PubMed Health⌕ Search

Biomedical subjects

M Z Atassi

Publications and source records attributed to M Z Atassi.

At least 37 records · Page 2Linked to original sources
PubMed1996

B-cell activation in vitro by helper T cells specific to region alpha 146-162 of Torpedo californica nicotinic acetylcholine receptor.

We have previously mapped the T and B cell epitopes on the alpha-subunit of acetylcholine receptor (AChR) in human myasthenia gravis (MG) and in experimental autoimmune MG-susceptible (C57BL/6 (B6)) and nonsusceptible mouse strains. In addition to regions recognized by both T and B cells, the AChR alpha-subunit has regions that are recognized solely by T cells. An exclusive T cell epitope within residues alpha 146-162 of Torpedo californica (t), tAChR, plays an important role in experimental autoimmune MG pathogenesis in B6 mice. To study its function, we established, from tAChR-primed B6 mice, two t alpha 146-162-specific T cell lines (P14Th) which comprised Th2-type cells because they secreted IL-4 but not IL-2. P14Th did not recognize the corresponding region on mouse (m) AChR (m alpha 146-162). They caused in vitro differentiation of tAChR-primed B cells into plasma cells that secreted anti-AChR Abs directed, in decreasing order, against the following tAChR alpha regions: t alpha 122-138 > t alpha 134-150 > t alpha 45-60 > t alpha 170-186 > t alpha 56-71. Little or no Ab response could be detected against peptides t alpha 182-198 or t alpha 146-162 itself. The major enhancement was in the Abs against region t alpha 122-150 (spanning the t alpha 122-138/t alpha 134-150 overlap) that is involved in ACh binding. These Abs cross-reacted completely with m alpha 122-150, the corresponding region on mAChR. Therefore, t alpha 146-162-specific T cells, although unable to recognize m alpha 146-162, are nevertheless pathogenic because they help B cells responding to a tAChR region that is conserved in mAChR and involved in ACh binding. These Abs cross-react with the corresponding effector-binding region of mAChR, thereby disrupting the normal physiologic function of the mouse receptor.

Amino Acid Sequence↗
PubMed1996

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855-1296 with antitoxin antibodies from three host species.

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (Hc, residues 860-1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the Hc of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855-873, 939-957, 1079-1097/1093-1111 overlap, 1191-1209/1205-1223 overlap, 1261-1279 and 1275-1296. In addition, peptides 883-901, 911-929, 995-1013, 1023-1041/1037-1055 overlap, 1121-1139, and 1149-1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869-887, 925-943, 981-999, 995-1013, 1051-1069, and 1177-1195. In addition, lower amounts of Abs were bound by peptides 911-929, 939-957, 967-985, and the overlaps 1121-1139/1135-1153 and 1247-1265/1261-1279/1275-1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869-887, 1051-1069, and 1177-1195, while peptides 939-957, 995-1013, 1093-1111, and 1275-1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.

Amino Acid Sequence↗
PubMed1995

Effect of amino acid substitutions within the region 62-76 of I-A beta b on binding with and antigen presentation of Torpedo acetylcholine receptor alpha-chain peptide 146-162.

Previous study has shown that reduced T cell response to peptide alpha 146-162 of Torpedo californica acetylcholine receptor (tAChR) in B6.C-H-2bm12 (bm12) mice, a mutant of C57BL/6 (B6) mice, correlated with its nonsusceptiblity to experimental autoimmune myasthenia gravis. There are three amino acid differences between the I-A beta b of the two strains (positions 67, 70, and 71). We synthesized peptides I-A beta b62-76 (peptide b6), I-A beta bm1262-76 (peptide bm), and three additional peptides, b6(67F), b6(70Q), and b6(71K), and determined their ability to bind peptide alpha 146-162 and the dissociation constants (Kd) of the binding. Peptide alpha 146-162 bound with a significantly higher affinity to peptide b6 than to peptides bm or b6(71K), suggesting that the lower affinity of peptide alpha 146-162 to I-Abm12 is a factor in the reduced response to this peptide by bm12 T cells. This was confirmed by measurement of the Kd values of the binding of peptide alpha 146-162 to the I-A molecules of B6 and bm12. Furthermore, APC of bm12 presented the peptide, or tAChR, poorly to peptide-specific or to tAChR-specific B6 T cells. The major effect is caused by the change of Thr-71 in I-A beta b to lysine in I-A beta bm12. However, APC of B6 also presented peptide alpha 146-162 much less efficiently to peptide-specific T cells of bm12. This demonstrated that these three amino acid changes also influence the T cell receptor recognition of peptide-MHC complex and that both B6 and bm12 T cells recognizing peptide alpha 146-162 or tAChR are under a high H-2 restriction.

Amino Acid Sequence↗
PubMed1995

Antibody and T-cell recognition of alpha-bungarotoxin and its synthetic loop-peptides.

Peptides representing the loops and surface regions of alpha-bungarotoxin (BgTX) and control peptide analogs in which these sequences were randomized were synthesized and used to map the recognition profiles of the antibodies and T-cells obtained after BgTX immunization. Also, the abilities of anti-peptide antibodies and T-cells to recognize the immunizing peptide and BgTX were determined. Three regions of BgTX were immunodominant by both rabbit and mouse anti-BgTX antibodies. These regions resided within loops L1 (residues 3-16), L2 (residues 26-41) and the C-terminal tail (residues 66-74) of the toxin. The regions recognized by BgTX-primed T-lymphocytes were mapped in five mouse strains: C57BL/6(H-2b), Balb/c (H-2d), CBA (H-2k), C3H/He (H-2k) and SJL (H-2s). The H-2b and H-2d haplotypes were high responders to BgTX, while the H-2k and H-2s were intermediate responders. The T-cell recognition profile of the peptides varied with the haplotype, consistent with Ir gene control of the responses to the individual regions. The submolecular specificities of antibodies and T-cells were compared in three of the mouse strains (C57BL/6, Balb/c and SJL). In a given mouse strain, there were regions that were strongly recognized by both antibodies and T-cells as well as regions that were predominantly recognized either by antibodies or by T-cells. The peptides were used as immunogens in their free form (i.e. without coupling to any carrier) in two of the mouse strains, Balb/c and SJL. In both mouse strains, the peptides gave strong antibody responses. Antibodies against peptide L2 showed the highest binding to intact BgTX. Antibodies against the other peptides exhibited lower binding activity to the intact toxin, and this activity was dependent on the peptide and the mouse strain. The response of peptide-primed T-cells to a given immunizing peptide was not related to whether this region was immunodominant with BgTX-primed T-cells. The ability of peptide-primed T-cells to recognize the intact toxin varied with the peptide and was dependent on the host strain. These results indicate that anti-peptide antibody and T-cell responses are also under genetic control and that their ability to cross-react with the parent toxin is not only dependent on the conformational exposure of the correlate region in intact BgTX.

Amino Acid Sequence↗
PubMed1995

Protection of mice against lethal viral infection by synthetic peptides corresponding to B- and T-cell recognition sites of influenza A hemagglutinin.

Previously, we reported 12 synthetic T- and B-cell recognition regions representing surface areas of the hemagglutinin (HA) of X31 influenza virus. In the present study, four of these peptides were examined in Balb/c mice for their ability to produce protective immunity against lethal infection with a dose equivalent to 10 LD50 of influenza virus. These peptides corresponded to the following sequences: 23-36 (HA1-1); 138-152 (HA1-3); 183-199 (HA1-6) and 1-11 (HA2-10). Each of the selected peptides, in their free form, evoked anti-peptide antibodies that cross-react with intact X31 virus. Two of the peptides, HA1-1 and HA1-3, also elicited virus-specific delayed type hypersensitivity (DTH) responses. These two peptides, when injected into mice, not only failed to protect the immunized mice against challenge with influenza virus, but in fact caused greater susceptibility to viral infection as compared to control animals that had been injected with saline. In contrast, peptides HA1-6 and HA2-10, which were unable to induce adequate virus-specific DTH responses, conferred 42-46% and 54-73% protection, respectively, compared to the control group that received only saline (P < 0.03 to P < 0.01).

Amino Acid Sequence↗
PubMed1994

Mapping of the polypeptide chain organization of the main extracellular domain of the alpha-subunit in membrane-bound acetylcholine receptor by antipeptide antibodies spanning the entire domain.

To study the organization of the polypeptide chain of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) alpha-subunit, we examined the ability of the native membrane-bound AChR of Torpedo californica (T-AChR) to bind a panel of antibodies against overlapping synthetic peptides which collectively encompassed this entire domain. Antibodies against the alpha-chain peptides alpha 1-16, alpha 89-104 and alpha 158-174 were able to bind to membrane-bound T-AChR. Other anti-peptide antibodies showed little or no binding to T-AChR in the membrane. It is concluded that regions alpha 1-16, alpha 89-104 and alpha 158-174 are highly exposed on the surface of the alpha subunit of membrane-bound AChR.

Amino Acid Sequence↗
PubMed1994

Analysis of exposed regions on the main extracellular domain of mouse acetylcholine receptor alpha subunit in live muscle cells by binding profiles of antipeptide antibodies.

To study the structural organization of the main extracellular domain of the nicotinic acetylcholine receptor (AChR) alpha subunit in live muscle cells, we examined the native membrane-bound receptors in cultured mouse skeletal muscle cells for their ability to bind a panel of antibodies against uniform-sized overlapping synthetic peptides which collectively represent this entire domain. The binding profile indicated that the regions alpha 23-49, alpha 78-126, alpha 146-174, and alpha 182-210 are accessible to binding with antibody. Residues alpha 23-49, alpha 78-126, and alpha 194-210 contain binding regions for alpha-neurotoxin and some myasthenia gravis autoantibodies. A comparison of this binding profile with the profile obtained for membrane-bound Torpedo californica AChR in isolated membrane fractions showed some similarities as well as significant differences between the subunit organization in the isolated membrane fraction and that in the membrane of live muscle cells. Regions alpha 89-104 and alpha 158-174, which are exposed in the isolated membrane fraction, are also exposed in the live cell. On the other hand, regions alpha 23-49, and alpha 182-210, which are exposed in the live cell, are not accessible in the isolated membrane and, furthermore, the region alpha 1-16, which has marginal accessibility in the cell, becomes highly accessible in the membrane isolates. The exposed regions defined by this study may be the primary targets for the initial autoimmune attack on the receptors in experimental autoimmune myasthenia gravis.

Amino Acid Sequence↗
PubMed1994

Mapping the extracellular topography of the alpha-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain.

The extracellular surface of the alpha-chain of Torpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptides alpha 1-16, alpha 89-104, alpha 158-174, alpha 262-276, and alpha 388-408. Lower, but significant, levels of reactivity were obtained with antibodies against peptides alpha 67-82, alpha 78-93, alpha 100-115, and alpha 111-126. On the other hand, free AChR bound high levels of antibodies against peptides alpha 34-49, alpha 78-93, alpha 134-150, alpha 170-186, and alpha 194-210. It also bound moderate levels of antibodies against peptides alpha 262-276 and alpha 388-408. Low, yet significant, levels of binding were exhibited by antibodies against peptides alpha 45-60, alpha 111-126, and alpha 122-138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).

Amino Acid Sequence↗
PubMed1994

Profile of the regions of acetylcholine receptor alpha chain recognized by T-lymphocytes and by antibodies in EAMG-susceptible and non-susceptible mouse strains after different periods of immunization with the receptor.

C57BL/6 (B6) mice develop a neuromuscular disease, experimental autoimmune myasthenia gravis (EAMG), after two or more immunizations with Torpedo californica acetylcholine receptor (AChR). To determine whether EAMG is related to recognition of particular region(s) on the main extracellular domain of the alpha chain (residues alpha 1-210) in prolonged immunization, we have examined the differences in the antibody and T cell recognition profiles of B6 and SJL (a strain that does not develop EAMG) mice after different periods and a number of immunizations with Torpedo AChR. In a given strain, antibodies and T cells recognized immunodominant regions, which may coincide or may be uniquely B cell or T cell determinants. Both B6 and SJL exhibited similar antibody recognition profiles after the second and through the fourth immunizations with AChR. Major differences between the two strains were found in their T cell recognition of regions in the second part (residues 100-210) of the main extracellular domain of the alpha chain. T cells of SJL recognized consistently only one region (111-126) within this part of the alpha chain, whereas in B6, T cell recognition of three peptides (111-126, 146-162 and 182-198) and next neighbor regions to them persisted throughout the period. Of these three peptides, 146-162 was an immunodominant peptide unique to B6, as the other two peptides (111-126 and 182-198) were also recognized by either T cells or antibodies in SJL. To study the role of the T cells recognizing region 146-162 in EAMG, a T cell line was generated against this region and the cells transferred into B6 mice followed by one Torpedo AChR injection. Enhancement of antibody production toward alpha chain peptides was observed as an influence of T cell transfer compared to profiles at 1 week. In addition, one out of three mice examined showed signs of EAMG. These results suggest the importance of T cells recognizing residues 146-162 in EAMG. It is concluded that the presence of persistent T cell responses to the second half (residues (100-210) of the main extracellular domain of the alpha chain is associated with the development of EAMG in B6 mice, while absence of these responses in SJL mice may enable them to escape the disease. The preservation of the immunodominance of peptide 146-162 in the T cell recognition of B6 is probably most important for the pathogenesis of EAMG in this strain.

Amino Acid Sequence↗
PubMed1993

Design of peptide enzymes (pepzymes): surface-simulation synthetic peptides that mimic the chymotrypsin and trypsin active sites exhibit the activity and specificity of the respective enzyme.

Two 29-residue peptides were prepared, one of which (ChPepz) was designed by surface-simulation synthesis to mimic the active site of alpha-chymotrypsin, and the other (TrPepz), which contained four substitutions relative to ChPepz, was fashioned after the active site of trypsin. Each peptide was cyclized by a disulfide bond. The ChPepz monomer effected hydrolysis of the ester group in N-benzoyl-L-tyrosine ethyl ester, an alpha-chymotrypsin substrate, with Km and kcat values that were comparable to those of alpha-chymotrypsin. ChPepz was completely inactivated by diisopropyl fluorophosphate (DIFP), L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK), or reduction of the disulfide bond. It had no catalytic activity on N-tosyl-L-arginine methyl ester, a trypsin substrate. On the other hand, TrPepz, which had no effect on N-benzoyl-L-tyrosine ethyl ester, hydrolyzed N-tosyl-L-arginine methyl ester with a Km value that was essentially identical to that of trypsin, but its kcat value was almost half that of trypsin. TrPepz was fully inactivated by reduction of the disulfide bond, by DIFP, or by phenylmethylsulfonyl fluoride but not by TPCK. It was also completely inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, and human alpha 1-antitrypsin. ChPepz and TrPepz hydrolyzed proteins (myoglobin and casein) to give panels of peptides that were similar to those of the same protein obtained with the respective enzyme. However, TrPepz was more efficient than trypsin at hydrolyzing the C bonds of two or more consecutive lysine and/or arginine residues. Like its esterase activity, the proteolytic activity of ChPepz was inhibited by either DIFP or TPCK whereas that of TrPepz was inhibited by either DIFP or phenylmethylsulfonyl fluoride but not by TPCK. Finally, ChPepz and TrPepz were each more active at low temperature than the respective enzyme. This ability to construct fully functional peptide enzymes (pepzymes) of chosen specificities should find many practical applications.

Amino Acid Sequence↗
PubMed1993

Congenital myasthenic syndromes: II. Syndrome attributed to abnormal interaction of acetylcholine with its receptor.

A 21-year-old woman had myasthenic symptoms since birth that responded poorly to anticholinesterase therapy. Tests for acetylcholine receptor (AChR) antibodies were negative. An intercostal muscle specimen was obtained to investigate the character of the neuromuscular transmission defect. There were no immune deposits at the endplates. The quantal content of the endplate potential was normal. Miniature endplate potentials and currents were very small, but the number of AChR per endplate was normal. On electron microscopy, the synaptic vesicles were of normal size, the junctional folds were intact, and the density and distribution of AChR on the folds was normal. The kinetic properties of AChR were studied by analysis of acetylcholine (ACh)-induced current noise. The mean single channel conductance was normal. The noise power spectrum was abnormal, containing two components of different time course. This could result from an abnormal interaction of ACh with AChR, or from two populations of AChR at the endplate. The second possibility is unlikely because if two populations of AChR were present at the endplate, then both would have to have low conductance to explain the small miniature endplate current, but the average conductance of the channels that did open was normal.

Acetylcholine↗
PubMed1993

Suppression of experimental autoimmune myasthenia gravis by epitope-specific neonatal tolerance to synthetic region alpha 146-162 of acetylcholine receptor.

A gene conversion event between Ebb and Abb in the B6.C-H-2bm12 (bm12) strain, which alters three amino acids in the C-terminal half of the first domain of Abb (Ile-67-->Phe; Arg-70-->Gln; Thr-71-->Lys) resulted in resistance to experimental autoimmune myasthenia gravis (EAMG) pathogenesis. To study the effect of bm12 mutation on the T-cell responses to epitopes of acetylcholine receptor (AChR)-alpha subunit, C57BL6 (B6) and bm12 mice were primed with Torpedo californica AChR, and the profiles of T-lymphocyte proliferation were determined with 18 synthetic overlapping peptides encompassing the entire extracellular portion of the AChR-alpha subunit. The proliferative responses of AChR-primed bm12 lymphocytes were markedly reduced to two (alpha 146-162 and alpha 182-198) of the three AChR peptides (alpha 111-126, alpha 146-162, and alpha 182-198) that are immunodominant in B6 mice. Thus, the Ab residues encompassing the region 67-71 determine the immunogenicity of two of the AChR-alpha subunit T-cell epitopes. To test the involvement of AChR-alpha chain epitopes within peptide alpha 146-162 in EAMG pathogenesis, B6 mice were neonatally tolerized with soluble peptide alpha 146-162, and subsequently immunized with AChR in complete Freund's adjuvant. Neonatal tolerance to AChR or to peptide alpha 146-162 reduced the incidence of clinical myasthenia gravis and suppressed serum anti-AChR antibodies. This indicates the involvement of T-cell epitopes within AChR-alpha subunit region alpha 146-162 in EAMG pathogenesis. Neonatal tolerance to peptide alpha 146-162 could have caused specific clonal deletion, and/or clonal anergy, and/or recruited suppressor cells to prevent clinical EAMG. Presumably, epitope(s) with AChR alpha 146-162, in the context of Ab encompassing region 67-71, stimulate specific T helper cells which interact with specific B cells to produce pathogenic antibodies, the primary culprit causing the end plate lesion in patients with myasthenia gravis.

Amino Acid Sequence↗
PubMed1993

Amino acid substitutions outside a preselected antigenic region in hemoglobin affect the binding to monoclonal antibodies obtained by immunization with the synthetic region.

It is often assumed that amino acid substitutions outside a protein antigenic site have no effect on the reactivity of a protein variant with antibodies, especially monoclonal antibodies (mAbs). Substitutions that exert an effect on the reactivity of a protein variant with mAbs are frequently considered to be within the antigenic site of the mAb. To test this assumption, two mAbs [IgG1(k) and IgG2a(k)] were prepared by immunization with a synthetic peptide corresponding to region 63-78 of the alpha chain of human hemoglobin (Hb). The peptide was used as an immunogen in its free form (i.e., without conjugation to a carrier), so that the results will not be made ambiguous by peptide modification nor by an immune response to sites spanning peptide and protein carrier. In addition to their reaction with human Hb, the mAbs were also studied with four primate Hbs which had no substitutions within region alpha 63-78 and only a limited number of substitutions which occurred outside of, and at considerable distances in three-dimensional (3D) structure from, this region. Inhibition studies revealed substantial differences in the binding affinities of some of the primate Hbs, relative to human Hb. Some of the substitutions caused major decreases in binding, although they were at considerable distances in the 3D structure from the indicated site residues. It is concluded that substitutions in a protein, even when distant from an antigenic site, can exert major influences on the protein's reactivity with anti-site mAbs.

Amino Acid Sequence↗
PubMed1993

Autoimmune T-cell recognition sites of human thyrotropin receptor in Graves' disease.

Five overlapping synthetic peptides representing two regions of thyrotropin (TSH) binding sites of human thyrotropin receptor (TSHR) (peptides 12-30, 24-44, 308-328, 324-344 and 339-364) were investigated for their ability to cause proliferation of peripheral blood lymphocytes (PBL) from eight patients with Graves' disease. The same experiment was done using PBL from four cases with Hashimoto's thyroiditis, two cases with subacute thyroiditis, two cases with rheumatoid arthritis (RA) and eight normal volunteers. PBL obtained from each patient with Graves' disease responded to one or more of peptides 12-30, 24-44, 308-328 and 324-344, while peptide 339-364 had no stimulating activity. The level of stimulating activity of each of the four aforementioned TSHR peptides varied from patient to patient. None of the five TSHR peptides caused the proliferation of PBL from patients with Hashimoto's thyroiditis, subacute thyroiditis, or RA and from normal volunteers. The results indicate that the proliferation of PBL by TSHR peptides is specific in patients with Graves' disease and that the regions of TSHR which are involved in the binding to TSH are also the target of autoimmune T-cell recognition in Graves' disease. The difference in T-cell response from patient to patient could be explained by genetic regulation toward each autodeterminant.

Adolescent↗