α-Neurotoxin binding to acetylcholine receptor: Localization of the full profile of the cobratoxin-binding regions on the α-chain ofTorpedo californica acetylcholine receptor by a comprehensive synthetic strategy

Eighteen consecutive uniform overlapping synthetic peptides that spanned the entire extracellular part (residues 1–210) of the α-chain ofTorpedo californica acetylcholine receptor were screened for binding activity of¹²⁵I-labeled cobratoxin. Five toxin-binding regions were localized within residues 1–10, 32–41, 100–115, 122–150, and 182–198. The five toxin-binding regions may be distinct sites or, alternatively, different faces in one or more sites.     

Inhibition of Botulinum Neurotoxin A Toxic Action In Vivo by Synthetic Synaptosome- and Blocking Antibody-Binding Regions

In previous studies, we showed that certain peptides of the H_N and H_C domains of the H-chain of BoNT/A bind to mouse brain synaptosomes (snps). There was either complete correspondence or overlap between peptides that bind snps and those that bind human or mouse blocking antibodies (Abs). An equimolar mixture of the overlapping peptides N5/N6/N7/N8 (residues 505–523/519–537/533–551/547–565) extended the survival time of the mice to 74 h (20%) relative to controls, which had a 50% survival time of 60 h. On the other hand, peptide N26 (residues 799–817) provided no protection (50% survival time, 58 h), but the overlapping peptide N25 (785–803) almost doubled the 50% survival time to 119 h. A mixture of the overlap N25/N26 provided an unexpected level of protection permitting 40% of the mice to survive a lethal BoNT/A dose. In the H_C domain, the overlap C23/C24 (1163–1181/1177–1195) provided no protection. Peptide C31 (1275–1296) also provided no significant protection. But an equimolar mixture of peptides C15/C16 (1051–1069/1065–1083) or peptides C18/C19/C20 (1093–1111/1107–1125/1121–1139) extended the 50% survival time by 41% (to 85 h) over controls (60 h) and was able to fully protect 20% of the mice which eventually recovered. Surprisingly, the mixture of the peptides C15/C16 and C18/C19/C20, which gave a 50% survival time of 75 h, was less protective than either peptides C15/C16 or peptides C18/C19/C20. The in vivo inhibitory activity of these peptides is discussed in relation to their location in the 3-dimensional structure of the toxin molecule and their membrane receptor binding.     

Analysis of exposed regions on the main extracellular domain of mouse acetylcholine receptor α 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) 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 23–49,78–126,146–174, and182–210 are accessible to binding with antibody. Residues23–49,78–126, and194–210 contain binding regions for-neurotoxin and some myasthenia gravis autoantibodies. A comparison of this binding profile with the profile obtained for membrane-boundTorpedo 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. Regions89–104 and158–174, which are exposed in the isolated membrane fraction, are also exposed in the live cell. On the other hand, regions23–49, and182–210, which are exposed in the live cell, are not accessible in the isolated membrane and, furthermore, the region1–16, 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.     

Prostaglandin Endoperoxide H Synthase-2 (PGHS-2) Variants and Risk of Obesity and Microvascular Dysfunction Among Tunisians: Relevance of rs5277 (306G/C) and rs5275 (8473T/C) Genetic Markers

The purpose of this study was to determine the impact of six PGHS-2 genetic variants on obesity development and microvascular dysfunction. The study included 305 Tunisian subjects (186 normal weights, 35 overweights and 84 obeses). PCR analyses were used for allelic discrimination between polymorphisms. Prostaglandin (PGE2, PGI2), leptin, and matrix metalloproteinase (MMP1, 2, 3, 9) levels were evaluated by ELISA. Fatty acid composition was performed by gas chromatography–mass spectrometry. Our results revealed that subjects carrying the PGHS-2 306CC (rs5277) and 8473CC (rs5275) genotypes present higher anthropometric values compared to wild-type genotypes (306GG, BMI (Kg/m²): 27.11?±?0.58; WC (cm): 93.09?±?1.58; 306CC, BMI: 33.83?±?2.46; WC: 109.93?±?5.41; 8473TT, BMI: 27.75?±?0.68; WC: 93.96?±?1.75; 8473CC, BMI: 33.72?±?2.2; WC: 117.89?±?2.94). A reduced microvascular reactivity and a higher PGE2 level were also found in individuals with the 306CC and 8473CC genotypes in comparison to 306GG and 8473TT carriers (306GG, Peak Ach-CVC (PU/mmHg): 0.46?±?0.03; PGE2 (pg/ml): 7933.1?±?702; 306CC, Peak Ach-CVC: 0.24?±?0.01; PGE2: 13,380.3?±?966.2; 8473TT, Peak Ach-CVC: 0.48?±?0.05; PGE2: 7086.41?±?700.31; 8473CC, Peak Ach-CVC: 0.23?±?0.01; PGE2: 13,175.7?±?1165.8). Fatty acid analysis showed a significant increase of palmitic acid (PA) (34.2?±?2.09 vs. 16.82%?±?1.76, P?<?0.001), stearic acid (SA) (25.76?±?3.29 vs. 9.05%?±?2.53, P?<?0.001), and linoleic acid (LA) (5.25?±?1.18 vs. 0.5%?±?0.09, P?<?0.001) levels in individuals carrying the PGHS-2 306CC genotype when compared to GG genotype individuals. Subjects with the 8473CC genotype showed also a significant increase of PA, SA ,and LA levels when compared to TT genotype carriers (PA: 38.02?±?1.51 vs. 12.65%?±?1.54, P?<?0.001; SA: 32.96?±?1.87 vs. 1.38%?±?0.56, P?<?0.001; LA: 26.84?±?2.09 vs. 3.7%?±?1.54, P?<?0.001). Logistic regression analysis revealed that PGHS-2 306CC and 8473CC variants are significantly associated with obesity status (OR 6.25, CI (1.8–21.6), P?=?0.004; OR 3.01, CI (1.13–8.52), P?=?0.03, respectively). Haplotypes containing the C₃₀₆:T₈₄₇₃ (OR 2.91; P?=?0.01) and G_306:C₈₄₇₃ (OR 5.25; P?=?0.002) combinations were associated with an enhanced risk for obesity development in the studied population. In conclusion, our results highlight that PGHS-2 306G/C and 8473T/C variants could be useful indicators of obesity development, inflammation, and microvascular dysfunction among Tunisians.

     

A peptide-based immunoassay for antibodies against botulinum neurotoxin A

Cervical dystonia (CD) is due to neck-muscle spasms that cause pain and involuntary contractions resulting in abnormal neck movements and posture. Symptoms can be relieved by injecting the affected muscle with a botulinum neurotoxin (BoNT, usually type A or type B). The therapeutic benefits are impermanent and toxin injections need to be repeated every 3-6 months. In a very small percentage of patients (less with BoNT/A than with BoNT/B) the treatment elicits blocking anti-toxin antibodies (Abs), which reduce or terminate the patient's responsiveness to further treatment. We have recently mapped (Dolimbek et al., 2006) the CD sera Ab-binding profile using a panel of 60, 19-residue peptides that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by 5 residues. Abs in CD sera bound to one or more of the peptides N25, C10, C15, C20, and C31. This suggested the possibility that binding to these peptides could be used for assay of Abs in CD sera. Data analysis reported here found that Ab binding to these regions showed very significant deviations from the control responses. Of these four peptides, C10 showed the most significant level of separation between patient and control groups (p = 5 x 10(-7)) and the theoretical resolution (i.e., ability to distinguish CD patients from control, see full definition under 'Statistical analysis' in Methods), 84%, was about 4% higher than the least resolved response, C31 (p = 6 x 10(-6), resolution 80%). Since the amounts of Abs bound to a given peptide varied with the patient and not all the patients necessarily recognized all four peptides, there was the possibility that binding to combinations of two or more peptides might give a better discriminatory capability. Using two peptides, C10 plus C31, the resolution improved to 87% (p = 4 x 10(-8)). These two peptides appeared to compliment each other and negate the lower resolution of C31. Combination of three peptides gave resolutions that ranged from 85 (N25 + C15 + C31; p = 2 x 10(-7)) to 88% (C10 + C15 + C31; p = 1 x 10(-8)). Finally, using the data of all four peptides, N25 + C10 + C15 + C31, gave a resolution of 86% (p = 1 x 10(-7)). Although these levels of resolution are somewhat lower than that obtained with whole BoNT/A (resolution 97%; p = 6 x 10(-12)), it may be concluded that the two-peptide combination C10 + C31, or the three-peptide combination C10 + C15 + C31 (affording resolutions of 87 and 88%, respectively) provide a good diagnostic, toxin-free procedure for assay of total specific anti-toxin Abs in BoNT/A-treated CD patients.     

Location of the synaptosome-binding regions on botulinum neurotoxin B

The regions of botulinum neurotoxin B (BoNT/B) involved in binding to mouse brain synaptosomes (snps) were localized. Sixty 19-residue overlapping peptides (peptide C31 consisted of 24 residues) encompassing BoNT/B H chain (residues 442-1291) were synthesized and used to inhibit binding of (125)I-labeled BoNT/B to snps. Synaptosome-binding regions were noncompeting and existed on both H(N) and H(C) domains of neurotoxin. At 37 °C, inhibitory activities on H(N) resided, in decreasing order, in peptides 638-656 (26.7%), 596-614 (18.2%), 512-530 (13.9%), 778-796 (13.8%), and 526-544 (11.6%). On H(C), activity resided in decreasing order in peptides 1170-1188 (44.6%), 1128-1146 (21.6%), 1184-1202 (18.6%), 1156-1174 (13.0%), 946-964 (11.8%), 1114-1132 (11.2%), 1100-1118 (6.2%), 876-894 (6.1%), 1268-1291 (4.6%), and 1226-1244 (4.3%). The 45 remaining H(N) and H(C) peptides had no activity. At 4 °C, peptide C24 (1170-1188) remained quite active (inhibiting, 31.2%), while activities of peptides N15, C21, and C25 were little under 10%. The snp-binding regions contained sites that bind synaptotagmin II and gangliosides. Despite the low degree of sequence homology, BoNT/B and BoNT/A display significant structural homology and appeared to bind in part to the same snp-binding regions. Binding of each labeled toxin to snps was inhibited ~50% by the other toxin, 70-72% by its correlate H(C), and by the H(C) of the other toxin [29% (BoNT/A by H(C) of B) or 32% (BoNT/B by H(C) of A)]. In the three-dimensional structure of BoNT/B, the greater part of H(C), one H(N) face, and part of the belt on the same side interact with snps. Thus, BoNT/B binds to snps through the H(C) head and employs regions on one H(N) face and the belt, reserving flexibility for the belt's unbound part to release the light chain. Most snp-binding regions coincide or overlap with blocking antibody (Ab)-binding regions explaining how such Abs prevent BoNT/B toxicity.     

Antigenic structure of human hemoglobin: Delineation of the antigenic site (site 2) within region 41–65 of the alpha chain by immunochemistry of synthetic peptides

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major “continuous” antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41–65. Ten peptides representing the α-chain regions 41–55, 51–65, 45–54, 45–56, 45–58, 45–60, 48–56, 49–56, 50–56, and 51–56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45–54, 45–45, 45–58, and 45–60). Binding studies revealed that maximum antibody activity resided in peptide 45–45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41–65 of the α chain comprises residues 49–56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.