Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.     

Effects of periodate oxidation and glycosidases on structural and functional properties of the acetylcholine receptor and the non-receptor,peripheral ν-polypeptide (Mr 43,000)

NaIO₄ oxidation, exo- and endo-glycosidase treatments and combinations thereof have been applied to acetylcholine receptor from Torpedo marmorata in its membrane-bound and detergent-solubilised forms. The effects of these chemical and enzymatic treatments are made apparent in the electrophoretic properties of the four receptor subunits (α, β, γ and δ) and of the non-receptor polypeptides, their thermal and proteolytic susceptibility, and the steady-state and kinetic parameters of receptor-toxin complex formation. The electrophoretic pattern of the membrane polypeptides is found to depend on the redox state of the membranes, presence or absence of the non-receptor peripheral ν-peptide (M_r 43,000), pH and temperature. Very low NaIO₄ concentrations (50 μM) suffice to prevent the penetration of the ν-peptide into NaDodSO₄ polyacrylamide gels. This effect could be abolished by N-ethylmaleimide alkylation of free sulphydryl groups, suggesting the involvement of easily oxidizable vicinal thiols in the aggregation of the peptide. Higher reagent concentrations resulted in the altered mobility and subsequent splitting of the receptor subunit carrying the ligand recognition site (α, M_r 40,000) into a doublet. In contrast, NaIO₄ treatment of the detergent-solubilized receptor aggregated the α-subunit, presumably via chemical groups hidden in the membrane but exposed in detergent. Only this subunit underwent such NaIO₄-dependent changes within the concentration range in which (a) an increase of the 13-S dimeric receptor species at the expense of the 9-S monomeric form was observed and (b) half-maximal quenching of the intrinsic fluorescence occurred (～2 mM NaIO₄).Neuraminidase digestion affected exclusively the γ- and δ-subunits of the receptor, suggesting the presence of substantial amounts of sialic acid residues in these subunits. β-Glucosidase and endoglycosidase D had no effect on the electrophoretic properties of receptor and non-receptor polypeptides. Neither NaIO₄ nor neuroaminidase treatments had any effect on the thermal sensitivity of the receptor. Similarly, the equilibrium and kinetic properties of receptor-α-neurotoxin complex formation were not modified by such treatment nor was the susceptibility to tryptic digestion. The thermal and proteolytic sensitivities were affected by acid pH (5.2) and β-glucosidase treatments. The latter enzymatic digestion reduced the α-toxin binding capacity of the receptor by 35% and increased the equilibrium dissociation constant by 2-fold.     

Method for Lyophilizing Brain Proteolipid Preparation That Increases Subsequent Solubilization by Detergent

Abstract: A frozen mixture of solubilized brain proteolipid proteins in chloroform-methanol is not sublimable in a vacuum. However, when 7 to 10 volumes of benzene were added to a chloroform-methanol solution containing 5 mg of proteolipid protein per ml, the proteolipid proteins remained in solution for a while and the frozen mixture was easily sublimated at 2 mm Hg. Before the addition of benzene, higher concentrations of protein required the acidification of the medium to avoid precipitation of proteolipid proteins. In contrast to what happens when proteolipid proteins are obtained by the evaporation of the organic mixture at room temperature, the protein obtained by lyophilization was soluble in aqueous solutions of ionic and nonionic detergents. Sodium dodecyl sulfate at 0.6 to 0.7% concentration completely solubilized the proteolipid protein obtained by lyophilization. With the nonionic detergents Lubrol WX and Triton X-100, a solubilization between 50 and 65% was achieved. Sodium deoxycholate was practically ineffective. Triton X-100 showed selectivity in solubilizing certain proteins. The role of lipids in the solubilization of proteolipid proteins with detergents is discussed.     

Monocyte-Derived Dendritic Cells from Patients with Dermatophytosis Restrict the Growth of Trichophyton rubrum and Induce CD4-T Cell Activation

Dermatophytes are the most common agents of superficial mycoses that are caused by mold fungi. Trichophyton rubrum is the most common pathogen causing dermatophytosis. The immunology of dermatophytosis is currently poorly understood. Recently, our group investigated the interaction of T. rubrum conidia with peritoneal mouse macrophages. We found that macrophages phagocytose T. rubrum conidia resulted in a down-modulation of class II major histocompatibility complex (MHC) antigens and in the expression of co-stimulatory molecules. Furthermore, it induced the production of IL-10, and T. rubrum conidia differentiated into hyphae that grew and killed the macrophages after 8 hrs of culture. This work demonstrated that dendritic cells (DCs) and macrophages, from patients or normal individuals, avidly interact with pathogenic fungus T. rubrum. The dermatophyte has two major receptors on human monocyte-derived DC: DC-SIGN and mannose receptor. In contrast macrophage has only mannose receptor that participates in the phagocytosis or bound process. Another striking aspect of this study is that unlike macrophages that permit rapid growth of T. rubrum, human DC inhibited the growth and induces Th activation. The ability of DC from patients to interact and kill T. rubrum and to present Ags to T cells suggests that DC may play an important role in the host response to T. rubrum infection by coordinating the development of cellular immune response.     

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.