Abstract
Proper protein glycosylation is critical to normal cardiomyocyte physiology. Aberrant glycosylation can alter protein localization, structure, drug interactions, and cellular function. The in vitro differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM) has become increasingly important to the study of protein function and to the fields of cardiac disease modeling, drug testing, drug discovery, and regenerative medicine. Here, we offer our perspective on the importance of protein glycosylation in hPSC-CM. Protein glycosylation is dynamic in hPSC-CM, but the timing and extent of glycosylation are still poorly defined. We provide new data highlighting how observed changes in hPSC-CM glycosylation may be caused by underlying differences in the protein or transcript abundance of enzymes involved in building and trimming the glycan structures or glycoprotein gene products. We also provide evidence that alternative splicing results in altered sites of glycosylation within the protein sequence. Our findings suggest the need to precisely define protein glycosylation events that may have a critical impact on the function and maturation state of hPSC-CM. Finally, we provide an overview of analytical strategies available for studying protein glycosylation and identify opportunities for the development of new bioinformatic approaches to integrate diverse protein glycosylation data types. We predict that these tools will promote the accurate assessment of protein glycosylation in future studies of hPSC-CM that will ultimately be of significant experimental and clinical benefit.
Highlights
This article is published as part of the Special Issue on Recent Progress with hPSCs for Drug Discovery, Organoids, Disease Models, and Cardiac Repair
An N-linked precursor glycan is transferred from a dolichol-phosphate onto asparagine residues within the consensus sequence [(Asp (N)-x-Ser (S)/Thr (T)/Cys (C)), where x can be any amino acid except proline] within the polypetide chain [9, 62]
23% of the glycans were shared among human pluripotent stem cells into cardiomyocytes (hPSC-CM) and primary cardiomyocytes, while different sialylation linkages were observed between the two sample types
Summary
Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 0A3, Canada. There can be varying levels of site occupancy and different combinations of glycan structures across the protein sequence (i.e., macroheterogeneity) (Fig. 1B) Beyond their canonical intracellular location within the ER-Golgi apparatus, some glycosyltransferases are secreted and function as extracellular enzymes. 20% of CDG have been associated with cardiac complications (reviewed in [61]), including defects in glycosylation enzymes that give rise to dilated cardiomyopathy, hypertrophic cardiomyopathy, and endocardial sclerosis [19, 39, 59, 71, 76, 100] These studies reveal how proper protein glycosylation is critical to normal cardiomyocyte function. Using xCGE‐LIF, Konze et al examined early myogenic and cardiomyogenic commitment by comparing glycans from hPSC-CM at differentiation days 0, 7, and 15 and from primary human cardiomyocytes [45] They reported 62 N-glycan structures and observed three structures unique to hPSC compared to hPSC-CM, including β1,3-linked galactose, α2,6-linked sialic acid, and complex fucosylation. Our group used porous graphitized carbon liquid chromatography electrospray tandem mass spectrometry (PGC-LC–ESI–MS/MS) to separate and
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