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A Mutation Linked with Autism Reveals a Common Mechanism of Endoplasmic Reticulum Retention for the α,β-Hydrolase Fold Protein Family *

A Mutation Linked with Autism Reveals a Common Mechanism of Endoplasmic Reticulum Retention for... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 14, pp. 9667–9676, April 7, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. A Mutation Linked with Autism Reveals a Common Mechanism of Endoplasmic Reticulum Retention for the ,-Hydrolase Fold Protein Family Received for publication, September 19, 2005, and in revised form, January 10, 2006 Published, JBC Papers in Press, January 24, 2006, DOI 10.1074/jbc.M510262200 ‡ ‡ ‡1 § ‡ ¶ Antonella De Jaco , Davide Comoletti , Zrinka Kovarik , Guido Gaietta , Zoran Radic´ , Oksana Lockridge , § ‡2 Mark H. Ellisman , and Palmer Taylor ‡ § From the Departments of Pharmacology and Neurosciences and National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California 92093-0636 and Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 A mutation linked to autistic spectrum disorders encodes an neuroligin-3 (NL-3) at position 451 near the C terminus of the extra- Arg to Cys replacement in the C-terminal portion of the extra- cellular domain (1). This mutation, identified in an affected twin set, has cellular domain of neuroligin-3. The solvent-exposed Cys causes been shown to give rise to an altered cellular phenotype. The protein is virtually complete retention of the protein in the endoplasmic largely retained intracellularly in the endoplasmic reticulum (ER), with reticulum when the protein is expressed in transfected cells. An little of the nascent protein reaching its cell membrane location (3, 4). In identical Cys substitution was reported for butyrylcholinesterase addition, the residual protein that reaches the cell surface has an altered through genotyping patients with post-succinylcholine apnea. affinity for its cognate partner, -neurexin (3). When Arg-451 (rat num- Neuroligin, butyrylcholinesterase, and acetylcholinesterase are bering, Arg-471) is mutated to a Thr or a Glu, the mutant NL is exported members of the ,-hydrolase fold family of proteins sharing normally but shows significantly lower affinity for -neurexins (3). Mass sequence similarity and common tertiary structures. Although spectrometry analysis of the secreted fraction of the R471C-NL3 these proteins have distinct oligomeric assemblies and cellular mutant indicated that the mutated protein had an unaltered disulfide dispositions, homologous Arg residues in neuroligin-3 bonding pattern (3). (Arg-451), in butyrylcholinesterase (Arg-386), and in acetylcho- Arg-451 is highly conserved within the NL family and across mam- linesterase (Arg-395) are conserved in all studied mammalian malian species. This residue is also conserved in many other ,-hydro- species. To examine whether an homologous Arg to Cys muta- lase fold proteins. Recently, a cysteine substitution for the conserved tion affects related proteins similarly despite their differing arginine in butyrylcholinesterase (BChE) was found as an infrequent capacities to oligomerize, we inserted homologous mutations in mutation in patient populations with post-succinylcholine apnea (5–7). the acetylcholinesterase and butyrylcholinesterase cDNAs. BChE appears as a soluble tetramer in the plasma but is synthesized in Using confocal fluorescence microscopy and analysis of oligosac- liver (8). The physiological function of BChE is not clear; nevertheless, charide processing, we find that the homologous Arg to Cys the enzyme has proven critical in inactivating various administered mutation also results in endoplasmic reticulum retention of the drugs such as succinylcholine (9). two cholinesterases. Small quantities of mutated acetylcholines- The cloning of the acetylcholinesterase (AChE) gene 2 decades ago terase exported from the cell retain activity but show a greater revealed a new protein family structurally distinct from other hydrolases of K , a much smaller k , and altered substrate inhibition. The the then known structures (10), and homology of AChE to a large domain in m cat nascent proteins associate with chaperones during processing, thyroglobulin suggested a new superfamily of proteins with diverse func- but the mutation presumably restricts processing through the tions extending beyond hydrolytic reactions (10). Subsequently a variety of endoplasmic reticulum and Golgi apparatus, because of local homologous hydrolases have been identified that fall in this family as do protein misfolding and inability to oligomerize. The mutation several proteins that lack hydrolase activity but function as adhesion pro- may alter the capacity of these proteins to dissociate from their teins (11–13). Among these, the neuroligins are the only ones of mamma- chaperone prior to oligomerization and processing for export. lian origin that have been identified and characterized to date (14, 15). The commonality of tertiary structure has enabled one to classify these proteins as members of the ,-hydrolase fold family (16). The neuroligins are a family of multidomain transmembrane- Several mutations that were found in human neuroligin 3 and 4 genes spanning proteins expressed on the postsynaptic side of the synapse appear to be associated with the autistic spectrum disorders (1, 2). One (14, 17, 18). The N-terminal extracellular region is homologous to of the more interesting mutations is an Arg to Cys mutation found in AChE and serves as the extracellular recognition portion of the mol- ecule. C-terminal to this recognition domain is a linking O-glycosy- * This work was supported by the Rotary Foundation Ambassadorial Scholar Fellowship lation-rich region followed by a transmembrane span and a cytoplas- (to A. D. J.), National Alliance for Autism Research Grant 843 (to D. C.), National Insti- mic region with recognition capacity for PDZ proteins. The tutes of Health, Division of Research Resources grant (to M. H. E.), and United States Public Health Service Grants R37-GM18360 and P42-ES 10337 (to P. T.). The costs of association of NL with -neurexin and perhaps -neurexin provides publication of this article were defrayed in part by the payment of page charges. This a potential trans-synaptic interaction. Accordingly, the neuroligins article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address: Institute for Medical Research and Occupational Health, P. O. 291, HR-10001 Zagreb, Croatia. The abbreviations used are: NL, neuroligin; AChE, acetylcholinesterase; BChE, butyryl- To whom correspondence should be addressed: Dept. of Pharmacology, University of cholinesterase; ER, endoplasmic reticulum; Endo H, endoglycosidase H; PNGase F, California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636. Tel.: 858-534-1366; N-glycosidase F, HEK, human embryonic kidney; GPI, glycosylphosphatidylinositol; Fax: 858-534-8248; E-mail: pwtaylor@ucsd.edu. RT, reverse transcriptase. APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9667 This is an Open Access article under the CC BY license. ER Retention of Cholinesterases presumably display a capacity to stimulate formation and matura- saline, and activity was measured on the surface of intact cells. Proteins tion of new excitatory and inhibitory synapses in the central nervous were extracted from cells in 20 mM sodium phosphate, pH 7.0, contain- system (19 –23). ing 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100 plus a mixture of Although the molecular details of the neuroligin-neurexin association protease inhibitors containing 5 g/ml each of pepstatin A, leupeptin, that underlie this function are not understood, a crystal structure of neur- and aprotinin, 10 g/ml bacitracin, and 0.01 mM benzamidine (33). exin has been reported (24), and the structure of neuroligin has been Cells were sonicated on ice for 10 min and then spun at 12,000 g for 10 inferred from its homology to AChE (25, 26). By constructing truncated, min. Supernatants containing all solubilized proteins were used for sub- soluble forms of neuroligin, we characterized the disulfide bonding and sequent experiments. glycosylation pattern of NL and demonstrated secondary structural fea- Assay of AChE and BChE Catalytic Activity—To avoid serum tures that are similar to AChE (26, 27). In contrast to NL, cholinesterases do AChE contamination, selected clones of transfected cells were main- not possess a transmembrane spanning region but rather associate with tained in serum-free Ultraculture medium supplemented with L-glu- membranes through either an attached glycophospholipid or by attach- tamine (Cambrex, Walkersville, MD). Media were collected at 48-h ment to structural subunits that associate with the plasma membrane or intervals, pooled for purification, and assayed for AChE or BChE basement membrane (13, 28). Moreover, the alternative splicing mecha- activity using 0.5 mM acetylthiocholine iodide or 5 mM butyrylthio- nism that controls the nature of the membrane attachment of AChE also choline iodide as substrates (34). Both media and cell-associated influences its degree of its oligomerization. Homomeric dimers and tetram- AChE activities were then normalized to the total cell protein con- ers have been identified, as have heteromeric associations between catalytic tent (35). To ascertain whether AChE expression could be enhanced and structural subunits (13, 29, 30). at low temperature, 80% confluent cells were cultured for 48 h at Accordingly, given the distinct assembly modes of this family of pro- 31 °C prior to harvesting. teins, we sought to determine whether a mutation at homologous resi- Gel Electrophoresis and Immunoblotting—Western blots were per- due positions in AChE and BChE would give rise to similar processing formed as described by Towbin et al. (36). Briefly, crude cell extracts (20 aberrations as those seen with the neuroligins. The data presented here g of total protein) or batch anti-FLAG immunoprecipitated proteins suggest that the mutation results in a common folding and processing from cell culture media were separated on 10% SDS-polyacrylamide gels deficiency. This particular mutation may also uncover mechanistic (Invitrogen). After transfer to a polyvinylidene difluoride membrane details essential for catalytic activity, subunit assembly, and oligomer- (Millipore, Bedford, MA), blocking of nonspecific binding was achieved ization of nascent proteins belonging to the ,-hydrolase fold family. by incubating the membrane with 50 mM Tris-HCl, pH 7.4, and 100 mM NaCl, containing 5% nonfat dry milk, and 0.1% Tween 20 for 1 h. Pri- EXPERIMENTAL PROCEDURES mary antibodies were diluted in blocking buffer as follows: anti-AChE Plasmids and Mutagenesis—A cDNA encoding mouse GPI-AChE polyclonal (37), 1:4000; anti-FLAG M2 monoclonal antibody (Sigma), was subcloned into a FLAG-tagged vector (Sigma) for detection and 1:1000; anti NL-1 monoclonal (Synaptic Systems, Goettingen, Ger- purification. The natural leader peptide of AChE was replaced by the many), 1:5000; anti-PTP-1B monoclonal (Calbiochem), 1:10,000; anti- pre-protrypsin leader peptide. The N-terminal FLAG is followed by a BChE monoclonal antibody, 1:1. Monoclonal antibody to human BChE linker peptide of 10 residues and by the AChE sequence beginning at was prepared by the Monoclonal Core Facility at the University of Glu-1 (sequence of the mature mouse AChE protein). Soluble mono- Nebraska Medical Center. The pure BChE protein used to form crystals meric mouse AChE (AChE-548) was constructed by introducing a stop (38) was injected into mice for production of antibodies. Human BChE codon at Cys-549 of mouse AChE-GPI as described previously (31). protein was truncated at residue 529 thereby missing residues 530–574. Arg-395 3 Cys (R395C) and Leu-386 3 Cys (L386C) mutations were It was also devoid of carbohydrate chains at Asn-17, -455, -481, and introduced using the QuikChange mutagenesis kit (Stratagene, San -486. Hybridoma clone C191 2.1.1 secreted the monoclonal antibody Diego, CA) and were subsequently subcloned into expression vectors. into culture medium. Antibody hybridization was detected with ECL Mutations were verified by automated sequencing. Plasmids were puri- (Pierce). fied using DEAE columns (Qiagen Inc., Valencia, CA). Rat NL1 and NL3 The proteasome inhibitor, lactacystin (10 M, Sigma), was applied to wild type constructs and the Arg to Cys mutants were described previ- the cells 24 h after transient transfection, and the altered cellular dispo- ously by Comoletti et al. (3). The pGS vector containing cDNA encod- sition of NL-3, AChE, and BChE was assessed after treatment for 24 h. ing human BChE was described previously (32). Mutagenesis intro- Total RNA Extraction, cDNA Preparation, and Real Time RT- duced the Cys mutation at Arg-386. A FLAG tag was added to the C PCR—Total RNA was extracted with TRIzol reagents (Invitrogen) from terminus of wild type BChE after Leu-574 (32). one 100-mm tissue culture dish of HEK-293 cells stably transfected with Cell Culture and Transfections—HEK-293 cells were maintained at wild type and mutant AChE constructs. DNase I turbo treatment 37 °C and 10% CO in Dulbecco’s modified Eagle’s medium, containing (Ambion, Austin, TX) was performed to avoid any genomic DNA con- 10% fetal bovine serum. Plasmids (10 g) carrying the neomycin resist- tamination. Four g of total RNA was reverse-transcribed into single- ance gene were transfected into HEK-293 cells with (Ca) (PO ) precip- 3 4 2 stranded cDNA using the Superscript First-strand System (Invitrogen). itation or FuGENE 6 (Roche Applied Science). Stably transfected cells One l of the resulting cDNA was subjected to real time RT-PCR using were selected with G418 (Geneticin, Sigma) as described elsewhere (27). Taqman primer/probe technology (Applied Biosystems, Foster City, Although initial studies were conducted on protein generated after CA). The primer/probe sets for detecting AChE (exon 3 to exon 4) and transient transfection, clonal cells were selected for studies of mRNA the endogenous reference gene -actin are, respectively, Mm004772 levels, enzyme activity at 31 and 37 °C, kinetic profiles, immunoblotting, 75_m1 and Mm00607939_s1. PCRs were run according to the manu- and immunofluorescence. Wild type and mutant AChEs were also puri- facturer’s instructions. All assays were run in duplicate. The quantity of fied on affinity columns to apparent homogeneity for the kinetic and turnover studies. BChE was studied only after transient transfection. the target mRNA was calculated using the Sequence Detector software Cell Surface Activity and Preparation of Cell Extracts—Cells were package version 1.7 (Applied Biosystems). AChE mRNA was normal- rinsed and harvested by removal from the dish in phosphate-buffered ized to -actin mRNA allowing for measurement of the relative expres- 9668 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases FIGURE 1. Proteins of the ,-hydrolase fold superfamily. Schematic drawing of the domain organization and regions of homology between the rat neuroligins, mouse acetyl- cholinesterase, and human butyrylcholinesterase proteins as reported in UniProtKB/Swiss-Prot (us.expasy.org/sprot/). Leader peptides (Lp), disulfide bonds (brackets), unpaired cysteines (SH), and N-linked (N) and O-linked (O) glycosylation sites and the stalk region (St) for NLs are highlighted. The approximate position of the naturally occurring arginine (R) that is mutated in the three proteins and a reference leucine (L) residue in AChE are shown in boldface type. Inverted triangles show the catalytic triad (Ser-203, His-447, and Glu-334 in AChE and Ser-198, His-438, and Glu-325 in BChE). Size of domains is not drawn to scale. sion level of AChE mRNA with the comparative C method (Applied and R386-BChE mutant proteins to be used for Endo H digestion. After Biosystems user’s manual). purification, E386C BChE was also concentrated. Analysis of Glycosylation—Cell lysates (10 g) from HEK-293 cells Immunohistochemical Localization and Confocal Microscopy—Parental expressing NL1 and AChE wild type and mutant proteins were incu- HEK-293, NL1, and AChE-GPI stably expressing cells were plated on poly- bated with 1500 units of endoglycosidase H (Endo H) or 250 units of D-lysine-coated glass coverslips and grown overnight in Dulbecco’s modi- N-glycosidase F (PNGase F) (New England Biolabs, Beverly, MA) at fied Eagle’s medium. Cells were fixed in 4% paraformaldehyde/phosphate- 37 °C for 3 h at pH 5.5 and 7.5, respectively. buffered saline for 20 min, washed, and labeled for immunofluorescence FLAG-tagged Wild type and R395C-AChE-548 Expression and (3). Briefly, anti-FLAG M2 monoclonal antibody (Sigma) and anti-calnexin Purification—Serum-free culture medium was collected from AChE- polyclonal antibody (Stressgen, Victoria, Canada) were mixed and diluted 548 wild type and R395C-expressing cells maintained in triple layer 1:500 and 1:200, respectively, with blocking buffer diluted 5-fold. Fluores- flasks at 37 °C and 5% CO . Enzymes were purified by passing the media cein isothiocyanate-conjugated anti-mouse antibody and Cy5-conjugated over an M2 anti-FLAG-affinity column (Sigma). The column was then anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) were washed with 10 mM Hepes buffer, pH 7.4, with 450 mM NaCl and eluted diluted 1:100 in the same buffer. Image processing employed an MRC-1024 using the FLAG peptide (Sigma) in 2.5-bed volumes of the Hepes buffer laser-scanning confocal system (Bio-Rad) coupled to a Zeiss Axiovert 35 M with NaCl containing 1 g/ml leupeptin. Proteins were concentrated to microscope. 4 M using Centriprep 30 (Millipore, Bedford, MA) and stored at 4 °C. Determination of Kinetic Parameters for the AChE Catalytic Purified proteins were used for AChE kinetics studies. Lower scale puri- Activity—AChE activity was measured over acetylthiocholine concen- fication, from around 20 ml of media, was done to purify R395C-AChE trations of 0.010 to 100 mM, and individual kinetic constants were APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9669 ER Retention of Cholinesterases TABLE 1 Expression of wild type and mutant mouse AChEs in HEK-293 cells a b AChE activity, mean  S.E. % control AChE gene product Media Lysates Media Lysates units/min/g cell protein % Monomeric, truncated Wild type AChE-548 1.30  0.17 0.016  0.003 100 100 R395C AChE-548 0.003 0.002  0.001 0.27 13.6 a b AChE activity, mean  S.E. % control AChE gene product Intact cell Lysates Intact cells Lysates GPI-linked dimer Wild type GPI dimer 0.019  0.003 0.091  0.017 100 100 R395C GPI dimer 0.008  0.001 0.018  0.006 42 20 Activity is normalized to total cell protein content. Values represent the mean of at least four experiments. Percent values have been calculated considering the activities for both AChE-548 and AChE-GPI constructs equal 100%. FIGURE 2. Protein and mRNA expression levels of wild type and recombinant AChE. A, immunoblot of secreted wild type (WT) and R395C AChE-548 immunoadsorbed with FLAG monoclonal antibody from culture medium of transfected clonal cells. An enhanced exposure reveals a minimal secreted fraction of the R395C AChE-548 protein compared with parental AChE-548. B, immunoblot of cellular lysates without -mercaptoethanol showing monomers and dimers of AChE. Immunoblots were probed with rabbit polyclonal antibody against AChE. Adaptor protein PTP1B was used as loading control. C, relative quantification by real time RT-PCR analysis of AChE mRNA normalized to human -actin. *, significantly different with p  0.05. assessed by nonlinear fitting of activities versus substrate concentration glycophospholipid tethering it to the outer leaflet of the plasma (39). The AChE catalytic turnover constant k was then determined by membrane (42, 43). cat normalizing V to the concentration of AChE active sites. The concen- In contrast to the wild type form of AChE-548, activity is not detected in tration of active sites was determined by titration of AChE activity using the culture medium when the R395C AChE-548 mutant is transfected into the covalent, “irreversible” organophosphate inhibitor, (S )-dimethyl- HEK-293 cells (Table 1). Measurement of cell-associated activity in the cell butyl methylphosphonothiocholine (39). lysates of AChE-548 and R395C AChE-548 reveals intracellular activity in both the wild type and in the Cys mutant, although the R395C mutant has RESULTS far lower activity than the wild type enzyme (Table 1). Expression of Cysteine-substituted AChE—The AChE gene was Expression of the cDNA encoding AChE with the requisite signal mutated so that the homologous Arg mutation in NL (Arg-4513 Cys) sequence for addition of a glycophospholipid anchor at its C terminus, was also converted to a Cys in AChE (R395C) (Fig. 1). To analyze the AChE-GPI, yields cells expressing AChE on the cell surface. Activity effect of oligomeric assembly on the processing and export of AChE and measurements on the intact cells show substantially reduced activity for its related proteins, two forms of mutated AChE were generated. The the R395C mutant of the GPI form of AChE (Table 1). This reduction is first is a truncated form of AChE terminating at residue 548 (AChE-548) also evident when the cells are extracted with Triton X-100 to reveal that is expressed as a soluble monomer. This form lacks sequence nec- total cellular activity, suggesting that transport of AChE-GPI to its cell essary to form disulfide-linked dimers or larger oligomers of AChE (40). surface location is compromised with the R395C mutation (Table 1). Similar monomeric species of AChE have been identified in situ. The Electrophoretic Analysis of Expressed AChE—Western blots to detect second form contained an amino acid sequence capable of forming a AChE protein production confirmed the activity measurements show- disulfide-linked dimer and the signal capable of adding a glycophospho- ing that virtually no R395C AChE-548 was exported into the media, lipid linkage at its C terminus after cellular processing, AChE-GPI (42). whereas expression of the parent AChE-548 showed robust secretion This form appears largely in hematopoietic cells as a dimer with the into the media (Fig. 2A). Western blots using cell lysates in nonreducing 9670 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases FIGURE 3. Co-localization of NL and AChE wild type and mutant proteins with calnexin. Repre- sentative immunofluorescence images of HEK-293 cells expressing FLAG-tagged wild type (WT) NL1 and -3, AChE-GPI wild type, and their Arg to Cys mutations. Images show the FLAG staining (left col- umn, red) and the ER marker calnexin staining (mid- dle column, green) and the merged view (yellow)of the two fluorescence signals in the presence of sap- onin.Mutatedproteinsco-localizewithcalnexinindi- cating that the protein is retained in the ER. Scale bar, 10 m. conditions (Fig. 2B) show slightly higher cellular levels of R395C AChE- subunit linkage (41). To examine whether introduction of a second 548 compared with the wild type enzyme indicating that the mutated cysteine near the C terminus might interfere with oligomerization, we R395C protein is synthesized but compromised in its capacity to be examined the electrophoretic migration of both R395C AChE-GPI and exported from the cell. The lower catalytic activity found for the cell the wild type form of AChE-GPI in nonreducing conditions. As is evi- lysates may reflect either inactivation of a metastable enzyme confor- dent in Fig. 2B, expression of the R395C AChE-GPI mutant yields an mation or altered catalytic properties of the mutated AChE. appreciable fraction of monomer, in contrast to the wild type AChE- The glycophospholipid forms of AChE, whether expressed in trans- GPI that is mainly expressed as a dimer. Other satellite bands are pres- fected cells or natively expressed on the erythrocyte surface, exist as ent in the lane of the R395C GPI-AChE mutant. This heterogeneity of disulfide-linked dimers with the C-terminal cysteine forming the inter- molecular forms may reflect accumulation of ubiquitinated species of APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9671 ER Retention of Cholinesterases the mutant protein and/or different intermediates of glycosylation examined the immunochemical localization of wild type and R395C processing. AChE-GPI compared with the ER residing protein, calnexin (Fig. 3). To evaluate if the higher protein expression seen in the cells stably trans- We observed in the immunohistochemical analysis that wild type fected with R395C AChE compared with the wild type construct was AChE-GPI and wild type NLs 1 and 3 are localized to the cell surface as resulting from different expression levels, mRNA levels were measured by expected. Minimal co-localization with the chaperone protein calnexin, real time reverse transcriptase (RT) (Fig. 2C). No significant differences in which is present endogenously, is evident. On the other hand, the the amount of AChE mRNA was found comparing R395C AChE-548 and R473C NL1, R471C NL3, and the R395C AChE-GPI mutants are wild type AChE-548, indicating that the higher protein amount found in retained in the endoplasmic reticulum and show clear co-localization the cells expressing the mutant protein is the result of a cellular retention with calnexin. Similar retention is seen with R395C AChE-548; how- mechanism. On the other hand R395C AChE-GPI shows reduced mRNA ever, it is more difficult to quantitate retention of this form of AChE expression compared with WT AChE-GPI (Fig. 2C). because the wild type and mutant are located in the ER during biosyn- Co-localization of NL and AChE with the ER Protein, Calnexin—Pre- thesis (data not shown). vious studies have shown that the R471C-NL3 mutation is retained in Analysis of Glycosylation in NL and AChE—To further investigate the endoplasmic reticulum with minimal mutant protein being the stage of processing of NL and AChE that the common mutation exported (3, 4). On the other hand, mutation to residues other than influences, we examined the sensitivity of NL and AChE to glycosidase cysteine at 471 did not result in cellular retention (3). To ascertain treatment. We found that the predominant portion of wild type NL1, whether this behavior is common to the ,-hydrolase fold family, we when extracted from the cell lysate, is resistant to Endo H treatment. Extracellular NL migrates to the higher molecular mass position found for the fully processed protein (Fig. 4A). This is to be expected because a recent characterization of the oligosaccharide content by mass spec- trometry shows primarily bi- and tri-antennary chains containing sev- eral terminal sialic acids (26). By contrast, the sensitivity of R473C NL1 to Endo H digestion is virtually complete, indicating the predominance of high mannose chains and the incomplete oligosaccharide processing of the mutant protein. Hence, with the mutation the majority of NL retained in the cell fails to reach the trans-Golgi stages of oligosaccha- ride processing. Analysis of the Endo H treatment of the cell lysates of the wild type form of AChE-GPI reveals that only a portion of the glycoprotein is digested (Fig. 4B). The total cell extract contains various biosynthetic fractions of the enzyme that transit through the organelles in addition to the processed protein on the cell surface. These intracellular fractions presumably have not yet acquired the fully processed oligosaccharides, whereas the fraction that is exposed at the cell surface contains oligo- FIGURE 4. Endo H and PNGase F sensitivity of NL1 and AChE wild type and mutant saccharides that should be processed completely. By contrast, R395C proteins. Lysates of HEK-293 cells, transiently transfected with wild type (WT)or AChE-GPI is completely sensitive to the Endo H treatment. The R395C mutated NL1 (A), AChE-GPI (B), and AChE-548 (C) were incubated with either Endo H or PNGase F () or mock-digested ()for3hat37 °C. Wild type and R395C-AChE-548 AChE-GPI mutant also migrates faster than wild type AChE-GPI proteins immunoadsorbed from conditioned medium were also incubated with Endo H because the oligosaccharides are largely unprocessed (Fig. 4B). PNGase (C). After digestion, samples were subjected to 10% SDS-PAGE and blotted with rabbit anti-AChE antibody for AChE detection. F treatment of wild type and mutant GPI-AChE reveals that both forms FIGURE 5. Catalytic parameters of purified wild type and mutants of AChE-548. Curves were generated over a range of acetylthiocholine con- centrations using the Ellman assay, and catalytic constants are calculated by nonlinear regression of AChE activity data as described previously (39). For the kinetic Scheme 1 and the corresponding equation (see Ref. 39), the following catalytic con- stants were determined. For the wild type AChE (O), K  0.043 0.009 mM; K  22 9mM; k m ss cat 5 1 1.4  10 min ; and b  0. For R395C-AChE (- - -), K  0.082  0.055 mM; K  4.2  1.8 mM; m ss 5 1 k  0.18  10 min ; and b  2.94  0.77. For cat L386C-AChE (), K  0.032  0.004 mM; K m ss 5 1 11 3mM; k  1.3 10 min ; and b 0.20 cat 0.04. 9672 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases are equally sensitive (Fig. 4B). This is to be expected because PNGase F tioning of the inserted Cys. By simultaneously examining the substrate does not have altered specificity for differentially processed N-linked dependence and titrating the number of active sites with an irreversible glycosylated sugars. organophosphate, we deconstructed the steady-state kinetics into the Although secretion of the mutant R395C AChE-548 is severely com- catalytic parameters for substrate hydrolysis and inhibition. Although promised, a small fraction of AChE becomes fully processed (thus the R395C AChE-548 retained activity, it exhibited marked differences becoming Endo H-resistant) and secreted as soluble entity (Fig. 4C). in catalytic parameters when compared with the wild type form (Fig. 5 This fraction was affinity-purified and was used for subsequent moni- and Scheme 1). In particular, hydrolysis was compromised at low sub- toring of the enzymatic activity. The wild type form of AChE-548 found strate concentration but showed substrate activation (b  1), rather in the cell lysates is completely sensitive to Endo H, and therefore the than the characteristic substrate inhibition (b  1) of AChE at high oligosaccharide pattern of the R395C mutant and wild type AChE-548 substrate concentration. A comparison of catalytic through-puts (k / cat forms cannot be distinguished in the cell lysates (Fig. 4C). K ) reveals a 13-fold difference between the R395C and wild type Catalytic Properties of the AChE Mutants—To understand whether AChE-548 resulting from a greater K and smaller k . By contrast, the m cat the mutated Cys affects the enzymatic function of the cholinesterases, reference mutation L386C AChE-548 shows virtually identical catalytic we compared the catalytic constants of the wild type and R395C AChE- parameters with the classical substrate inhibition characteristic of the 548-purified enzymes. A second Cys mutation near the C terminus mammalian AChEs (39). (L386C) was employed as a reference to assess the importance of posi- Temperature Dependence of AChE Expression—Because the reten- tion of AChE in the ER and the altered kinetic parameters of secreted AChE may reflect incomplete or abnormal folding of the enzyme, we examined expression at lower temperatures to minimize misfolding (44). Although the lower temperature diminishes expression of the wild type AChE-548, relative activities are enhanced for R395C AChE-548 in both the cell lysate and medium (Fig. 6, A and C). Protein levels appear to be slightly reduced in the wild type and mutant lysates (Fig. 6B), but only expression at 31 °C shows appreciable mutant protein in the media (Fig. 6D). Insufficient quantities of mutant enzyme precluded a com- plete kinetic analysis of AChE produced at 31 °C. However, an exami- nation of activity at low and high substrate concentration suggests that expression at low temperature yields the same concentration profile as SCHEME 1 the respective enzyme produces at 37 °C. Hence expression at 31 °C FIGURE 6. Temperature dependence of AChE expression. AChE activity levels measured by Ellman assay and immunoblot analysis of cellular lysates (A and B) and media (C and D) from HEK-293 stable cell lines expressing soluble AChE-548 wild type (WT) and the R395C mutation. Following a medium change, cells were cultured in Ultraculture medium at 37 and 31 °C for 48 h. Immunoblots were probed with rabbit polyclonal antibody against AChE and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9673 ER Retention of Cholinesterases does not affect the substrate dependence but rather alters the expres- sion levels of fully active enzyme. Expression of Wild Type and R386C BChE—BChE is known to have a single splice option giving rise to a soluble tetramer of identical catalytic subunits that assembles as dimer of two disulfide-linked dimers (32, 45). This form closely resembles the dominant splice option of AChE expressed in brain and muscle. As seen with the two forms of AChE, the Cys substitution for Arg in BChE impaired secretion of the protein. Also, a diminished amount of enzyme activity in the cell lysates was evident (Fig. 7A). Similar reductions in cellular and exported BChE are evident in comparing immunoreactive protein (Fig. 7B). Endo H treat- ment of the cell lysates shows a similar result to that observed for the soluble AChE-548 form for the wild type and mutant BChE proteins, both resulting in sensitivity to the enzymatic digestion. Endo H diges- tion of the purified proteins from the media shows a shift after Endo H treatment of a far smaller magnitude than in the lysate. This suggests that a small fraction of the nine N-linked oligosaccharides may still retain high mannose composition. Degradation via the Proteasome Pathway of NL, BChE—To examine the influence of the degradation pathway of the Arg to Cys mutant proteins retained in the ER, lactacystin was used to block degradation via the proteasome pathway. Lactacystin causes an increase in cellular levels for both the wild type and Arg to Cys mutant in the NL3 and BChE proteins but has a more pronounced effect upon R451C-NL3 and R386C-BChE (Fig. 8, A and C). This suggests that a greater fraction of the mutant proteins is degraded intracellularly via the proteasome path- way. Similarly, lactacystin increases cellular levels of AChE-548; curi- ously, the differential effect on the mutation was not evident. This may be due to the truncated monomeric subunit being more susceptible to proteasome degradation, because it lacks a capacity for protection by oligomerization. Cells exposed to 0.2% Me SO, the solvent required for lactacystin, show unaltered expression, as shown for BChE (Fig. 8C). DISCUSSION We demonstrate here that single point mutation of a conserved Arg found in the neuroligin gene of an autistic twin set (1) and in patients with post-succinylcholine apnea (5–7) causes a similar defect in protein expression for both NL3 and BChE. Demonstration of processing defi- ciencies in AChE and BChE, as ,-hydrolase fold family members related to the neuroligins, carries the advantage that catalytic parame- ters of the folded protein can be monitored during the intracellular processing and extracellular secretion steps of protein biosynthesis. Velan et al. (46) have examined AChE biosynthesis through methionine pulse-chase labeling and immunoprecipitation. The initial monomeric spe- FIGURE 7. Expression analysis of cellular and secreted recombinant BChE. A, BChE cies containing exon 6 formed intracellularly was not exported from the activity is measured in lysate and medium from HEK-293 cells transiently transfected cell; rather dimerization and formation of the disulfide linkage are required with BChE wild type (WT) and R386C expression plasmids. B, BChE expression levels by Western blot analysis of lysate and medium probing with mouse anti-BChE antibody and for progression through the trans-Golgi, with concomitant oligosaccharide an antibody to the cellular protein PTP-1B as a loading control. C, immunoblot after Endo processing and subsequent secretion from the cell. Of particular interest is H treatment of lysate and medium for BChE wild type and R386C immunoprecipitated their observation that mutation of the C-terminal cysteine responsible for from growth media. Media samples were immunoadsorbed with a FLAG antibody prior to elution for gel electrophoresis. The R386C required additional concentration (5-fold) the inter-subunit disulfide bond (Cys-580) leads to secretion of the mono- for detection. meric form of AChE and that secreted AChE also contains Endo H-resist- ant or terminally processed oligosaccharides (47). Accordingly, Cys-580 in mammalian AChE was proposed to be part of the oligomerization signal. mammalian cholinesterase with the intent of attaching labels to the free Similarly, our results indicate that the introduction of a cysteine at an cysteine side chain (48, 49). These studies have shown that cysteines exposed but abnormal position near the C terminus may be the aberrant introduced in the N-terminal two-thirds of AChE, at least up through signal for association of a chaperone protein rendering the ,-hydrolase residue 386, do not appear to alter secretion. Moreover, Torpedo AChE fold protein unable to proceed efficiently through processing and secretion. contains a free cysteine at position 231. Thus, it would appear that only Consequently, the protein accumulates in the endoplasmic reticulum and is Cys residues residing near the C terminus give rise to the assembly and more susceptible to proteosomal degradation. secretion deficiency. This was also shown for NL1 where the unpaired Extensive cysteine substitution mutagenesis has been conducted on cysteine at the homologous position (Cys-286) did not interfere with 9674 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases expression nor rescue the deficiency in expression found in the neuroli- neuroligin extracellular domain alone, examined by analytical ultracen- gin mutant (3). trifugation, dimerizes (27). By contrast, AChE devoid of the cysteine in There are some distinguishing structural features between members the C-terminal region, as in AChE-548 or in a naturally occurring spe- of the ,-hydrolase fold family. The cholinesterases contain a four- cies containing a retained intron between exons 4 and 5, is normally helix bundle near the C terminus that has the requisite proximity to be secreted and behaves as a monomer in dilute solution. Formation of essential for dimerization and the proper placement of the disulfide- tetrameric forms of AChE and BChE occurs noncovalently between linking cysteines. Although NL by sequence homology is believed to disulfide-linked dimers (9). have the same helices (25), it does not contain a cysteine C-terminal to Accordingly, the cysteine mutations originally identified in human NL the four-helix bundle found in the cholinesterase sequence. Yet, the and BChE affect processing and cellular export irrespective of the oligo- meric assembly of members of the ,-hydrolase fold family. A possible mechanism for the diminished secretion of the Arg to Cys mutant involves the attachment of a chaperone protein to the introduced cysteine forming a complex that is not immediately dissociable when the partnering subunit comes into proximity. This complex can accumulate and become routed to the proteasome for degradation. Analysis of the chaperone shuttling mech- anism through immunoprecipitation may shed further light on the aber- rant mechanism seen in the naturally occurring Cys mutations. However, abnormal processing and intracellular retention and degradation are not solely responsible for diminished NL adhesion activity or cholinesterase catalytic activity. Rather Cys mutants, when secreted, have compromised intrinsic activity despite the normal disulfide bond interactions (26) (Fig. 5 and Scheme 1). Irrespective of the fractional contributions of the above two proposed mechanisms for diminished activity, losses of expression are not com- plete, and it may be possible through the use of particular stabilizing agents to alter the folding and/or enhance expression of the mutant protein (50). Interestingly, the truncated monomeric form of AChE-548 present within the cells shows complete sensitivity to Endo H. Hence, no differ- ence can be detected between the mutant and native AChE by this approach. This may, in part, be due to the difference in processing rate between the ER and the Golgi apparatus. Once the proteins complete the primary processing in the ER and enter the Golgi, final maturation and secretion could happen at a sufficient rate that no intermediate stages are detectable. A similar finding was reported previously by Kerem et al. (47), who observed that removal of a cysteine near the C terminus of human AChE not only resulted in the secretion of mono- mers but that the monomers were Endo H-resistant. Hence, the absence of a disulfide bond, as is the case for AChE-548, results in secretion and carbohydrate processing by a default or other pathway. The changes in catalytic parameters for the enzyme with the introduced Cys are sur- FIGURE 8. Western blot of lysates expressing wild type or Arg to Cys mutant pro- prising because Arg-395, where the sulfhydryl is introduced (Fig. 9), is teins after treatment with proteasome inhibitor, lactacystin (10 M). NL3 (A), AChE- 548 (B), and BChE (C). Me SO (0.2%) was used to dissolve lactacystin. C, Me SO-treated 2 2 22 Å removed from the active center serine and about 17 Å from the cells are included as a control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) other two catalytic triad residues (Glu-334 and His-447). Arg-395 is, and PTP-1B were used as loading controls. Lanes are labeled as follows: C, control; L, lactacystin; D,Me SO (DMSO) treatment. however, in the immediate vicinity of Trp-442 that is linked to Met-443 FIGURE 9. Position of Arg to Cys mutation in three-dimensional structures of ,-hydrolase fold proteins. A, homology model of NL-1 (27). B, crystal structure of mouse AChE (53). C, crystal structure of human BChE (39). The mutated Arg residue in the three proteins appears in structures as a black stick. The centrally located catalytic ser- ine, located at the base of the active center gorge of AChE and BChE some 18 –20Å from the surface, is similarly indicated. APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9675 ER Retention of Cholinesterases Sudhof, T. C. (1995) Cell 81, 435–443 and on the same loop as His-447. As oxidation of Met-443 is known to 15. Scholl, F. G., and Scheiffele, P. (2003) Trends Neurosci. 26, 618–624 significantly compromise AChE activity (37), small perturbations in 16. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Trp-442 position because of the absence of the positive charge in R395C Doctor, B. P. (1993) Protein Sci. 2, 366–382 could indirectly affect the alignment of the catalytic triad resulting in 17. Ichtchenko, K., Nguyen, T., and Sudhof, T. C. (1996) J. Biol. Chem. 271, 2676–2682 reduction of k for R395C. On the other hand, the molecular basis for 18. Song, J. Y., Ichtchenko, K., Sudhof, T. C., and Brose, N. (1999) Proc. Natl. Acad. Sci. cat U. S. A. 96, 1100–1105 the shift from substrate inhibition to substrate activation for the AChE 19. Scheiffele, P., Fan, J., Choih, J., Fetter, R., and Serafini, T. (2000) Cell 101, 657–669 is not understood, being generated with several independent side chain 20. Dean, C., Scholl, F. G., Choih, J., DeMaria, S., Berger, J., Isacoff, E., and Scheiffele, P. modifications in the AChE active center gorge (39, 51). (2003) Nat. Neurosci. 6, 708–716 A molten globule state of AChE has also been described (52). 21. Chubykin, A. A., Liu, X., Comoletti, D., Tsigelny, I., Taylor, P., and Sudhof, T. C. Although it could be considered as a precursor to the fully folded native (2005) J. Biol. Chem. 280, 22365–22374 22. Varoqueaux, F., Jamain, S., and Brose, N. (2004) Eur. J. Cell Biol. 83, 449–456 form of the enzyme, it is devoid of activity rather than having markedly 23. Graf, E. R., Zhang, X., Jin, S. X., Linhoff, M. W., and Craig, A. M. (2004) Cell 119, reduced activity and altered catalytic parameters as seen for the R395C 1013–1026 mutant AChE. 24. Rudenko, G., Nguyen, T., Chelliah, Y., Sudhof, T. C., and Deisenhofer, J. (1999) Cell The arginine position associated with a natural mutation in neuroligin 99, 93–101 25. Tsigelny, I., Shindyalov, I. N., Bourne, P. E., Sudhof, T. C., and Taylor, P. (2000) implicated in autism is conserved in many proteins in the ,-hydrolase Protein Sci. 9, 180–185 fold family. When mutated to Cys, we demonstrate that it gives rise to 26. Hoffman, R. C., Jennings, L. L., Tsigelny, I., Comoletti, D., Flynn, R. E., Sudhof, T. C., retention of homologous proteins in the cell. The majority of protein is and Taylor, P. (2004) Biochemistry 43, 1496–1506 incompletely processed and is likely shuttled to the proteasome for degra- 27. Comoletti, D., Flynn, R., Jennings, L. L., Chubykin, A., Matsumura, T., Hasegawa, H., dation. This is evident irrespective of whether the protein is a dimer as in Sudhof, T. C., and Taylor, P. (2003) J. Biol. Chem. 278, 50497–50505 28. Massoulie´, J. (2000) in Cholinesterases and Cholinesterase Inhibitors (Giacobini, E., neuroligin or a monomer, dimer, or tetramer as in the case of AChE or ed) pp. 81–101, Martin Dunitz Ltd., London BChE. Chaperone association may be an initial step of oligomerization, and 29. Massoulie´, J., Anselmet, A., Bon, S., Krejci, E., Legay, C., Morel, N., and Simon, S. introduction of an abnormal cysteine may interfere with chaperone associ- (1998) J. Physiol. (Paris) 92, 183–190 ation and subsequent oligomerization of the,-hydrolase fold family pro- 30. Silman, I., and Sussman, J. L. (2005) Curr. Opin. Pharmacol. 5, 293–302 31. Marchot, P., Ravelli, R. B., Raves, M. L., Bourne, Y., Vellom, D. C., Kanter, J., Camp, S., teins. Small quantities of folded proteins containing the mutation are Sussman, J. L., and Taylor, P. (1996) Protein Sci. 5, 672–679 exported from the cell, but their conformation is altered and catalytic or 32. Blong, R. M., Bedows, E., and Lockridge, O. (1997) Biochem. J. 327, 747–757 adhesive activities are compromised. It will be of interest to ascertain 33. Duval, N., Massoulie´, J., and Bon, S. (1992) J. Cell Biol. 118, 641–653 whether altering intracellular redox parameters can enhance export and 34. Ellman, G. L., Courtney, K. D., Jr., Andres, V., and Featherstone, R. M. (1961) Bio- proper folding of the mutated proteins. chem. Pharmacol. 7, 88–95 35. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254 36. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, Acknowledgments—We thank Shannon Weiman and Meghan Miller for 4350–4354 excellent assistance and Shelley Camp for helpful discussions. 37. Jennings, L. L., Malecki, M., Komives, E. A., and Taylor, P. (2003) Biochemistry 42, 11083–11091 38. Nicolet, Y., Lockridge, O., Masson, P., Fontecilla-Camps, J. C., and Nachon, F. (2003) REFERENCES J. Biol. Chem. 278, 41141–41147 1. Jamain, S., Quach, H., Betancur, C., Rastam, M., Colineaux, C., Gillberg, I. C., Soder- 39. Radic´, Z., Pickering, N. A., Vellom, D. C., Camp, S., and Taylor, P. (1993) Biochemistry strom, H., Giros, B., Leboyer, M., Gillberg, C., and Bourgeron, T. (2003) Nat. Genet. 32, 12074–12084 34, 27–29 40. Li, Y., Camp, S., Rachinsky, T. L., Getman, D., and Taylor, P. 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(2003) EMBO J. 22, 1–12 9676 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

A Mutation Linked with Autism Reveals a Common Mechanism of Endoplasmic Reticulum Retention for the α,β-Hydrolase Fold Protein Family *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2006 Elsevier Inc.
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0021-9258
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10.1074/jbc.m510262200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 14, pp. 9667–9676, April 7, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. A Mutation Linked with Autism Reveals a Common Mechanism of Endoplasmic Reticulum Retention for the ,-Hydrolase Fold Protein Family Received for publication, September 19, 2005, and in revised form, January 10, 2006 Published, JBC Papers in Press, January 24, 2006, DOI 10.1074/jbc.M510262200 ‡ ‡ ‡1 § ‡ ¶ Antonella De Jaco , Davide Comoletti , Zrinka Kovarik , Guido Gaietta , Zoran Radic´ , Oksana Lockridge , § ‡2 Mark H. Ellisman , and Palmer Taylor ‡ § From the Departments of Pharmacology and Neurosciences and National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California 92093-0636 and Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 A mutation linked to autistic spectrum disorders encodes an neuroligin-3 (NL-3) at position 451 near the C terminus of the extra- Arg to Cys replacement in the C-terminal portion of the extra- cellular domain (1). This mutation, identified in an affected twin set, has cellular domain of neuroligin-3. The solvent-exposed Cys causes been shown to give rise to an altered cellular phenotype. The protein is virtually complete retention of the protein in the endoplasmic largely retained intracellularly in the endoplasmic reticulum (ER), with reticulum when the protein is expressed in transfected cells. An little of the nascent protein reaching its cell membrane location (3, 4). In identical Cys substitution was reported for butyrylcholinesterase addition, the residual protein that reaches the cell surface has an altered through genotyping patients with post-succinylcholine apnea. affinity for its cognate partner, -neurexin (3). When Arg-451 (rat num- Neuroligin, butyrylcholinesterase, and acetylcholinesterase are bering, Arg-471) is mutated to a Thr or a Glu, the mutant NL is exported members of the ,-hydrolase fold family of proteins sharing normally but shows significantly lower affinity for -neurexins (3). Mass sequence similarity and common tertiary structures. Although spectrometry analysis of the secreted fraction of the R471C-NL3 these proteins have distinct oligomeric assemblies and cellular mutant indicated that the mutated protein had an unaltered disulfide dispositions, homologous Arg residues in neuroligin-3 bonding pattern (3). (Arg-451), in butyrylcholinesterase (Arg-386), and in acetylcho- Arg-451 is highly conserved within the NL family and across mam- linesterase (Arg-395) are conserved in all studied mammalian malian species. This residue is also conserved in many other ,-hydro- species. To examine whether an homologous Arg to Cys muta- lase fold proteins. Recently, a cysteine substitution for the conserved tion affects related proteins similarly despite their differing arginine in butyrylcholinesterase (BChE) was found as an infrequent capacities to oligomerize, we inserted homologous mutations in mutation in patient populations with post-succinylcholine apnea (5–7). the acetylcholinesterase and butyrylcholinesterase cDNAs. BChE appears as a soluble tetramer in the plasma but is synthesized in Using confocal fluorescence microscopy and analysis of oligosac- liver (8). The physiological function of BChE is not clear; nevertheless, charide processing, we find that the homologous Arg to Cys the enzyme has proven critical in inactivating various administered mutation also results in endoplasmic reticulum retention of the drugs such as succinylcholine (9). two cholinesterases. Small quantities of mutated acetylcholines- The cloning of the acetylcholinesterase (AChE) gene 2 decades ago terase exported from the cell retain activity but show a greater revealed a new protein family structurally distinct from other hydrolases of K , a much smaller k , and altered substrate inhibition. The the then known structures (10), and homology of AChE to a large domain in m cat nascent proteins associate with chaperones during processing, thyroglobulin suggested a new superfamily of proteins with diverse func- but the mutation presumably restricts processing through the tions extending beyond hydrolytic reactions (10). Subsequently a variety of endoplasmic reticulum and Golgi apparatus, because of local homologous hydrolases have been identified that fall in this family as do protein misfolding and inability to oligomerize. The mutation several proteins that lack hydrolase activity but function as adhesion pro- may alter the capacity of these proteins to dissociate from their teins (11–13). Among these, the neuroligins are the only ones of mamma- chaperone prior to oligomerization and processing for export. lian origin that have been identified and characterized to date (14, 15). The commonality of tertiary structure has enabled one to classify these proteins as members of the ,-hydrolase fold family (16). The neuroligins are a family of multidomain transmembrane- Several mutations that were found in human neuroligin 3 and 4 genes spanning proteins expressed on the postsynaptic side of the synapse appear to be associated with the autistic spectrum disorders (1, 2). One (14, 17, 18). The N-terminal extracellular region is homologous to of the more interesting mutations is an Arg to Cys mutation found in AChE and serves as the extracellular recognition portion of the mol- ecule. C-terminal to this recognition domain is a linking O-glycosy- * This work was supported by the Rotary Foundation Ambassadorial Scholar Fellowship lation-rich region followed by a transmembrane span and a cytoplas- (to A. D. J.), National Alliance for Autism Research Grant 843 (to D. C.), National Insti- mic region with recognition capacity for PDZ proteins. The tutes of Health, Division of Research Resources grant (to M. H. E.), and United States Public Health Service Grants R37-GM18360 and P42-ES 10337 (to P. T.). The costs of association of NL with -neurexin and perhaps -neurexin provides publication of this article were defrayed in part by the payment of page charges. This a potential trans-synaptic interaction. Accordingly, the neuroligins article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Present address: Institute for Medical Research and Occupational Health, P. O. 291, HR-10001 Zagreb, Croatia. The abbreviations used are: NL, neuroligin; AChE, acetylcholinesterase; BChE, butyryl- To whom correspondence should be addressed: Dept. of Pharmacology, University of cholinesterase; ER, endoplasmic reticulum; Endo H, endoglycosidase H; PNGase F, California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636. Tel.: 858-534-1366; N-glycosidase F, HEK, human embryonic kidney; GPI, glycosylphosphatidylinositol; Fax: 858-534-8248; E-mail: pwtaylor@ucsd.edu. RT, reverse transcriptase. APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9667 This is an Open Access article under the CC BY license. ER Retention of Cholinesterases presumably display a capacity to stimulate formation and matura- saline, and activity was measured on the surface of intact cells. Proteins tion of new excitatory and inhibitory synapses in the central nervous were extracted from cells in 20 mM sodium phosphate, pH 7.0, contain- system (19 –23). ing 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100 plus a mixture of Although the molecular details of the neuroligin-neurexin association protease inhibitors containing 5 g/ml each of pepstatin A, leupeptin, that underlie this function are not understood, a crystal structure of neur- and aprotinin, 10 g/ml bacitracin, and 0.01 mM benzamidine (33). exin has been reported (24), and the structure of neuroligin has been Cells were sonicated on ice for 10 min and then spun at 12,000 g for 10 inferred from its homology to AChE (25, 26). By constructing truncated, min. Supernatants containing all solubilized proteins were used for sub- soluble forms of neuroligin, we characterized the disulfide bonding and sequent experiments. glycosylation pattern of NL and demonstrated secondary structural fea- Assay of AChE and BChE Catalytic Activity—To avoid serum tures that are similar to AChE (26, 27). In contrast to NL, cholinesterases do AChE contamination, selected clones of transfected cells were main- not possess a transmembrane spanning region but rather associate with tained in serum-free Ultraculture medium supplemented with L-glu- membranes through either an attached glycophospholipid or by attach- tamine (Cambrex, Walkersville, MD). Media were collected at 48-h ment to structural subunits that associate with the plasma membrane or intervals, pooled for purification, and assayed for AChE or BChE basement membrane (13, 28). Moreover, the alternative splicing mecha- activity using 0.5 mM acetylthiocholine iodide or 5 mM butyrylthio- nism that controls the nature of the membrane attachment of AChE also choline iodide as substrates (34). Both media and cell-associated influences its degree of its oligomerization. Homomeric dimers and tetram- AChE activities were then normalized to the total cell protein con- ers have been identified, as have heteromeric associations between catalytic tent (35). To ascertain whether AChE expression could be enhanced and structural subunits (13, 29, 30). at low temperature, 80% confluent cells were cultured for 48 h at Accordingly, given the distinct assembly modes of this family of pro- 31 °C prior to harvesting. teins, we sought to determine whether a mutation at homologous resi- Gel Electrophoresis and Immunoblotting—Western blots were per- due positions in AChE and BChE would give rise to similar processing formed as described by Towbin et al. (36). Briefly, crude cell extracts (20 aberrations as those seen with the neuroligins. The data presented here g of total protein) or batch anti-FLAG immunoprecipitated proteins suggest that the mutation results in a common folding and processing from cell culture media were separated on 10% SDS-polyacrylamide gels deficiency. This particular mutation may also uncover mechanistic (Invitrogen). After transfer to a polyvinylidene difluoride membrane details essential for catalytic activity, subunit assembly, and oligomer- (Millipore, Bedford, MA), blocking of nonspecific binding was achieved ization of nascent proteins belonging to the ,-hydrolase fold family. by incubating the membrane with 50 mM Tris-HCl, pH 7.4, and 100 mM NaCl, containing 5% nonfat dry milk, and 0.1% Tween 20 for 1 h. Pri- EXPERIMENTAL PROCEDURES mary antibodies were diluted in blocking buffer as follows: anti-AChE Plasmids and Mutagenesis—A cDNA encoding mouse GPI-AChE polyclonal (37), 1:4000; anti-FLAG M2 monoclonal antibody (Sigma), was subcloned into a FLAG-tagged vector (Sigma) for detection and 1:1000; anti NL-1 monoclonal (Synaptic Systems, Goettingen, Ger- purification. The natural leader peptide of AChE was replaced by the many), 1:5000; anti-PTP-1B monoclonal (Calbiochem), 1:10,000; anti- pre-protrypsin leader peptide. The N-terminal FLAG is followed by a BChE monoclonal antibody, 1:1. Monoclonal antibody to human BChE linker peptide of 10 residues and by the AChE sequence beginning at was prepared by the Monoclonal Core Facility at the University of Glu-1 (sequence of the mature mouse AChE protein). Soluble mono- Nebraska Medical Center. The pure BChE protein used to form crystals meric mouse AChE (AChE-548) was constructed by introducing a stop (38) was injected into mice for production of antibodies. Human BChE codon at Cys-549 of mouse AChE-GPI as described previously (31). protein was truncated at residue 529 thereby missing residues 530–574. Arg-395 3 Cys (R395C) and Leu-386 3 Cys (L386C) mutations were It was also devoid of carbohydrate chains at Asn-17, -455, -481, and introduced using the QuikChange mutagenesis kit (Stratagene, San -486. Hybridoma clone C191 2.1.1 secreted the monoclonal antibody Diego, CA) and were subsequently subcloned into expression vectors. into culture medium. Antibody hybridization was detected with ECL Mutations were verified by automated sequencing. Plasmids were puri- (Pierce). fied using DEAE columns (Qiagen Inc., Valencia, CA). Rat NL1 and NL3 The proteasome inhibitor, lactacystin (10 M, Sigma), was applied to wild type constructs and the Arg to Cys mutants were described previ- the cells 24 h after transient transfection, and the altered cellular dispo- ously by Comoletti et al. (3). The pGS vector containing cDNA encod- sition of NL-3, AChE, and BChE was assessed after treatment for 24 h. ing human BChE was described previously (32). Mutagenesis intro- Total RNA Extraction, cDNA Preparation, and Real Time RT- duced the Cys mutation at Arg-386. A FLAG tag was added to the C PCR—Total RNA was extracted with TRIzol reagents (Invitrogen) from terminus of wild type BChE after Leu-574 (32). one 100-mm tissue culture dish of HEK-293 cells stably transfected with Cell Culture and Transfections—HEK-293 cells were maintained at wild type and mutant AChE constructs. DNase I turbo treatment 37 °C and 10% CO in Dulbecco’s modified Eagle’s medium, containing (Ambion, Austin, TX) was performed to avoid any genomic DNA con- 10% fetal bovine serum. Plasmids (10 g) carrying the neomycin resist- tamination. Four g of total RNA was reverse-transcribed into single- ance gene were transfected into HEK-293 cells with (Ca) (PO ) precip- 3 4 2 stranded cDNA using the Superscript First-strand System (Invitrogen). itation or FuGENE 6 (Roche Applied Science). Stably transfected cells One l of the resulting cDNA was subjected to real time RT-PCR using were selected with G418 (Geneticin, Sigma) as described elsewhere (27). Taqman primer/probe technology (Applied Biosystems, Foster City, Although initial studies were conducted on protein generated after CA). The primer/probe sets for detecting AChE (exon 3 to exon 4) and transient transfection, clonal cells were selected for studies of mRNA the endogenous reference gene -actin are, respectively, Mm004772 levels, enzyme activity at 31 and 37 °C, kinetic profiles, immunoblotting, 75_m1 and Mm00607939_s1. PCRs were run according to the manu- and immunofluorescence. Wild type and mutant AChEs were also puri- facturer’s instructions. All assays were run in duplicate. The quantity of fied on affinity columns to apparent homogeneity for the kinetic and turnover studies. BChE was studied only after transient transfection. the target mRNA was calculated using the Sequence Detector software Cell Surface Activity and Preparation of Cell Extracts—Cells were package version 1.7 (Applied Biosystems). AChE mRNA was normal- rinsed and harvested by removal from the dish in phosphate-buffered ized to -actin mRNA allowing for measurement of the relative expres- 9668 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases FIGURE 1. Proteins of the ,-hydrolase fold superfamily. Schematic drawing of the domain organization and regions of homology between the rat neuroligins, mouse acetyl- cholinesterase, and human butyrylcholinesterase proteins as reported in UniProtKB/Swiss-Prot (us.expasy.org/sprot/). Leader peptides (Lp), disulfide bonds (brackets), unpaired cysteines (SH), and N-linked (N) and O-linked (O) glycosylation sites and the stalk region (St) for NLs are highlighted. The approximate position of the naturally occurring arginine (R) that is mutated in the three proteins and a reference leucine (L) residue in AChE are shown in boldface type. Inverted triangles show the catalytic triad (Ser-203, His-447, and Glu-334 in AChE and Ser-198, His-438, and Glu-325 in BChE). Size of domains is not drawn to scale. sion level of AChE mRNA with the comparative C method (Applied and R386-BChE mutant proteins to be used for Endo H digestion. After Biosystems user’s manual). purification, E386C BChE was also concentrated. Analysis of Glycosylation—Cell lysates (10 g) from HEK-293 cells Immunohistochemical Localization and Confocal Microscopy—Parental expressing NL1 and AChE wild type and mutant proteins were incu- HEK-293, NL1, and AChE-GPI stably expressing cells were plated on poly- bated with 1500 units of endoglycosidase H (Endo H) or 250 units of D-lysine-coated glass coverslips and grown overnight in Dulbecco’s modi- N-glycosidase F (PNGase F) (New England Biolabs, Beverly, MA) at fied Eagle’s medium. Cells were fixed in 4% paraformaldehyde/phosphate- 37 °C for 3 h at pH 5.5 and 7.5, respectively. buffered saline for 20 min, washed, and labeled for immunofluorescence FLAG-tagged Wild type and R395C-AChE-548 Expression and (3). Briefly, anti-FLAG M2 monoclonal antibody (Sigma) and anti-calnexin Purification—Serum-free culture medium was collected from AChE- polyclonal antibody (Stressgen, Victoria, Canada) were mixed and diluted 548 wild type and R395C-expressing cells maintained in triple layer 1:500 and 1:200, respectively, with blocking buffer diluted 5-fold. Fluores- flasks at 37 °C and 5% CO . Enzymes were purified by passing the media cein isothiocyanate-conjugated anti-mouse antibody and Cy5-conjugated over an M2 anti-FLAG-affinity column (Sigma). The column was then anti-rabbit antibody (Jackson ImmunoResearch, West Grove, PA) were washed with 10 mM Hepes buffer, pH 7.4, with 450 mM NaCl and eluted diluted 1:100 in the same buffer. Image processing employed an MRC-1024 using the FLAG peptide (Sigma) in 2.5-bed volumes of the Hepes buffer laser-scanning confocal system (Bio-Rad) coupled to a Zeiss Axiovert 35 M with NaCl containing 1 g/ml leupeptin. Proteins were concentrated to microscope. 4 M using Centriprep 30 (Millipore, Bedford, MA) and stored at 4 °C. Determination of Kinetic Parameters for the AChE Catalytic Purified proteins were used for AChE kinetics studies. Lower scale puri- Activity—AChE activity was measured over acetylthiocholine concen- fication, from around 20 ml of media, was done to purify R395C-AChE trations of 0.010 to 100 mM, and individual kinetic constants were APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9669 ER Retention of Cholinesterases TABLE 1 Expression of wild type and mutant mouse AChEs in HEK-293 cells a b AChE activity, mean  S.E. % control AChE gene product Media Lysates Media Lysates units/min/g cell protein % Monomeric, truncated Wild type AChE-548 1.30  0.17 0.016  0.003 100 100 R395C AChE-548 0.003 0.002  0.001 0.27 13.6 a b AChE activity, mean  S.E. % control AChE gene product Intact cell Lysates Intact cells Lysates GPI-linked dimer Wild type GPI dimer 0.019  0.003 0.091  0.017 100 100 R395C GPI dimer 0.008  0.001 0.018  0.006 42 20 Activity is normalized to total cell protein content. Values represent the mean of at least four experiments. Percent values have been calculated considering the activities for both AChE-548 and AChE-GPI constructs equal 100%. FIGURE 2. Protein and mRNA expression levels of wild type and recombinant AChE. A, immunoblot of secreted wild type (WT) and R395C AChE-548 immunoadsorbed with FLAG monoclonal antibody from culture medium of transfected clonal cells. An enhanced exposure reveals a minimal secreted fraction of the R395C AChE-548 protein compared with parental AChE-548. B, immunoblot of cellular lysates without -mercaptoethanol showing monomers and dimers of AChE. Immunoblots were probed with rabbit polyclonal antibody against AChE. Adaptor protein PTP1B was used as loading control. C, relative quantification by real time RT-PCR analysis of AChE mRNA normalized to human -actin. *, significantly different with p  0.05. assessed by nonlinear fitting of activities versus substrate concentration glycophospholipid tethering it to the outer leaflet of the plasma (39). The AChE catalytic turnover constant k was then determined by membrane (42, 43). cat normalizing V to the concentration of AChE active sites. The concen- In contrast to the wild type form of AChE-548, activity is not detected in tration of active sites was determined by titration of AChE activity using the culture medium when the R395C AChE-548 mutant is transfected into the covalent, “irreversible” organophosphate inhibitor, (S )-dimethyl- HEK-293 cells (Table 1). Measurement of cell-associated activity in the cell butyl methylphosphonothiocholine (39). lysates of AChE-548 and R395C AChE-548 reveals intracellular activity in both the wild type and in the Cys mutant, although the R395C mutant has RESULTS far lower activity than the wild type enzyme (Table 1). Expression of Cysteine-substituted AChE—The AChE gene was Expression of the cDNA encoding AChE with the requisite signal mutated so that the homologous Arg mutation in NL (Arg-4513 Cys) sequence for addition of a glycophospholipid anchor at its C terminus, was also converted to a Cys in AChE (R395C) (Fig. 1). To analyze the AChE-GPI, yields cells expressing AChE on the cell surface. Activity effect of oligomeric assembly on the processing and export of AChE and measurements on the intact cells show substantially reduced activity for its related proteins, two forms of mutated AChE were generated. The the R395C mutant of the GPI form of AChE (Table 1). This reduction is first is a truncated form of AChE terminating at residue 548 (AChE-548) also evident when the cells are extracted with Triton X-100 to reveal that is expressed as a soluble monomer. This form lacks sequence nec- total cellular activity, suggesting that transport of AChE-GPI to its cell essary to form disulfide-linked dimers or larger oligomers of AChE (40). surface location is compromised with the R395C mutation (Table 1). Similar monomeric species of AChE have been identified in situ. The Electrophoretic Analysis of Expressed AChE—Western blots to detect second form contained an amino acid sequence capable of forming a AChE protein production confirmed the activity measurements show- disulfide-linked dimer and the signal capable of adding a glycophospho- ing that virtually no R395C AChE-548 was exported into the media, lipid linkage at its C terminus after cellular processing, AChE-GPI (42). whereas expression of the parent AChE-548 showed robust secretion This form appears largely in hematopoietic cells as a dimer with the into the media (Fig. 2A). Western blots using cell lysates in nonreducing 9670 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases FIGURE 3. Co-localization of NL and AChE wild type and mutant proteins with calnexin. Repre- sentative immunofluorescence images of HEK-293 cells expressing FLAG-tagged wild type (WT) NL1 and -3, AChE-GPI wild type, and their Arg to Cys mutations. Images show the FLAG staining (left col- umn, red) and the ER marker calnexin staining (mid- dle column, green) and the merged view (yellow)of the two fluorescence signals in the presence of sap- onin.Mutatedproteinsco-localizewithcalnexinindi- cating that the protein is retained in the ER. Scale bar, 10 m. conditions (Fig. 2B) show slightly higher cellular levels of R395C AChE- subunit linkage (41). To examine whether introduction of a second 548 compared with the wild type enzyme indicating that the mutated cysteine near the C terminus might interfere with oligomerization, we R395C protein is synthesized but compromised in its capacity to be examined the electrophoretic migration of both R395C AChE-GPI and exported from the cell. The lower catalytic activity found for the cell the wild type form of AChE-GPI in nonreducing conditions. As is evi- lysates may reflect either inactivation of a metastable enzyme confor- dent in Fig. 2B, expression of the R395C AChE-GPI mutant yields an mation or altered catalytic properties of the mutated AChE. appreciable fraction of monomer, in contrast to the wild type AChE- The glycophospholipid forms of AChE, whether expressed in trans- GPI that is mainly expressed as a dimer. Other satellite bands are pres- fected cells or natively expressed on the erythrocyte surface, exist as ent in the lane of the R395C GPI-AChE mutant. This heterogeneity of disulfide-linked dimers with the C-terminal cysteine forming the inter- molecular forms may reflect accumulation of ubiquitinated species of APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9671 ER Retention of Cholinesterases the mutant protein and/or different intermediates of glycosylation examined the immunochemical localization of wild type and R395C processing. AChE-GPI compared with the ER residing protein, calnexin (Fig. 3). To evaluate if the higher protein expression seen in the cells stably trans- We observed in the immunohistochemical analysis that wild type fected with R395C AChE compared with the wild type construct was AChE-GPI and wild type NLs 1 and 3 are localized to the cell surface as resulting from different expression levels, mRNA levels were measured by expected. Minimal co-localization with the chaperone protein calnexin, real time reverse transcriptase (RT) (Fig. 2C). No significant differences in which is present endogenously, is evident. On the other hand, the the amount of AChE mRNA was found comparing R395C AChE-548 and R473C NL1, R471C NL3, and the R395C AChE-GPI mutants are wild type AChE-548, indicating that the higher protein amount found in retained in the endoplasmic reticulum and show clear co-localization the cells expressing the mutant protein is the result of a cellular retention with calnexin. Similar retention is seen with R395C AChE-548; how- mechanism. On the other hand R395C AChE-GPI shows reduced mRNA ever, it is more difficult to quantitate retention of this form of AChE expression compared with WT AChE-GPI (Fig. 2C). because the wild type and mutant are located in the ER during biosyn- Co-localization of NL and AChE with the ER Protein, Calnexin—Pre- thesis (data not shown). vious studies have shown that the R471C-NL3 mutation is retained in Analysis of Glycosylation in NL and AChE—To further investigate the endoplasmic reticulum with minimal mutant protein being the stage of processing of NL and AChE that the common mutation exported (3, 4). On the other hand, mutation to residues other than influences, we examined the sensitivity of NL and AChE to glycosidase cysteine at 471 did not result in cellular retention (3). To ascertain treatment. We found that the predominant portion of wild type NL1, whether this behavior is common to the ,-hydrolase fold family, we when extracted from the cell lysate, is resistant to Endo H treatment. Extracellular NL migrates to the higher molecular mass position found for the fully processed protein (Fig. 4A). This is to be expected because a recent characterization of the oligosaccharide content by mass spec- trometry shows primarily bi- and tri-antennary chains containing sev- eral terminal sialic acids (26). By contrast, the sensitivity of R473C NL1 to Endo H digestion is virtually complete, indicating the predominance of high mannose chains and the incomplete oligosaccharide processing of the mutant protein. Hence, with the mutation the majority of NL retained in the cell fails to reach the trans-Golgi stages of oligosaccha- ride processing. Analysis of the Endo H treatment of the cell lysates of the wild type form of AChE-GPI reveals that only a portion of the glycoprotein is digested (Fig. 4B). The total cell extract contains various biosynthetic fractions of the enzyme that transit through the organelles in addition to the processed protein on the cell surface. These intracellular fractions presumably have not yet acquired the fully processed oligosaccharides, whereas the fraction that is exposed at the cell surface contains oligo- FIGURE 4. Endo H and PNGase F sensitivity of NL1 and AChE wild type and mutant saccharides that should be processed completely. By contrast, R395C proteins. Lysates of HEK-293 cells, transiently transfected with wild type (WT)or AChE-GPI is completely sensitive to the Endo H treatment. The R395C mutated NL1 (A), AChE-GPI (B), and AChE-548 (C) were incubated with either Endo H or PNGase F () or mock-digested ()for3hat37 °C. Wild type and R395C-AChE-548 AChE-GPI mutant also migrates faster than wild type AChE-GPI proteins immunoadsorbed from conditioned medium were also incubated with Endo H because the oligosaccharides are largely unprocessed (Fig. 4B). PNGase (C). After digestion, samples were subjected to 10% SDS-PAGE and blotted with rabbit anti-AChE antibody for AChE detection. F treatment of wild type and mutant GPI-AChE reveals that both forms FIGURE 5. Catalytic parameters of purified wild type and mutants of AChE-548. Curves were generated over a range of acetylthiocholine con- centrations using the Ellman assay, and catalytic constants are calculated by nonlinear regression of AChE activity data as described previously (39). For the kinetic Scheme 1 and the corresponding equation (see Ref. 39), the following catalytic con- stants were determined. For the wild type AChE (O), K  0.043 0.009 mM; K  22 9mM; k m ss cat 5 1 1.4  10 min ; and b  0. For R395C-AChE (- - -), K  0.082  0.055 mM; K  4.2  1.8 mM; m ss 5 1 k  0.18  10 min ; and b  2.94  0.77. For cat L386C-AChE (), K  0.032  0.004 mM; K m ss 5 1 11 3mM; k  1.3 10 min ; and b 0.20 cat 0.04. 9672 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases are equally sensitive (Fig. 4B). This is to be expected because PNGase F tioning of the inserted Cys. By simultaneously examining the substrate does not have altered specificity for differentially processed N-linked dependence and titrating the number of active sites with an irreversible glycosylated sugars. organophosphate, we deconstructed the steady-state kinetics into the Although secretion of the mutant R395C AChE-548 is severely com- catalytic parameters for substrate hydrolysis and inhibition. Although promised, a small fraction of AChE becomes fully processed (thus the R395C AChE-548 retained activity, it exhibited marked differences becoming Endo H-resistant) and secreted as soluble entity (Fig. 4C). in catalytic parameters when compared with the wild type form (Fig. 5 This fraction was affinity-purified and was used for subsequent moni- and Scheme 1). In particular, hydrolysis was compromised at low sub- toring of the enzymatic activity. The wild type form of AChE-548 found strate concentration but showed substrate activation (b  1), rather in the cell lysates is completely sensitive to Endo H, and therefore the than the characteristic substrate inhibition (b  1) of AChE at high oligosaccharide pattern of the R395C mutant and wild type AChE-548 substrate concentration. A comparison of catalytic through-puts (k / cat forms cannot be distinguished in the cell lysates (Fig. 4C). K ) reveals a 13-fold difference between the R395C and wild type Catalytic Properties of the AChE Mutants—To understand whether AChE-548 resulting from a greater K and smaller k . By contrast, the m cat the mutated Cys affects the enzymatic function of the cholinesterases, reference mutation L386C AChE-548 shows virtually identical catalytic we compared the catalytic constants of the wild type and R395C AChE- parameters with the classical substrate inhibition characteristic of the 548-purified enzymes. A second Cys mutation near the C terminus mammalian AChEs (39). (L386C) was employed as a reference to assess the importance of posi- Temperature Dependence of AChE Expression—Because the reten- tion of AChE in the ER and the altered kinetic parameters of secreted AChE may reflect incomplete or abnormal folding of the enzyme, we examined expression at lower temperatures to minimize misfolding (44). Although the lower temperature diminishes expression of the wild type AChE-548, relative activities are enhanced for R395C AChE-548 in both the cell lysate and medium (Fig. 6, A and C). Protein levels appear to be slightly reduced in the wild type and mutant lysates (Fig. 6B), but only expression at 31 °C shows appreciable mutant protein in the media (Fig. 6D). Insufficient quantities of mutant enzyme precluded a com- plete kinetic analysis of AChE produced at 31 °C. However, an exami- nation of activity at low and high substrate concentration suggests that expression at low temperature yields the same concentration profile as SCHEME 1 the respective enzyme produces at 37 °C. Hence expression at 31 °C FIGURE 6. Temperature dependence of AChE expression. AChE activity levels measured by Ellman assay and immunoblot analysis of cellular lysates (A and B) and media (C and D) from HEK-293 stable cell lines expressing soluble AChE-548 wild type (WT) and the R395C mutation. Following a medium change, cells were cultured in Ultraculture medium at 37 and 31 °C for 48 h. Immunoblots were probed with rabbit polyclonal antibody against AChE and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9673 ER Retention of Cholinesterases does not affect the substrate dependence but rather alters the expres- sion levels of fully active enzyme. Expression of Wild Type and R386C BChE—BChE is known to have a single splice option giving rise to a soluble tetramer of identical catalytic subunits that assembles as dimer of two disulfide-linked dimers (32, 45). This form closely resembles the dominant splice option of AChE expressed in brain and muscle. As seen with the two forms of AChE, the Cys substitution for Arg in BChE impaired secretion of the protein. Also, a diminished amount of enzyme activity in the cell lysates was evident (Fig. 7A). Similar reductions in cellular and exported BChE are evident in comparing immunoreactive protein (Fig. 7B). Endo H treat- ment of the cell lysates shows a similar result to that observed for the soluble AChE-548 form for the wild type and mutant BChE proteins, both resulting in sensitivity to the enzymatic digestion. Endo H diges- tion of the purified proteins from the media shows a shift after Endo H treatment of a far smaller magnitude than in the lysate. This suggests that a small fraction of the nine N-linked oligosaccharides may still retain high mannose composition. Degradation via the Proteasome Pathway of NL, BChE—To examine the influence of the degradation pathway of the Arg to Cys mutant proteins retained in the ER, lactacystin was used to block degradation via the proteasome pathway. Lactacystin causes an increase in cellular levels for both the wild type and Arg to Cys mutant in the NL3 and BChE proteins but has a more pronounced effect upon R451C-NL3 and R386C-BChE (Fig. 8, A and C). This suggests that a greater fraction of the mutant proteins is degraded intracellularly via the proteasome path- way. Similarly, lactacystin increases cellular levels of AChE-548; curi- ously, the differential effect on the mutation was not evident. This may be due to the truncated monomeric subunit being more susceptible to proteasome degradation, because it lacks a capacity for protection by oligomerization. Cells exposed to 0.2% Me SO, the solvent required for lactacystin, show unaltered expression, as shown for BChE (Fig. 8C). DISCUSSION We demonstrate here that single point mutation of a conserved Arg found in the neuroligin gene of an autistic twin set (1) and in patients with post-succinylcholine apnea (5–7) causes a similar defect in protein expression for both NL3 and BChE. Demonstration of processing defi- ciencies in AChE and BChE, as ,-hydrolase fold family members related to the neuroligins, carries the advantage that catalytic parame- ters of the folded protein can be monitored during the intracellular processing and extracellular secretion steps of protein biosynthesis. Velan et al. (46) have examined AChE biosynthesis through methionine pulse-chase labeling and immunoprecipitation. The initial monomeric spe- FIGURE 7. Expression analysis of cellular and secreted recombinant BChE. A, BChE cies containing exon 6 formed intracellularly was not exported from the activity is measured in lysate and medium from HEK-293 cells transiently transfected cell; rather dimerization and formation of the disulfide linkage are required with BChE wild type (WT) and R386C expression plasmids. B, BChE expression levels by Western blot analysis of lysate and medium probing with mouse anti-BChE antibody and for progression through the trans-Golgi, with concomitant oligosaccharide an antibody to the cellular protein PTP-1B as a loading control. C, immunoblot after Endo processing and subsequent secretion from the cell. Of particular interest is H treatment of lysate and medium for BChE wild type and R386C immunoprecipitated their observation that mutation of the C-terminal cysteine responsible for from growth media. Media samples were immunoadsorbed with a FLAG antibody prior to elution for gel electrophoresis. The R386C required additional concentration (5-fold) the inter-subunit disulfide bond (Cys-580) leads to secretion of the mono- for detection. meric form of AChE and that secreted AChE also contains Endo H-resist- ant or terminally processed oligosaccharides (47). Accordingly, Cys-580 in mammalian AChE was proposed to be part of the oligomerization signal. mammalian cholinesterase with the intent of attaching labels to the free Similarly, our results indicate that the introduction of a cysteine at an cysteine side chain (48, 49). These studies have shown that cysteines exposed but abnormal position near the C terminus may be the aberrant introduced in the N-terminal two-thirds of AChE, at least up through signal for association of a chaperone protein rendering the ,-hydrolase residue 386, do not appear to alter secretion. Moreover, Torpedo AChE fold protein unable to proceed efficiently through processing and secretion. contains a free cysteine at position 231. Thus, it would appear that only Consequently, the protein accumulates in the endoplasmic reticulum and is Cys residues residing near the C terminus give rise to the assembly and more susceptible to proteosomal degradation. secretion deficiency. This was also shown for NL1 where the unpaired Extensive cysteine substitution mutagenesis has been conducted on cysteine at the homologous position (Cys-286) did not interfere with 9674 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006 ER Retention of Cholinesterases expression nor rescue the deficiency in expression found in the neuroli- neuroligin extracellular domain alone, examined by analytical ultracen- gin mutant (3). trifugation, dimerizes (27). By contrast, AChE devoid of the cysteine in There are some distinguishing structural features between members the C-terminal region, as in AChE-548 or in a naturally occurring spe- of the ,-hydrolase fold family. The cholinesterases contain a four- cies containing a retained intron between exons 4 and 5, is normally helix bundle near the C terminus that has the requisite proximity to be secreted and behaves as a monomer in dilute solution. Formation of essential for dimerization and the proper placement of the disulfide- tetrameric forms of AChE and BChE occurs noncovalently between linking cysteines. Although NL by sequence homology is believed to disulfide-linked dimers (9). have the same helices (25), it does not contain a cysteine C-terminal to Accordingly, the cysteine mutations originally identified in human NL the four-helix bundle found in the cholinesterase sequence. Yet, the and BChE affect processing and cellular export irrespective of the oligo- meric assembly of members of the ,-hydrolase fold family. A possible mechanism for the diminished secretion of the Arg to Cys mutant involves the attachment of a chaperone protein to the introduced cysteine forming a complex that is not immediately dissociable when the partnering subunit comes into proximity. This complex can accumulate and become routed to the proteasome for degradation. Analysis of the chaperone shuttling mech- anism through immunoprecipitation may shed further light on the aber- rant mechanism seen in the naturally occurring Cys mutations. However, abnormal processing and intracellular retention and degradation are not solely responsible for diminished NL adhesion activity or cholinesterase catalytic activity. Rather Cys mutants, when secreted, have compromised intrinsic activity despite the normal disulfide bond interactions (26) (Fig. 5 and Scheme 1). Irrespective of the fractional contributions of the above two proposed mechanisms for diminished activity, losses of expression are not com- plete, and it may be possible through the use of particular stabilizing agents to alter the folding and/or enhance expression of the mutant protein (50). Interestingly, the truncated monomeric form of AChE-548 present within the cells shows complete sensitivity to Endo H. Hence, no differ- ence can be detected between the mutant and native AChE by this approach. This may, in part, be due to the difference in processing rate between the ER and the Golgi apparatus. Once the proteins complete the primary processing in the ER and enter the Golgi, final maturation and secretion could happen at a sufficient rate that no intermediate stages are detectable. A similar finding was reported previously by Kerem et al. (47), who observed that removal of a cysteine near the C terminus of human AChE not only resulted in the secretion of mono- mers but that the monomers were Endo H-resistant. Hence, the absence of a disulfide bond, as is the case for AChE-548, results in secretion and carbohydrate processing by a default or other pathway. The changes in catalytic parameters for the enzyme with the introduced Cys are sur- FIGURE 8. Western blot of lysates expressing wild type or Arg to Cys mutant pro- prising because Arg-395, where the sulfhydryl is introduced (Fig. 9), is teins after treatment with proteasome inhibitor, lactacystin (10 M). NL3 (A), AChE- 548 (B), and BChE (C). Me SO (0.2%) was used to dissolve lactacystin. C, Me SO-treated 2 2 22 Å removed from the active center serine and about 17 Å from the cells are included as a control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) other two catalytic triad residues (Glu-334 and His-447). Arg-395 is, and PTP-1B were used as loading controls. Lanes are labeled as follows: C, control; L, lactacystin; D,Me SO (DMSO) treatment. however, in the immediate vicinity of Trp-442 that is linked to Met-443 FIGURE 9. Position of Arg to Cys mutation in three-dimensional structures of ,-hydrolase fold proteins. A, homology model of NL-1 (27). B, crystal structure of mouse AChE (53). C, crystal structure of human BChE (39). The mutated Arg residue in the three proteins appears in structures as a black stick. The centrally located catalytic ser- ine, located at the base of the active center gorge of AChE and BChE some 18 –20Å from the surface, is similarly indicated. APRIL 7, 2006• VOLUME 281 • NUMBER 14 JOURNAL OF BIOLOGICAL CHEMISTRY 9675 ER Retention of Cholinesterases Sudhof, T. C. (1995) Cell 81, 435–443 and on the same loop as His-447. As oxidation of Met-443 is known to 15. Scholl, F. G., and Scheiffele, P. (2003) Trends Neurosci. 26, 618–624 significantly compromise AChE activity (37), small perturbations in 16. Cygler, M., Schrag, J. D., Sussman, J. L., Harel, M., Silman, I., Gentry, M. K., and Trp-442 position because of the absence of the positive charge in R395C Doctor, B. P. (1993) Protein Sci. 2, 366–382 could indirectly affect the alignment of the catalytic triad resulting in 17. Ichtchenko, K., Nguyen, T., and Sudhof, T. C. (1996) J. Biol. Chem. 271, 2676–2682 reduction of k for R395C. On the other hand, the molecular basis for 18. Song, J. Y., Ichtchenko, K., Sudhof, T. C., and Brose, N. (1999) Proc. Natl. Acad. Sci. cat U. S. A. 96, 1100–1105 the shift from substrate inhibition to substrate activation for the AChE 19. Scheiffele, P., Fan, J., Choih, J., Fetter, R., and Serafini, T. (2000) Cell 101, 657–669 is not understood, being generated with several independent side chain 20. Dean, C., Scholl, F. G., Choih, J., DeMaria, S., Berger, J., Isacoff, E., and Scheiffele, P. modifications in the AChE active center gorge (39, 51). (2003) Nat. Neurosci. 6, 708–716 A molten globule state of AChE has also been described (52). 21. Chubykin, A. A., Liu, X., Comoletti, D., Tsigelny, I., Taylor, P., and Sudhof, T. C. Although it could be considered as a precursor to the fully folded native (2005) J. Biol. Chem. 280, 22365–22374 22. Varoqueaux, F., Jamain, S., and Brose, N. (2004) Eur. J. Cell Biol. 83, 449–456 form of the enzyme, it is devoid of activity rather than having markedly 23. Graf, E. R., Zhang, X., Jin, S. X., Linhoff, M. W., and Craig, A. M. (2004) Cell 119, reduced activity and altered catalytic parameters as seen for the R395C 1013–1026 mutant AChE. 24. Rudenko, G., Nguyen, T., Chelliah, Y., Sudhof, T. C., and Deisenhofer, J. (1999) Cell The arginine position associated with a natural mutation in neuroligin 99, 93–101 25. Tsigelny, I., Shindyalov, I. N., Bourne, P. E., Sudhof, T. C., and Taylor, P. (2000) implicated in autism is conserved in many proteins in the ,-hydrolase Protein Sci. 9, 180–185 fold family. 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(2003) EMBO J. 22, 1–12 9676 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 14 •APRIL 7, 2006

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Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Apr 7, 2006

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