Rodger, A. | bioRxiv Science

bioRxiv2023

Activation of cannabinoid CB2 receptors by (-)-cannabichromene but not (+)-cannabichromene

IntroductionCannabichromene (CBC) is a minor constituent of Cannabis that is a selective cannabinoid CB2 receptor agonist and activator of TRPA1. To date, it has not been shown whether (-)-CBC, (+)-CBC (or both) can mediate these effects. In this study we investigate the activity of the CBC enantiomers at CB1, CB2 and TRPA1 in vitro. Materials and MethodsCBC enantiomers were purified from synthetic CBC by chiral chromatography, and their optical activity was confirmed by spectroscopy. Human CB1 and CB2 receptor activity was measured using a fluorescent assay of membrane potential in stably transfected AtT20 cells. TRPA1 activation was measured using a fluorescent assay of intracellular calcium in stably transfected HEK293 cells. Results(-)-CBC activated CB2 with an EC50 of 1.5 {micro}M, to a maximum of 60 % of CP55940. (+)-CBC did not activate CB2 at concentrations up to 30 {micro}M. Only 30 {micro}M (-)- CBC produced detectable activation of CB1, (+)-CBC was inactive. Both (-)-CBC and (+)- CBC activated TRPA1; at 30 {micro}M (-)-CBC produced an activation 50% of that of the reference agonist cinnamaldehyde (300 {micro}M), 30 {micro}M (+)-CBC activated TRPA1 to 38% of the cinnamaldehyde maximum. DiscussionIt is unclear whether (-)-CBC is the sole or even the predominant enantiomer of CBC enzymatically synthesized in Cannabis. This study shows that (-)-CBC is the active isomer at CB2 receptors, while both isomers activate TRPA1. The results suggest that medicinal preparations of CBC that target cannabinoid receptors would be most effective when (-)-CBC is the dominant isomer.

pharmacology and toxicology↗

bioRxiv2020

Hyper-Truncated N-Acetyl-β-D-Glucosamine Decorations Augment the Activity and Inhibition Potential of Neutrophil Granule Myeloperoxidase

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remain elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a previously unreported low-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility, and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, structural modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity. SignificanceMyeloperoxidase (MPO) is an important microbicidal glycoprotein critical for fighting pathogens. We report, for the first time, the intriguingly complex relationship between glycobiology and MPO immune function by demonstrating that uncommon and strategically positioned hyper-truncated glycans both elevate the activity and the inhibition potential of this pathogen-combating enzyme. We have used a multifaceted approach employing integrated biomolecular analytics to generate new insights into the sugar code of MPO. The findings described in this study improve our understanding of key innate immune processes and may guide future glycoengineering efforts aiming to generate therapeutically relevant recombinant MPO products with tuneable activity and inhibition potential tailored to biomedical applications involving persisting and severe pathogen infections.

biochemistry↗