Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related

Hart, John E.; Clarke, I. J.; Risbridger, Gail P.; Ferneyhough, Ben; Vega-Hernández, Mónica

doi:10.7717/peerj.3833

Cited by 14 publications

(51 citation statements)

References 29 publications

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“…The probability of a nine-residue partial anagram of EPL001's 11 residues (i.e. minus NNI) occurring by chance in a typical ovine protein is about 1 in 146,000, as previously calculated 1 . Going further, EPL001 and sSgII-9 have the same initial residue: methionine.…”

Section: Any Further Responses From the Reviewers Can Be Found At Thesupporting

confidence: 72%

“…Preabsorption, i.e. mixing of the antibody with antigen prior to application of the antibody, to block staining, has been achieved in western blotting with the C-terminal EPL001 peptide but not with the N-terminal peptide, in regard to aqueous extract of rat hypothalamus purified using an immunoaffinity column 1 . Both the western blotting and the immunopurification used G530.…”

Section: Any Further Responses From the Reviewers Can Be Found At Thementioning

confidence: 99%

“…The deployment of epitope mapping is described here in a factor hunt. 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 . But what exactly were the antibodies seeing endogenously?…”

Section: Introductionmentioning

confidence: 99%

“…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 (aa) sequence data could be obtained before the target molecule was lost to view. In this prior work 1 an unambiguous sequence obtained by automated step-wise Edman degradation (Applied Biosystems/PROCISE, Foster City, CA, US) was the N-terminal 14 amino acids MKPLTGKVKEFNNI, synthesized as a peptide designated EPL001. The preceding purification run provided the partially similar ML/KPLTGQAMEF, while a following run delivered the highly similar MKPLT/GKVKxFNNI.…”

Section: Introductionmentioning

confidence: 99%

“…The 14 residues MKPLTGKVKEFNNI were synthesized as EPL001, as described. A goat anti-EPL001 antibody was raised 1 and designated G530. 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 .…”

Section: Introductionmentioning

confidence: 99%

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Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing

et al. 2019

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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 1MKPLTGKVKEFNNI14. 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 9KEFNNI14. 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.

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Section: Any Further Responses From the Reviewers Can Be Found At Thesupporting

confidence: 72%

Section: Any Further Responses From the Reviewers Can Be Found At Thementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing

et al. 2019

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Interaction of arsenic with gap junction protein connexin 43 alters gap junctional intercellular communication

Hussain

Sarma

Babu

et al. 2018

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Chronic exposure to Arsenic pollution in ground water is one of the largest environmental health disasters in the world. The toxicity of trivalent Arsenicals primarily happens due to its interaction with sulfhydryl groups in proteins. Arsenic binding to the protein can change the conformation of the protein and alter its interactions with other proteins leading to tissue damage. Therefore, much importance has been given to the studies of Arsenic bound proteins, for the purpose of understanding the origins of toxicity and to explore therapeutics. Here we study the dynamic effect of Arsenic on Connexin 43 (Cx43), a protein that forms the gap junctions, whose alteration deeply perturbs the cell-to-cell communication vital for maintaining tissue homeostasis. In silico molecular modelling and in vitro studies comparing Arsenic treated and untreated conditions show distinct results. Gap junction communication is severely disrupted by Arsenic due to reduced availability of unaltered Cx43 in the membrane bound form. In silico and Inductively Coupled Plasma Mass Spectrometry studies revealed the interaction of Arsenic to the Cx43 preferably occurs through surface exposed cysteines, thereby capping the thiol groups that form disulfide bonds in the tertiary structure. This leads to disruption of Cx43 oligomerization, and altered Cx43 is incompetent for transportation to the membrane surface, often forming aggregates primarily localizing in the endoplasmic reticulum. Loss of functional Cx43 on the cell surface have a deleterious effect on cellular homeostasis leading to selective vulnerability to cell death and tissue damage.

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Hexapeptides from a mammalian inhibitory hormone activate and inactivate nematode reproduction

Hart¹,

Mohan

Davies

et al. 2021

Preprint

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Increased reproduction (x3) of the entomopathogenic nematode Steinernema siamkayai occurred when exposed to one synthetic peptide (IEPVFT), while the fecundity of worms exposed to hexamer (KLKMNG) was reduced (x0.5). These hexamers were opposite ends of a 14 amino acid (aa) synthetic peptide KLKMNGKNIEPVFT (EPL030). The bioactivity of the hexamers is surprising it is a scrambled-sequence control of another peptide, MKPLTGKVKEFNNI (EPL001) which are bioinformatically obscure. EPL001 emerged from a physicochemical fractionation aimed at finding a postulated hormone that is reproductively related and tissue-mass reducing and has antiproliferative effects on human prostate cancer cells and rat bone marrow cells in vitro. Intracerebroventricular infusion of EPL001 in sheep was associated with elevated growth hormone in peripheral blood and reduced prolactin. The highly dissimilar EPL001 and EPL030 nonetheless have the foregoing biological effects in common in mammalian systems, while being divergently pro- and anti-fecundity respectively in the nematode Caenorhabditis elegans. Immunoprecipitation of EPL001 using an anti-EPL001 antibody suggests it encodes the sheep neuroendocrine prohormone secretogranin II (sSgII). Using bespoke bioinformatics with six sSgII residues deduced bioactivity to key aa: MKPLTGKVKEFNNI. Peptides more potent as cell inhibitors than EPL001 suggest a stereospecific bimodular tri-residue signature (i.e. simultaneous accessibility for binding of two specific trios of aa side chains, MKP & VFN). An evolutionarily conserved receptor is conceptualised as having dimeric binding sites, each with ligand-matching bimodular stereocentres. Sequence analysis and computational modelling suggest the activity of the control peptide EPL030 and its N- and C-terminal hexapeptide progeny is due the novel hormonal motif MKPVFN.

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Mysterious inhibitory cell regulator investigated and found likely to be secretogranin II related

Cited by 14 publications

References 29 publications

Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing

Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing

Interaction of arsenic with gap junction protein connexin 43 alters gap junctional intercellular communication

Hexapeptides from a mammalian inhibitory hormone activate and inactivate nematode reproduction

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