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J M Chatel

Publications and source records attributed to J M Chatel.

At least 19 recordsLinked to original sources
PubMed2005

Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Animals↗
PubMed2003

Various factors (allergen nature, mouse strain, CpG/recombinant protein expressed) influence the immune response elicited by genetic immunization.

BACKGROUND: Genetic immunization is a very promising therapeutic approach for allergy treatment. In the present study we investigate the influence of the nature of the allergen, the mouse strain, and the relative amount of CpG to expressed recombinant protein on immune responses using two major peanut allergens, Ara h 1 and Ara h 4. METHODS: The cDNA of Ara h 1 and of an isoform of Ara h 4 were cloned and inserted in pcDNA3. Antigen specific IgG1, IgG2a and IgE were followed after genetic immunization with 100 microg of these clones in mouse strain SKH-Hr1 or BALB/c and with 1 microg of the clones+99 blank plasmid in SKH-Hr1. RESULTS: Genetic immunization in SKH-Hr1 with Ara h 1 elicited a classical Th1 type response, but Ara h 4 elicited a mixed Th1/Th2 response with high IgG1 and even IgE in some mice. In BALB/c both plasmids produced a high IgG1 level. Decreasing the amount of plasmid injected did not change the immune response profile. However, increasing the amount of CpG administered relative to the recombinant Ara h 4 protein expressed reversed the Th1/Th2 response pattern in SKH-Hr1 mice. CONCLUSIONS: Immune responses after genetic immunization are strongly influenced by the nature of the allergen, the mouse strain, and the ratio of CpG to recombinant protein expressed.

Allergens↗
PubMed2002

[Prevention of an IgE response to bovine beta-lactoglobulin by gene immunization in mice].

The therapeutic routes for the treatment of allergy have as their objective to prevent or diminish the specific IgE responsible for the appearance of the allergic reaction. This allergic reaction survives in the genetically predisposed subject and results in a dis-equilibrium of the "Th1-Th2 balance" by increasing the Th2 response. This Th2 response induces the production of IgE against environmental antigens, which from that time on become allergens. In this context, use of gene immunisation seems to be very interesting. The immunisation consists of the injection of an expression vector of bacterial origin, containing the cDNA of a protein of interest. Different studies have shown that the injection of such a plasmid into mice initiates a specific immune response of Th1 (IgG2a) type, stopping all further response of the Th2 (IgG1 and IgE) type. This "non-allergic" response is due to the intrinsic properties of the bacterial ADN, notably the presence of sequences of immunostimulants, the adjuvant of the Th1 response. We have sought to show such a preventative effect in the case of a food protein, bovine beta-lactoglobulin (BLG), a major allergen of cow's milk. Firstly, we made a expression plasmid that contained the cDNA of BLG. Intramuscular administration of this plasmid to mice allowed expression of the BLG in the native form at the site of the injection. The primary response induced by this type of immunisation is characterised by a mixed IgG1/IgG2a response and an absence of anti-BLG IgE. In addition, pre-immunisation of the mice with a plasmid that contained the cDNA of BLG prevented all further sensitisation with the protein administered by the intra-peritoneal route in the presence of alum, an adjuvant of the Th2 response. It appeared further that the preventative effect is dependent on the latent time between gene and protein immunisation. Prevention of the anti-BLG IgE response seems to result in an active inhibition by the cytokines such as interferon-gamma and interleukin 10, rather than a reduction in the production of type Th2 cytokines. Use of efficacious non-pathogenic vectors, that may be administered by the digestive route, could envisage a specific protection in the case of severe food allergies.

Allergens↗
PubMed2001

Induction of mucosal immune response after intranasal or oral inoculation of mice with Lactococcus lactis producing bovine beta-lactoglobulin.

The bovine beta-lactoglobulin (BLG) is a major cow's milk allergen. Here, we evaluated the immune response against BLG induced in mice, using the organism Lactococcus lactis, which has GRAS ("generally regarded as safe") status, as a delivery vehicle. The cDNA of the blg gene, encoding BLG, was expressed and engineered for either intra- or extracellular expression in L. lactis. Using a constitutive promoter, the yield of intracellular recombinant BLG (rBLG) was about 20 ng per ml of culture. To increase the quantity of rBLG, the nisin-inducible expression system was used to produce rBLG in the cytoplasmic and extracellular locations. Although the majority of rBLG remained in the cytoplasm, the highest yield (2 microg per ml of culture) was obtained with a secreting strain that encodes a fusion between a lactococcal signal peptide and rBLG. Whatever the expression system, the rBLG is produced mostly in a soluble, intracellular, and denatured form. The BLG-producing strains were then administered either orally or intranasally to mice, and the immune response to BLG was examined. Specific anti-BLG immunoglobulin A (IgA) antibodies were detected 3 weeks after the immunization protocol in the feces of mice immunized with the secreting lactococcal strain. Specific anti-BLG IgA detected in mice immunized with lactococci was higher than that obtained in mice immunized with the same quantity of pure BLG. No specific anti-BLG IgE, IgA, IgG1, or IgG2a was detected in sera of mice. These recombinant lactococcal strains constitute good vehicles to induce a mucosal immune response to a model allergen and to better understand the mechanism of allergy induced by BLG.

Administration, Intranasal↗
PubMed2001

Genetic immunisation with bovine beta-lactoglobulin cDNA induces a preventive and persistent inhibition of specific anti-BLG IgE response in mice.

BACKGROUND: Various studies have shown that DNA immunisation with gene allergen induces a non-allergic response. METHODS: We applied this new type of vaccination to bovine beta-lactoglobulin (BLG), a major cow's milk allergen, using a plasmid that allows the production of a partially secreted protein. Specific antibodies and cytokines were quantified in different immunisation protocols. RESULTS: The primary response in mice immunised with BLG-encoding plasmid (pBLG) is of the Th1 type. Restricted recognition of a native form of BLG in pBLG mice contrasted with a broader range of recognition in BLG-in-alum-immunised mice, notwithstanding the fact that alum favours the presentation of a native form of the antigen. We also demonstrated an inhibitory effect of pDNA immunisation on the Th2 response induced by a subsequent immunisation using BLG adsorbed on alum. However, this preventive effect is highly dependent on the time of pre-administration of the pBLG, with an optimal effect when pDNA immunisation occurred at least 21 days before protein administration. This preventive effect resulted concomitantly in the inhibition of BLG-specific IgE, in the induction of specific IgG2a, and in the decrease of the specific IgG1/IgG2 ratio. It is accompanied by an increase in IFNgamma and IL-10 secretion. Moreover, the preventive effect was shown to be persistent even after a booster immunisation with alum-adsorbed BLG. The Th1 orientation of the response is very likely due to the presentation of the protein in the Th1 environment due to plasmid immunostimulatory sequences, as intramuscular injection of BLG itself leads to a weak Th2 response and had no preventive effect on a subsequent sensitisation. CONCLUSION: This study further demonstrates the potential use of DNA immunisation for prevention of IgE response, but the window of action seems to be very restricted if we are to inhibit an established Th2 response efficiently.

Allergens↗
PubMed2000

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface.

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

Acetylcholinesterase↗
PubMed2000

Evaluation of a high IgE-responder mouse model of allergy to bovine beta-lactoglobulin (BLG): development of sandwich immunoassays for total and allergen-specific IgE, IgG1 and IgG2a in BLG-sensitized mice.

An animal model of food allergy represents an important tool for studying the mechanisms of induction and repression of an allergic reaction, as well as for the development of an immunotherapy to prevent or minimize such an adverse reaction. IgE and IgG1 (Th2 response) vs. IgG2a (Th1 response) are good markers for the induction of an allergic response in mice. Nevertheless, while the total serum concentrations of these isotypes are easy to measure using classical sandwich immunoassays, this is not the case for allergen-specific isotypes. To develop an animal model of allergy to bovine beta-lactoglobulin (BLG), we set up quantitative assays for total and for allergen-specific IgE, IgG1 and IgG2a. Microtiter plates coated either with anti-isotype antibodies (Abs) or with allergen were used for Ab capture, while anti-isotype Fab' fragments coupled to acetylcholinesterase were used for visualization. These assays of anti-BLG specific Abs are original in two ways. First, assay calibration is performed using anti-BLG specific mAbs, thus allowing good quantification of the different isotypes and subclasses of serum antibodies. Second, the detection of all anti-BLG specific Abs, i.e., those recognizing both the native and denatured forms of the protein, is achieved through indirect coating of BLG using biotin-streptavidin binding. The present assays are quantitative, specific to the isotype (cross-reactivity <0.5%), very sensitive (detection limit in the 10 pg/ml range), and reproducible (coefficient of variation less than 10%). Applied to the humoral response in mice sensitized with BLG adsorbed on alum, these assays proved to be a very useful tool for monitoring high IgE-responder mice following BLG immunization, and for an immunotherapy directed at polarizing the immune response.

Adsorption↗
PubMed1999

Expression of a lipocalin in prokaryote and eukaryote cells: quantification and structural characterization of recombinant bovine beta-lactoglobulin.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation.

Animals↗
PubMed1998

Two-site enzyme immunometric assays for determination of native and denatured beta-lactoglobulin.

Two enzyme immunometric assays suitable for measuring native and denatured beta-lactoglobulin (BLg) have been developed. The assays were performed in 96-well microtitre plates and were based on the use of pairs of monoclonal antibodies specific to either the native form or the reduced and carboxymethylated form of BLg (RCM-BLg). Detection limits of 30 and 200 pg/ml were obtained for the native BLg and the RCM-BLg assay, respectively, with very low or negligible cross-reactivity of the other milk proteins and tryptic fragments of BLg. The validity of the assays in different media such as cow's milk and cow's milk products, saline buffer or serum was supported by recovery experiments. The assays were first applied to the determination of BLg and RCM-BLg in PBS and in raw skimmed milk. The ability of the RCM-BLg assay to detect heat-denatured BLg was confirmed by a kinetic study of BLg heat-denaturation in the two media. During heat treatment, the decrease in the concentration of native BLg was associated with an increase in denatured BLg specifically detected by the RCM-BLg assay. By selecting an appropriate monoclonal antibody which failed to recognize caprine BLg, we were able to establish a modified sandwich immunoassay permitting very sensitive detection of cow's milk in goat's milk.

Animals↗
PubMed1997

Acetylcholinesterases from Elapidae snake venoms: biochemical, immunological and enzymatic characterization.

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.

Acetylcholinesterase↗
PubMed1996

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen.

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Allergens↗
PubMed1994

Evolution of acetylcholinesterase transcripts and molecular forms during development in the central nervous system of the quail.

We studied the expression of acetylcholinesterase (AChE) in the nervous system (cerebellum, optic lobes and neuroretina) of the quail at different stages of development, from embryonic day 10 (E10) to the adult. Analyzing AChE mRNAs and AChE molecular forms, we observed variations in the following: (a) production of multiple mRNA species (4.5 kb, 5.3 kb, and 6 kb); (b) translation and/or stability of the AChE protein; (c) production of active and inactive AChE molecules; (d) production of amphiphilic and nonamphiphilic AChE forms; and (e) proportions of tetrameric G4, dimeric G2, and monomeric G1 forms. The large transcripts present distinct temporal patterns and disappear in the adult, which possesses only the 4.5-kb mRNA; these changes are unlikely to be related to those observed for the AChE protein, because all transcripts seem to encode the same catalytic subunit (type T). In addition, the levels of mRNA and AChE are not correlated in the three regions, especially at the adult stage. The proportion of inactive AChE was found to be markedly higher at the hatching period (E16) than at earlier stages (E10 and E13) or in the adult. The G4 form is predominant already at E10, and in the adult its proportion reaches 80% of the activity in the cerebellum and optic lobes, and 65-70% in the neuroretina. This form is largely nonamphiphilic in embryonic tissues, but it becomes progressively more amphiphilic with development. Thus, the different processing and maturation steps appear to be regulated in an independent manner and potentially correspond to physiologically adaptative mechanisms.

Acetylcholinesterase↗
PubMed1994

Two-site immunoradiometric assay of chicken acetylcholinesterase: active and inactive molecular forms in brain and muscle.

Several monoclonal antibodies were raised against chicken acetylcholinesterase (AChE; EC 3.1.1.7). Some of these antibodies react with quail AChE but not with AChEs from nonavian vertebrates or invertebrates and not with butyrylcholinesterase. They may be classified in several mutually compatible groups, i.e., that can bind simultaneously to the monomeric form of AChE. Most antibodies recognize a peptidic domain that does not exist in mammalian AChE and that may be digested by trypsin without loss of activity or dissociation of quaternary structure. The only exception is the antibody C-131, which is conformation dependent and preferentially recognizes active AChE. We have set up two-site immunoradiometric assays, using an immobilized capture antibody, C-6 or C-131, and a radiolabeled antibody, 125I-C-54. The C-6/C-54 assay quantifies the totality of inactive and active AChE subunits: It detects 10(-3) Ellman unit (approximately 40 pg of protein) and yields a linear response up to at least 25 10(-3) Ellman units. An analysis of gradient fractions, using C-6/C-54 and C-131/C-54 assays as well as activity determination, shows that the A12 and G4 forms are exclusively composed of active subunits, whereas inactive molecules cosediment with the active G2 and G1 forms. Both active and inactive G2 and G1 forms are amphiphilic, as indicated by the influence of detergents on their sedimentation coefficients and Stokes radii. In brain, the proportion of inactive forms decreases from 40% at embryonic day 11 (E11) to 20% at birth [day 1 (D1)]. In muscle, we observed no inactive AChE at E11 and a small proportion of inactive G1 at D1.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1993

A conformation-dependent monoclonal antibody against active chicken acetylcholinesterase.

We show that the C-131 monoclonal antibody, directed against chicken AChE, recognizes active chicken AChE, but not the SDS-denatured or heat-inactivated protein. Previous results indicated that C-131 only binds to the active enzyme, and not to inactive molecules which also occur in the embryonic chicken brain. In contrast with C-131, other monoclonal antibodies obtained in the same series, such as C-6 and C-54, also recognize denatured or inactive AChE. It is noteworthy that these antibodies all seem to react with a trypsin-sensitive peptide which is present in chicken but not in mammalian or Torpedo AChE, whereas the C-131 antibody binds trypsin-modified as well as intact molecules. These results show that C-131 is highly conformation-dependent, specific for active AChE. They confirm our previous conclusion that active and inactive molecules arise from different folding processes.

Acetylcholinesterase↗
PubMed1993

Existence of an inactive pool of acetylcholinesterase in chicken brain.

We analyzed acetylcholinesterase (AcChoEase; EC 3.1.1.7) activity and AcChoEase immunoreactive protein in chicken brain by using five monoclonal antibodies raised against chicken AcChoEase. Four of them specifically recognized AcChoEase catalytic subunits in Western blots and one, C-131, recognized only enzymatically active AcChoEase. We observed considerable differences in the ratio of immunoreactive protein to catalytic activity in various fractions, indicating the existence of inactive AcChoEase protein. This inactive AcChoEase component was more abundant in a low-salt-soluble extract than in a subsequent detergent-soluble extract. On the basis of the ratio between activity and immunoreactivity, we calculated that the inactive component represents about 30% of the total AcChoEase subunits in chicken brain. The immunoreactive AcChoEase protein sedimented in sucrose gradients like the active molecular forms; the G1 and G2 peaks contained inactive molecules, whereas the G4 peak appeared to contain only active AcChoEase. The bulk of inactive AcChoEase reacted with the organophosphate cholinesterase inhibitor O-ethyl S-[2-(diisopropylamino)ethyl]methylphosphonothioate (MTP) but was found to bind the active site affinity ligand N-methylacridinium poorly and was not recognized by the active-form-specific monoclonal antibody, C-131. In addition, most of this fraction is sensitive to endoglycosidase H and binds the lectin wheat germ agglutinin poorly, suggesting that it was not processed in the Golgi apparatus. From these observations, we propose that the active and inactive AcChoEase components are differently folded.

Acetylcholinesterase↗
PubMed1992

Molecular forms of acetylcholinesterase in dystrophic (mdx) mouse tissues.

We analyzed the activity of acetylcholinesterase (AChE) and its molecular forms in the tissues of normal and dystrophic (mdx) mice, at different developmental stages. We studied the brain, the heart and the serum, in addition to four predominantly fast-twitch muscles (tibialis, plantaris, gastrocnemius and extensor digitorum longus (EDL)) and the slow-twitch, soleus muscle. We found no difference between mdx and control mice in the AChE activity of the brain and the heart. The skeletal muscles affected by the disease undergo active degeneration counterbalanced by regeneration between 3 and 14 weeks after birth. The distribution of AChE patches associated with neuromuscular junctions was abnormally scattered in mdx muscles, and in some cases (tibialis and soleus), the number of endplates was more than twice that of normal muscles. There were only minor differences in the concentration and pattern of AChE molecular forms during the acute phase of muscle degeneration and regeneration. After this period, however, we observed a marked deficit in the membrane-bound G4 molecular form of AChE in adult mdx tibialis, gastrocnemius and EDL but not in the plantaris or in the soleus, as compared with their normal counterparts. Whereas the amount of AChE markedly decreased in the serum of normal mice during the first weeks of life, it remained essentially unchanged in the serum of mdx mice. It is likely that this excess of AChE activity in serum originates from the muscles. A deficit in muscle G4 was also reported in other forms of muscular dystrophy in the mouse and chicken. Since it is not correlated to the acute phase of the disease in mdx and also occurs in genetically different dystrophies, it probably represents a secondary effect of the dystrophy.

Acetylcholinesterase↗
PubMed1991

Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells.

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.

Acetylcholinesterase↗