Exciting New Developments and Emerging Themes in Glycosaminoglycan Research

There have been some exciting new technological advances, many demonstrating the benefit of large, international collaborations that are well supported by our friendly and collegiate field. We saw a new take on heparan sulfate (HS) sequencing ¹ using “shotgun” ion mobility mass spectrometry sequencing helping to position elusive 3-O-sulfate groups and to investigate structure–function relationships. In the often confusing and highly competitive area of glycosaminoglycan (GAG)-functionalized biomaterials, decellularized preparations from lung were shown to be unable to bind key matrix-associated growth factors without replenishment with specific GAGs, ² providing useful clarity for future studies. We saw the identification of a key element in the coordinated regulation of GAG synthesis, the transcription factor ZNF263, ³ suggesting a future for directed bioengineering of designer GAGs. Moving on to the role of GAGs in regulating cell signaling, there were many exciting new stories. To highlight just one, important new detail on the role of HS in vascular remodeling and maturation emerged, with important roles in the angiogenic processes active in diseases such as cancer and diabetic retinopathy. HS was demonstrated to support the assembly of cell surface receptor complexes of the angiopoietin–Tie pathway. Sulfated GAGs were identified as ligands of the orphan receptor Tie1, adding to the small but growing number of examples where GAGs directly engage and control downstream signaling from cell surface receptors. ⁴

Importantly, our community also responded to the call for improved understanding of SARS-CoV-2. There have been more publications than can be mentioned here, and there are likely to be many more in the coming months and years, but for me two studies stood out: first, the demonstration that viral binding and infection is enhanced by the involvement of cell surface HS in addition to angiotensin-converting enzyme 2 (ACE-2) ⁵ with HS interacting with the receptor-binding domain adjacent to the ACE-2 binding site; next, the use of ligand-binding assays and computational ligand docking to highlight the potential of non-anticoagulant heparin-based interventions for COVID-19 patients. ⁶ The scene is set for an exciting ride, hopefully providing much-needed insight into the virus, both in terms of infection and the progression of disease and with the additional hope for the identification of therapeutics that can be deployed rapidly.

Let’s move on to this collection of review articles. Let’s start from (nearly) the very beginning. Zimmer, Barycki, and Simpson provide a detailed update on the current understanding of how the activity of the enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH), acting early in the synthetic pathway of sulfated and non-sulfated GAGs, is regulated at multiple levels to prioritize metabolite distribution between downstream pathways. ⁷ The authors review recent studies of the importance of oligomerization (the poetically described “trimer of dimers”) and conformational dynamics, including site-directed mutagenesis to unpick the role of subunit–subunit interactions. Clinical relevance was also picked up with discussions of the impact of known clinically observed mutations and of the potential for use of UGDH as a field effect biomarker in prostate cancer.

Staying with metabolism but moving outside of the cell (or at least to the plasma membrane), Caon et al. ⁸ introduce us to the role of HA synthases (HASes), particularly HAS2, as nutrient sensors. HA biosynthesis, requiring glucose and glutamine-utilizing pathways, is described as a dynamic process closely influenced by the cell’s metabolic status, tied to the availability of N-acetylglucosamine generated by the hexosamine biosynthetic pathway. The authors also provide further insight on an area that is receiving a lot of current interest, that of the mechanistic link between hyperglycemic conditions and increased plasma and cell-associated HA. In summing up, the authors also point to the exciting potential for taking advantage of the cellular regulation of HA metabolism for targeted therapeutics.

Moving from metabolic regulation of HA on to the function of HA and its receptor, CD44, Queisser, Mellema, and Petrey introduce us to the glycocalyx. ⁹ This specialized extracellular matrix, rich in HA and other GAGs, is located between blood and the endothelial surface and has critical roles in homeostatic vascular function. In contrast to the role of metabolic flux in regulating HAS activity discussed above, here the authors introduce the role that HA and the proteins to which it binds plays as mechanosensory transducers, transmitting extracellular signals into the cell. The authors discuss the multiple disease states in which HA and its receptors play a role, suggesting that improved understanding of these interactions will provide novel therapeutic opportunities.

Inter-α-trypsin inhibitor (IaI) is a fascinating molecule with a bit of something for everyone, having myriad roles in cell regulation and matrix integrity in health and disease. ¹⁰ Structurally, IaI is composed of the chondroitin sulfate proteoglycan (CSPG) bikunin complexed with two heavy chain (HC) proteins, HC1 and HC2, covalently attached to the chondroitin sulfate (CS) chain. Lord et al provide a much-needed update of IaI function, explaining how contrasting activities likely reflect the temporal tissue context and specific composition of IaI family members. Other than its role in providing HCs for HA complexation, multiple studies have now also demonstrated interactions of IaI with additional and varied extracellular matrix components, as has additionally been shown for the constituent HCs themselves. When reviewing the known roles of IaI, the authors walk us through multiple tissue systems, carefully highlighting roles in normal tissue homeostasis as well as disease. Finally, we are provided with an update on the use of IaI family members in the diagnosis of diseases and disorders as diverse as cancer and preeclampsia as well as their application as therapeutics.

CSPGs are further explored by Mencio et al. ¹¹ who discuss and update our understanding of their role in neural development. In a recurring theme across these reviews, the authors highlight how specific sulfation patterns within GAGs can be associated with both inhibition and promotion of an effect, here neural regeneration after injury, with the outcome being time- and position-dependent. Discussing neuronal migration, we are introduced to how CSPGs work in concert with Semaphorin 3A to direct migrating interneurons away from the striatum and tangentially to the cortex. This is in contrast to the role of multiple CS/HS-binding chemoattractive factors, making the role of prostaglandins in neural crest migration complex and finely balanced. The same theme is returned to many times, with contrasting roles for HS and CS in the complex interplay between attractive and repulsive forces that regulates how sensory axons of the trigeminal nerve get to their targets. In a detailed overview of the role of CSPGs in synapase formation, stabilization, and myelination, we are introduced to perineuronal nets (PNNs) and their regulation of neuronal plasticity. Again, here we see association between CSPGs and HA with targeting of either of these destroying PNNs. In their summary, the authors highlight the widespread, complex, and sometimes contradictory roles of CSPGs, suggesting the need for more in-depth understanding.

To probe the complexities of HS biosynthesis, Filipek-Górniok et al. ¹² turn to zebrafish with a discussion of the benefits and drawbacks of this model system. Due to a whole-genome duplication in teleost fish, zebrafish has more genes for HS biosynthetic enzymes and core proteins than mammals, complicating investigations. However, an increasing number of studies are taking advantage of the many benefits of the system, and multiple useful mutants are now available. Forward genetic screens have identified mutants in UGDH as well as other early-acting factors in heparan sulfate proteoglycan synthesis, typically linked with consistent phenotypes of cartilage defects, particularly abnormal jaw and pharyngeal structures. Mutants specifically targeting HS biosynthesis (ext2 and extl3) display complex phenotypes, and interestingly, extl3 mutants were found to have elevated levels of CS, reminiscent of what had previously been seen in Chinese hamster ovary or embryonic stem cells with reduced or altered HS production. That this wasn’t also seen in the ext2 mutants is suggested to be linked to the role of Extl3 in HS synthesis, controlling the committing step between HS and CS/dermatan sulfate. Looking to the future, the authors highlight the potential benefits of CRISPR/Cas9 gene editing technology and how it may be possible to build a “living library” of fish/larva of GAG mutants for studies of biosynthesis, gene function, and pathology.