Excavating proteoglycan structure-function relationships: modern approaches to capture the interactions of ancient biomolecules

Abstract

Proteoglycans are now well regarded as key facilitators of cell biology. Although a majority of their interactions and functions are attributed to the decorating glycosaminoglycan chains, there is a growing appreciation for the roles of the proteoglycan core protein and for considering proteoglycans as replete protein-glycan conjugates. This appreciation, seeded by early work in proteoglycan biology, is now being advanced and exalted by modern approaches in chemical glycobiology. In this review, we discuss up-and-coming methods to unearth the fine-scale architecture of proteoglycans that modulate their functions and interactions. Crucial to these efforts is the production of chemically defined materials, including semisynthetic proteoglycans and the in situ capture of interacting proteins. Together, the integration of chemical biology approaches promises to expedite the dissection of the structural heterogeneity of proteoglycans and deliver refined insight into their functions.

Keywords: chemical biology; glycobiology; glycosaminoglycans; interactome; proteoglycan.

Graphical abstract

Figure 1.

Modern chemical biology approaches to investigate proteoglycan biology. A: chemoenzymatic and total synthesis strategies result in homogenous yet short GAG oligosaccharides, which do not span the lengths or recapitulate the heterogeneity and domain structure of native GAGs. B: synthetic xylosides (bottom) can hijack GAG biosynthesis by mimicking xylosylated core proteins and serving as a substrate for the β4GalT7 galactosyltransferase for elongation. These xylosides can be functionalized with chemical moieties [i.e., azide (2, 3)] for biorthogonal conjugation to surfaces and/or proteins. C: PGs, GAG-binding proteins (GAGBPs), and GAG disaccharides can be modified to enable capture of interaction partners. Proximity labeling with enzymes (e.g., APEX2) tags proteins within a short radius, capturing both direct and indirect interactors. Appending a photoreactive moiety [i.e., benzophenone (top) or diazirine (bottom)] captures interactors of proteins or GAGs (depicted). Induced proximity capture, which requires the modification of both the GAG and associated GAGBP, permits cross linking of GAG to GAGBP complexes. D: polypeptide mimics, such as those derived from the syndecan-1 peptide SSTN_92-119 (yellow), permit access to PG glycoconjugates via covalent attachment with GAG oligosaccharides. GAGs, glycosaminoglycans; GAGBPs, GAG-binding proteins; PGs, proteoglycans; SDC1, syndecan-1.

Figure 2.

Uncharted opportunities in exploring proteoglycan structure-function relationships. A: the usage of proximity labeling approaches (e.g., APEX2, depicted) permits the identification of the collection of proteoglycans within a cell using GAGBPs. On the contrary, APEX2-PG fusion proteins can allow the identification of both GAG- and core protein-dependent interactions. Furthermore, these interactors can be derivatized to create small molecule modulators of PG glycosylation. B: the expansion of PG mimetics to include both HS and CS/DS GAGs will progress de novo PGs toward replicating native hybrid PGs, further enabling exploration of structure-activity relationships. C: xylose is a unique sugar in mammalian cells, primarily found in GAG core tetrasaccharide structures (GlcA-Gal-Gal-Xyl). The creation of a derivative with a functional handle for enrichment (e.g., azide, depicted) could allow for pull-down of GAG-modified proteins, including crucially, part-time PGs. D: through derivatization of PGs/GAGBPs or small molecules, molecular glue technologies could be used to modulate PG expression or structure in vitro. This could include the targeted degradation of disease-associated PG glycoforms using proteolysis targeting chimeras that recruit E3 ligase (depicted), or modulation of structure by recruiting GAG modifying enzymes (e.g., sulfatases to remove sulfate groups, or heparanase to cleave HS chains). The combination of these approaches would allow for a global view of the complete proteoglycan repertoire and characterization of their interactions and molecular mechanisms within a chosen biological setting, such as synapses. CS/DS, chondroitin sulfate/dermatan sulfate; GAGs, glycosaminoglycans; GAGBPs, GAG-binding proteins; HS, heparan sulfate; MOE, metabolic oligosaccharide engineering; PGs, proteoglycans.