Neuropeptides are the largest class of signaling molecules used by nervous systems.  Most active forms of neuropeptides have been subjected to post-translational modifications that include C-terminal amidation, N-terminal pyroglutamate formation, disulfide bond formation and sulfation of tyrosine residues.  Much work has focused on the identification/characterization of crustacean neuropeptides because of the ability to use crustaceans as model systems for the investigation of peptidergic control of rhythmic behavior. Crustaceans provide an ideal model system to better understand neuropeptide modulation, as these systems as less complex and can be studied when removed from the animal and kept in saline.  Glycosylation is a less well understood post-translational modification that may play a role in modulating cell signaling, and recent work in our laboratory has identified a number of peptides from neural tissue extracts that are glycosylated.  This study reports on work to characterize neuropeptides modified by O-linked glycosylation using beta-elimination reactions coupled with chemical labelling to facilitate neuropeptide sequencing and localization of the site of glycosylation.  Work has focused on <i>H. americanus</i> sinus glands, which have been extracted, fractionated using high pH reversed-phase chromatography, and probed for the production of fragment ions characteristic of glycosylation using MS/MS techniques on a Q-TOF system.  Following the analysis of whole tissue extraction, the extracts were subjected to beta-elimination and chemical labelling with amines.  Experiments using peptides analyzed with and without enzymatic digestion highlighted challenges associated with peptide degradation during beta-elimination, particularly for peptides rich in serine and threonine residues.  We report on work on the detection and characterization of glycosylated variants of four CHH precursor-related peptides (CPRPs) present in the sinus gland.  While other members of the CHH family of peptides are known to play a role in processes that include glucose regulation, molting, and ovary development, the specific role of the CPRP peptides has yet to be determined. Characterization of the type, site, and nature of the glycosylation should provide insights into possible roles that glycosylation may play in the crustacean nervous system.

Characterization of O-linked glycosylated neuropeptides from the lobster, <i>Homarus americanus</i>, employing beta-elimination and peptide labelling

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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Characterization of O-linked glycosylated neuropeptides from the lobster, Homarus americanus, employing beta-elimination and peptide labelling