Abstract
Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI 14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI 14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.
Highlights
Polyclonal antisera raised in rabbits and a goat to a synthetic peptide have provided neuroendocrine immunohistochemistry (IHC) images in mammals and in the embryo of the fruit fly Drosophila melanogaster[1]
An antibody to secretogranin II (SgII) did not produce any labelling of mouse, rat and human cerebral blood vessels; labelling was observed in the mouse small intestine, this bore no relation to G530 labelling
This report describes the unusual situation where the identity is unclear of an endogenous antigen of an antibody raised to a synthetic peptide, itself of problematic sequence
Summary
Polyclonal antisera (hereafter ‘antibodies’) raised in rabbits and a goat to a synthetic peptide have provided neuroendocrine immunohistochemistry (IHC) images in mammals (human, sheep, rat) and in the embryo of the fruit fly Drosophila melanogaster[1]. A peptide of 7-8 kDa (polyacrylamide gel electrophoresis, MALDI-TOF MS) was found in sheep jugular vein plasma subjected to fractionation via ultrafiltration and guided by assays in vivo (internal organ reduction in rats) and in vitro (reduced division of rat bone marrow cells), but scant amino acid sequence data could be obtained before the target molecule was lost to view. Apart from being used in IHC, G530 was deployed in an antigen capture campaign featuring immunoprecipitation (IP) with liquid chromatography-mass spectroscopy (LC-MS), using two main feedstocks: aqueous extract of rat hypothalamus and fruit fly embryo material[1] The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A
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