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

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 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.

Hexapeptides from a mammalian inhibitory hormone activate and inactivate nematode reproduction

“…EPL001 is a found sequence, as described in the Introduction. As a 14mer synthetic peptide it has been represented elsewhere as a minimized ball and stick model in silico (Howlett et al, 2019). EPL001 amounts to a 20% N-terminal fragment of Candidate 7500, an approximately 70 aa candidate polypeptide from gel electrophoresis and MS for a postulated inhibitory hormone, yet EPL001 displays on its own in vivo the two crucial features of the sought-for hormone: anti-organotrophic activity (dose-dependently) and reproductive modulation.…”

“…Along the string there is 236 NNI 238, the motif deemed crucial for full binding of the anti-EPL001 antibody when exposed C-terminally. Epitope mapping gave rise to the deduction that the sought-for sSgII proteoform was the product of reverse peptide splicing within a single SgII molecule or splicing between two separate SgII molecules (Howlett et al, 2019). In the relevant location of intracellular secretory vesicles, another granin protein, chromogranin A, has been reported to be involved with insulin in an exotic form of peptide fusion splicing (Baker et al, 2018).…”

“…The readings are 'picked' in (spiral) surface accessible order, MKP, LTG, KVK and EFNNI (Fig. 1), such that EPL001 encodes the source molecule's spatial configuration, explaining why Immunoprecipitation in the inhibitory hormone project delivered SgII relatedness (Hart et al, 2017); bioinformatic NNI-omics led to the second sorting domain of sSgII (Howlett et al, 2019); whence the anagrammatical sequence grid of Fig. 1, linking EPL001 and sSgII-14, for which there would appear to be no literature precedent.…”

“…The lead candidates to emerge from this exercise were (a) a rat protein derived by splice variation from the mammalian secretory vesicle protein secretogranin II (SgII, UniProt P10362) and (b) an SgII homologue in the fruit fly (UniProt Q9W2X8). The binding site (epitope) on EPL001 of the anti-EPL001 antibody and the epitope on any endogenous antigen were investigated using mammalian IHC as the readout (Howlett et al, 2019). This epitope mapping yielded results consistent with the mammalian endogenous antigen being SgII related and emphasized the importance of the aa motif NNI in EPL001 and in the endogenous antigen.…”

“…Yet preliminary molecular modelling in silico (RPN) drew the eye to EPL001’s C-terminal FNNI as a biologically relevant motif, as alluded to elsewhere (Hart, 2008), suggesting the involvement of the Beale 4’s FN . EPL001’s C-terminal KE FN NI is the epitope of the anti-EPL001 antibody (G530) and also probably the SgII-related endogenous epitope (Howlett et al, 2019), indicating surface accessibility for the antibody in both cases. The epitope residues within EPL001 are ranged in the correct order across the foot of the sequence grid.…”