Abstract:SynopsisThe N-terminal maleoyl-0-alanyl derivative of human gastrin-[2-17] has been synthesized as a model compound to investigate the usefulness of such peptide derivatives for their mild and selective conjugation, via reaction with thiol groups, to high-molecular-weight carrier molecules to produce antigens, as well as to radioiodinable or fluorogenic molecules to prepare tracers for immunoassays. In this context the examined enzyme substrate-gastrin conjugate was found to exhibit the full immunoreactivity o… Show more
“…The BOC-protected AH−PamMal reagent ( 2 ) was constructed to serve as a phospholipid membrane anchor that could be conveniently modified with amine reactive reporter groups. BOC was chosen as the amino acyl protecting group since it could be removed under acidic conditions that would not alter the maleimide group ( , ). 2 Lipid modification of the N-terminal extracellular domain of the human thrombin receptor.…”
Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces.
“…The BOC-protected AH−PamMal reagent ( 2 ) was constructed to serve as a phospholipid membrane anchor that could be conveniently modified with amine reactive reporter groups. BOC was chosen as the amino acyl protecting group since it could be removed under acidic conditions that would not alter the maleimide group ( , ). 2 Lipid modification of the N-terminal extracellular domain of the human thrombin receptor.…”
Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces.
“…HG17-[13-171 and HG17-[14-17] (51). The antigastrin antisera obtained in the present study recognize the CCKpeptides CCK33-[24-331, CCK33-[22-331, and the analogue [Thr", Nle"]-CCK33- [25][26][27][28][29][30][31][32][33] only with affinities in the range of 1-4%. However, these binding potencies are again about 10 times higher than the cross-reactivities detected for the gastrin fragments HGI 7-[ 14-17] and HGl7-[13-171.…”
Section: Hg17-[2-171 and Hg34-[1-14]-hp-hgl7-[2-17] Asmentioning
confidence: 99%
“…In our previous studies of the effects of chemically well-defined conjugates on the immune responses we have selected human-little-gastrin (HG 17) in the nonsulfated form I as model hapten (25)(26)(27)(28). Selective linkage of the HG17-[2-171 at its N-terminus via the rnaIeinirnide/thiol procedure to the single surfaceexposed cysteine-107 of baker's yeast iso-1 -cytochrome c as carrier, led in immunization experiments to anti-gastrin antibodies of gastrin receptor-like specificity.…”
As core molecule for the multiple attachment of antigenic peptides we have selected the human IgGl hinge fragment 225–232/225′‐232′. Two types of conjugates of this double‐chain bis‐cystinyl hinge‐peptide were prepared i) by linking its C‐termini to [NIe15]‐human‐little‐gastrin‐[2‐17] and ii) by elongating the resulting hinge‐peptide/[NIe15]‐little‐gastrin‐[2‐17] conjugate at the two N‐termini with the human big‐gastrin sequence 1–14 to produce the big‐gastrin‐[l‐14]/hinge‐peptide/little‐gastrin‐[2‐17] conjugate. For the synthesis of these peptide structures both the route via the preformed double‐chain bis‐cystinyl peptide and the route via suitably protected monomeric bis‐cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis‐cysteinyl hinge‐peptide 225–232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti‐little‐gastrin antibody titers comparable to those induced by the iso‐I‐cytochrome c/little‐gastrin‐[2‐17] conjugate as carrier‐hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor‐like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little‐gastrin‐specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little‐gastrin 2–17 and the big‐gastrin 1–14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.
“…Moreover the linkage thus obtained is more flexible and stable in aqueous solutions (17). For these reasons, we chose succinimidyl 3-lV-maleimidopropionate (MPS) (9,13) possessing two CH2 only, to get a spacer resembling as closely as possible the SPPS spacer, the weakest immunogenic one.…”
We have previously shown that the carrier polytuftsin obtained by polycondensation of tuftsin, a naturally occurring macrophage activator, increases significantly the antibody response against a linked B-epitope. In the present work, we have studied the influence of different cross-linking reagents on the quality of the conjugation and on the immune response, at both B-cell and T-cell levels. We observed that the cross-linking method used for coupling the B-epitope to the carrier influences the immune response. A hypothesis is put forward to explain the differences observed.
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