@article {121922, title = {Contryphan sequence diversity: Messy N-terminus processing, effects on chromatographic behaviour and mass spectrometric fragmentation.}, journal = {J Proteomics}, year = {2023}, month = {2022 Dec 29}, pages = {104805}, abstract = {

Contryphans, peptides containing a single disulfide bond, are found abundantly in cone snail venom. The analysis of a large dataset of available contryphan sequences permits a classification based on the occurrence of proline residues at positions 2 and 5 within the macrocyclic 23-membered disulfide loop. Further sequence diversity is generated by variable proteolytic processing of the contryphan precursor proteins. In the majority of contryphans, presence of Pro at position 2 and a D-residue at position 3 leads to a slow conformational dynamics, manifesting as anomalous chromatographic profiles during LC analysis. LC-MS analysis of diverse contryphans suggests that elution profiles may be used as a rapid diagnostic for the presence of the Pro2-Xxx3 motif. Natural sequences from C.inscriptus and C.frigidus together with synthetic analogs permit the delineation of the features necessary for abnormal chromatographic behaviour. A diagnostic for the presence of Pro at position 5 is obtained by the observation of non-canonical fragment ions, generated by N-C bond cleavage at the dehydroalanine residue formed by disulfide cleavage. Anomalous LC profiles supports Pro at position 2, while non-canonical mass spectral fragments established Pro at position 5, providing a rapid method for contryphan analysis from LC-ESI-MS/MS profiles of crude Conus venom. SIGNIFICANCE: Contryphans are peptides, widely distributed in cone snail venom, which display extensive sequence diversity. Heterogeneity of proteolytic processing of contryphan precursor proteins, together with post-translational modifications contributes to contryphan diversity. Contryphans, identified by a combination of mass spectrometry and transcriptomic analysis, are classified on the basis of sequence features, primarily the number of proline residues within the disulfide loop. Conformational diversity arises in contryphans by cis-trans isomerization of Cys-Pro bonds, resulting in characteristic chromatographic profiles, permitting identification even in crude venom mixtures. Rapid identification of contryphans in cone snail peptide libraries is also facilitated by diagnostic mass spectral fragments arising by non-canonical cleavage of the N-C bond at Cys(6).

}, issn = {1876-7737}, doi = {10.1016/j.jprot.2022.104805}, author = {Vijayasarathy, M and Kumar, Sanjeev and Venkatesha, M A and Balaram, P} } @article {73044, title = {Cone snail analogs of the pituitary hormones oxytocin/vasopressin and their carrier protein neurophysin. Proteomic and transcriptomic identification of conopressins and conophysins.}, journal = {Biochim Biophys Acta Proteins Proteom}, year = {2020}, month = {2020 Feb 10}, pages = {140391}, abstract = {

Transcriptomic analysis of cone snail venom duct tissue has permitted the identification of diverse conopressin/conophysin precursor sequences from seven distinct conus species. Multiple precursor isoforms are present in C.monile, C.lividus and C.loroisii. Aqueous extracts of the venom duct tissue from C.monile yield a band, at ~ 15-20 kDa on SDS-PAGE. In-gel trypsin digestion, followed by mass spectrometry establishes the presence of two distinct conopressin/conophysin isoforms that differ at position 8 in the predicted conopressin nonapeptide sequence. Mass spectrometric analysis of aqueous extracts revealed the presence of four conopressin related peptides, whose sequences could be deduced from MS/MS fragmentation patterns. The four sequences determined in this study are CFIRNCPKG*, CFIRNCPEG*, CFIRNCPK* and CFIRNCPE* (* indicates amide), which were further confirmed by comparison with chemically synthesized peptides. A conophysin with a mass of 9419.7 Da was also detected, corresponding to one of the isoforms revealed by the transcriptome data. Complete conservation of fourteen Cys residues and the key residues involved in peptide hormone binding is established by comparison of conophysin sequences, with the crystallographically characterized sequence of bovine neurophysin, in complex with vasopressin. A survey of available sequences for oxytocin/vasopressin peptides in both vertebrates and invertebrates establishes the conopressins as a distinct group in this family. C-terminal amidated, truncated conopressin analogs may arise by alternate post-translational processing.

}, issn = {1878-1454}, doi = {10.1016/j.bbapap.2020.140391}, author = {Kumar, Sanjeev and Vijayasarathy, M and Venkatesha, M A and Sunita, P and Balaram, P} } @article {53177, title = {Cone snail prolyl-4-hydroxylase α-subunit sequences derived from transcriptomic data and mass spectrometric analysis of variable proline hydroxylation in C. amadis venom.}, journal = {J Proteomics}, volume = {194}, year = {2019}, month = {2019 Mar 01}, pages = {37-48}, abstract = {

Putative prolyl-4-hydroxylase (P4H) α-subunit sequences have been extracted by mining transcriptomic data obtained from seven cone snail species C. amadis, C. monile, C. araneosus, C. miles, C. litteratus, C. frigidus, and C. ebraeus. Sequences ranging from 518 to 559 residues have been compared with representative animal P4H sequences. The α-subunit consists of an N-terminus double domain, involved in dimerization and substrate binding, while the C-terminus contains the catalytic domain. Definitive functional annotation of the cone snail sequences has been achieved by an analysis of conserved residues responsible for catalytic function, specific conformational features, and subunit interactions, using two independent structures of the double domain, and the catalytic domain, previously reported in the literature. The variability of proline hydroxylation in conotoxins is illustrated by a mass spectrometric analysis of C. amadis venom. Site specific hydroxylation and the presence of peptides with multiple proline residues, resistant to modification, suggests that sequence and conformational effects may determine the substrate specificity of the Conus prolyl-4-hydroxylases. SIGNIFICANCE: Proline hydroxylation is a widely observed post translational modification, with collagen being the pre-eminent example. Hydroxylation of proline is also widely observed in conotoxins, which are a major component of marine cone snail venom. This paper describes newly identified prolyl-4-hydroxylase sequences, using transcriptome data from seven Conus species. The predicted functional annotation of prolyl-4-hydroxylase sequences was carried out using two available crystal structures of independent domains. The mass spectrometric characterisation of proline/hydroxyproline containing peptides in C. amadis venom confirms sequence specific hydroxylation in Conus venom as shown previously by others.

}, issn = {1876-7737}, doi = {10.1016/j.jprot.2018.12.028}, author = {Vijayasarathy, M and Balaram, P} } @article {45452, title = {Mass spectrometric identification of bromotryptophan containing conotoxin sequences from the venom of C.~amadis.}, journal = {Toxicon}, volume = {144}, year = {2018}, month = {2018 Mar 15}, pages = {68-74}, abstract = {

Four 30 residue conotoxin have been identified from the venom of C.\ amadis. MS/MS analysis of crude venom subjected to global reduction/alkylation yielded fragmentation patterns, which permitted searching and matching with a database of putative mature toxin sequences obtained from transcriptomic analysis. Of the four sequences identified, Am3408(Am6.1b), Am3452(Am6.1c), Am3136(Am6.2a) and Am3214(Am6.2b), three contain bromotryptophan residues, while an additional post translational modification, gamma carboxylation of glutamic acid, is present in Am3408(Am6.1b)/3452(Am6.1c). The conotoxins belong to the O1/O2 gene superfamily and possess cysteine framework VI/VII. While, the cysteine patterns show a similarity to omega conotoxins, the three C.\ amadis peptides are highly negatively charged and possess a significant content of hydrophobic residues.

}, issn = {1879-3150}, doi = {10.1016/j.toxicon.2018.02.005}, author = {Vijayasarathy, M and Balaram, P} }