LSLLFFLFLLHSGLPWL. In mutants rm1-f, rm5-f and rm13-f, the FLAG epitope was added at the C terminus (not illustrated). Open table in a new tab Table IIChimeric rat/Torpedo GPI addition signals, composite constructs, and deletions between the α10 helix and the ω siteNameω − 1/ω + 3C-terminal sequenceActivityGlypiationSecreted activityScore%%%rrLHGEAAPSPAHGEAAPRPGPALSLSLLFFLFLLHSGLPWL100641006.7rtLHGEAASPAHGEAAPRPGPALSLSIIFYVLFSILYLIFY108571392.3trLLSSSGTDGELSSSGTSSSKGALSLSLLFFLFLLHSGLPWL1164718119.8ttLLSSSGTDGELSSSGTSSSKG ALSLS IIFYVLFSILYLIFY965017515.4rrSHGEAAPSPAHGEAAPRPGP–––––LLFFLFLLHSGLPWL5152400.9rtSHGEAAPSPAHGEAAPRPGP–––––IIFYVLFSILYLIFY97521222.1trSLSSSGTDGELSSSGTSSSKG LLFFLFLLHSGLPWL50334014ttSLSSSGTDGELSSSGTSSSKGIIFYVLFSILYLIFY1105514915.2Nameω − 1/ω + 3Sequence [.....]CommentsActivityGlypiationSecreted activityScore%%%ccLLSPSPQEVLPLEIKPTEPSPILS8468100−1.7ccL*LTPSPQEVLPLEIKPTEPSPILTSuppression of preceding ω1300.2−13.3ccSLSPSP–––––––––––––––––SDeletion in cmL(S)494923−1.8ccS*LTPSP–––––––––––––––––TSuppression of preceding ω13010−13.1Chimeric constructs are shown at the top. The GPI addition signal is composed of intervening regions (located between the cysteine and the hydrophobic region) and hydrophobic regions from rat (r) orTorpedo (t). They are called rr, rt, tr, and tt, either long (L) or short (S), depending on the deletion of five residues (ALSLS) at the boundary between the rat spacer and hydrophobic region or insertion of the same residues in Torpedo (doubly underlined), thus elongating the hydrophobic region. These chimeric peptides were added downstream of Cys6 from the rat H peptide, so that the formation of an intersubunit disulfide bond should be identical in all cases. The Torpedo regions are shown in italics. The sequence of the wild type H peptide of Torpedo AChE, with numbering of Torpedo AChE (the possible natural ω sites are underlined) is as follows:536AC¯DGELSSS¯GTSSSKGIIFYVLFSILYLIFY566Composite constructs are shown at the bottom. The long and short composite constructs (ccL, ccS) were partly derived from the QN/HC protein, in which the N-terminal region of ColQ was fused to the Torpedo GPI addition signal (26Duval N. Krejci E. Grassi J. Coussen F. Massoulié J. Bon S. EMBO J. 1992; 11: 3255-3261Crossref PubMed Scopus (39) Google Scholar). The sequence of these constructs is as follows: ATEVPCTETNIL [.....] PSPTPSPKGIIFYVLFSILYLIFY, the brackets containing the peptides shown in the table. In ccL and ccS, the functional ω site is a serine, as demonstrated by its replacement with a threonine (ccL* and ccS*). Open table in a new tab Table IIIDeletions between helix α10 and the ω site and corresponding truncated constructsNameω − 1/ω + 3C-terminal sequenceGlypiationScore%rm35HNGGRLPKLLSATATEVPCTCPSPAHNGGRPGPALPLSLLFFLFLLHSGLPWL756.7rm42HNGGRLPKLLSATATEV––––––––HNGGRPGPALPLSLLFFLFLLHSGLPWL906.3D1HNGGRLPKLLSA–––––––––––––HNGGRPGPALPLSLLFFLFLLHSGLPWL854.6D2HNGGRLPKLL–––––––––––––––HNGGRPGPALPLSLLFFLFLLHSGLPWL932.6D3HNGGRLPK–––––––––––––––––HNGGRPGPALPLSLLFFLFLLHSGLPWL962.9NameC-terminal sequenceCommentsCellular activitySecreted activityS1..LPKLLHN-stopSame mature protein as for D2, but without a GPI100100S2..LPKLL-stopSame as preceding, with leucines 536–537 but without HN8788S3..LPKHN-stopSame mature protein as for D3, but without a GPI7840S4..LPK-stopMature protein without leucines 536–5377038The sequence shown at the top starts at Leu528(Torpedo numbering) and thus includes the end of helix α10, which is shown in italics (LPKL). The mutants contain an ω site asparagine with the same immediate environment, at various distances from the catalytic domain. The bottom of the table shows truncated constructs, expressed as controls for the effect of deletions at the C terminus of the catalytic domain. The cellular and secreted activities produced by the truncated constructs are expressed as percentage of S1, which corresponds to the mature D2 enzyme, but without a GPI anchor. For all truncated mutants, the cellular activity represented less than 5% of the secreted activity, 2 days after transfection. Open table in a new tab The C-terminal peptides of mutants rm1 to rm44 are derived from the rat H peptide, except that the third residue before the C terminus is a proline rather than an arginine, as in mice (48Li Y. Camp S. Rachinsky T.L. Getman D. Taylor P. J. Biol. Chem. 1991; 266: 23083-23090Abstract Full Text PDF PubMed Google Scholar). The sequence of the wild type H peptide of rat AChE (the cysteines are doubly underlined, the ω site is underlined, and the hydrophobic region is shown in boldface type) is as follows.Numbering of the mature AChE is indicated on the line (Torpedo numbering), and numbering of the H peptide is indicated below. In the second column, the predicted ω site position is underlined. In the top of Table I, mutants rm1 to rm12 contain point mutations in the wild type GPI-addition signal. The columns on the right indicate the level of cellular activity (cell act.), as a percentage of the wild type, the proportion of GPI-anchored AChE in the cell extracts (Glyp.), the level of secreted activity as a percentage of the wild type (Secr. act.), and the score given by the big-PI algorithm. In the middle part, mutants rm13 to rm34 differ only in the 15–19 interval, the rest of the C-terminal peptide being identical: ATEVPCTCPSPAHP [.....] PGPALPLSLLFFLFLLHSGLPWL. The bottom of the table shows mutants rm35 to rm44 which contain deletions (dashes) or insertions (doubly underlined). All of these mutants share the same N- and C-terminal segments: ATEV [.....]LSLLFFLFLLHSGLPWL. In mutants rm1-f, rm5-f and rm13-f, the FLAG epitope was added at the C terminus (not illustrated). Chimeric constructs are shown at the top. The GPI addition signal is composed of intervening regions (located betw