CRISPR screens and lectin microarrays identify novel high mannose N-glycan regulators
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Abstract
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.
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In contrast, excess high mannose epitopes can over-activate complement pathways through interaction with mannose-binding lectin 7,8 and promote cancer metastasis
It would be very interesting to see whether changes in the presentation of the high-mannose glycans mediated by knockdown of either TM9SF3 or CCDC22 can lead to immune lectin engagement or immune cell activation. Have you thought at all about doing some sort of immune receptor binding assay (i.e. via flow cytometry with an immune lectin-Fc fusion) or an immune activation assay with macrophages or dendritic cells?
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Interestingly, these scattered, smaller Golgi structures observed in our TM9SF3 and CCDC22 knockdown cells are reminiscent of Golgi satellites or outposts in dendrites of neurons, where localized glycosylation events can occur in response to neuronal excitation26,38, suggesting that such regulation of Golgi morphology and function may be a general mechanism by which cells control glycosylation.
I was curious about tissue expression of TM9SF3 and CCDC22 because you mentioned that the Golgi structures you observed in your imaging experiments was reminiscent of Golgi satellites observed in neurons. Interestingly, it seems that TM9SF3 levels are lower in brain tissue than most others in the body, but this is not the case for CCDC22. I’d be curious to know whether this corresponds to glycomic profile changes that match the data you present …
Interestingly, these scattered, smaller Golgi structures observed in our TM9SF3 and CCDC22 knockdown cells are reminiscent of Golgi satellites or outposts in dendrites of neurons, where localized glycosylation events can occur in response to neuronal excitation26,38, suggesting that such regulation of Golgi morphology and function may be a general mechanism by which cells control glycosylation.
I was curious about tissue expression of TM9SF3 and CCDC22 because you mentioned that the Golgi structures you observed in your imaging experiments was reminiscent of Golgi satellites observed in neurons. Interestingly, it seems that TM9SF3 levels are lower in brain tissue than most others in the body, but this is not the case for CCDC22. I’d be curious to know whether this corresponds to glycomic profile changes that match the data you present for A549s.
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Together, these results suggest that the fragmented cis- and medial-Golgi and the reduction in TGN in TM9SF3 knockdown cells may impede the trafficking of glycoproteins through Golgi compartments for glycan remodeling, resulting in a glycan repertoire enriched in high and oligomannose structures
I’m very curious about the hypothesis that TM9SF3 leads to increased high-mannose presentation via TGN bypass. Have you looked at colocalization of high-mannose structures (i.e. using fluorescent GNA or HHL) with the TGN to see if it is reduced in TM9SF3 KD cells?
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The CCC complex negatively regulates Golgi function and complex glycan formation
I found it really interesting how a similar Golgi effect can produce very divergent outcomes on the presentation of N-glycans! I was especially interested in CCDC22’s effect, since it is not annotated to have any Golgi localization. Have you explored whether CCDC22 or any of its interacting partners localize to the Golgi under any circumstances, or else how its knockdown affects Golgi structure and glycosylation pathways?
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