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Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system

Date: 2015-09-01

Creator: Patsy S. Dickinson, Sienna C. Kurland, Xuan Qu, Brett O. Parker, Anirudh, Sreekrishnan, Molly A. Kwiatkowski, Alex H. Williams, Alexandra B. Ysasi, Andrew E. Christie

Access: Open access

Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family.

Characterization of O-Linked Glycosylated Neuropeptides in the American Lobster (Homarus americanus): The Use of Peptide Labeling Following Beta Elimination

Date: 2020-01-01

Creator: Edward Myron Bull

Access: Open access

Neuropeptides are a class of small peptides that govern various neurological functions, and the American lobster (Homarus americanus) provides a model system for their characterization. Neuropeptides are commonly post-translationally modified (PTM), and one common PTM is glycosylation. Past research in the Stemmler lab has found glycosylated neuropeptides in H. americanus; however, the extent and biological role of this modification has not been well characterized. This study was undertaken to determine the number of glycosylated peptides in the sinus glands of H. americanus and to develop an approach to tag the site of glycosylation using beta-elimination chemistry. LC-MS paired with high pH reverse phase fractionation was used to survey for glycosylated neuropeptides and beta elimination with an amine tag was used as an approach to characterize the site of glycosylation. Our results indicate that high pH fractionation is a useful approach to simplify complex mixtures of neuropeptides and improve glycopeptide detection. Efforts to use beta elimination and tagging to characterize glycosylated neuropeptides have been less successful. Beta elimination of full length peptides resulted in peptide degradation. An approach utilizing chymotrypsin to reduce peptide size coupled with beta elimination and labeling with 2-dimethylaminoethanethiol showed less evidence for degradation, and this approach yielded data isolating two potential serine residues for the site of glycosylation; however, the data was not sufficient to distinguish the two sites. Work to optimize reaction conditions using a glycopeptide standard showed that multiple isomeric products were formed during beta elimination. With the goal of optimizing reaction conditions, future work will further examine reaction kinetics to eventually apply the approach to the entire sinus gland