CRISPR screens and lectin microarrays identify novel high mannose N-glycan regulators.

Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genes but the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes pooled CRISPR screens and lectin microarrays to uncover and characterize regulators of cell surface glycosylation. We applied this approach to study the regulation of high mannose glycans - the starting structure of all asparagine(N)-linked-glycans. We used CRISPR screens to uncover the expanded network of genes controlling high mannose surface levels, followed by lectin microarrays to fully measure the complex effect of select regulators on glycosylation globally. Through this, we elucidated how two novel high mannose regulators - TM9SF3 and the CCC complex - control complex N-glycosylation via regulating Golgi morphology and function. Notably, this method allowed us to interrogate Golgi function in-depth and reveal that similar disruption to Golgi morphology can lead to drastically different glycosylation outcomes. Collectively, this work demonstrates a generalizable approach for systematically dissecting the regulatory network underlying glycosylation.

An internally quenched peptide as a new model substrate for rhomboid intramembrane proteases.

Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.