Biology subjects

Kwok, J. C. F.

Publications and source records attributed to Kwok, J. C. F..

3 recordsLinked to original sources

bioRxiv2022

A quantitative method for sizing hyaluronan and other glycosaminoglycans on surfaces

Hyaluronan (HA) is a major component of peri- and extra-cellular matrices and plays important roles in many biological processes such as cell adhesion, proliferation and migration. The abundance, size distribution and presentation of HA dictate its biological effects and are also useful indicators of pathologies and disease progression. Methods to assess the molecular mass of free-floating HA and other glycosaminoglycans (GAGs) are well established. In many biological and technological settings, however, GAGs are displayed on surfaces, and methods to obtain the size of surface-attached GAGs are lacking. Here, we present a method to size HA that is end-attached to surfaces. The method is based on the quartz crystal microbalance with dissipation monitoring (QCM-D) and exploits that the softness and thickness of films of grafted HA increase with HA size. These two quantities are sensitively reflected by the ratio of the dissipation shift ({Delta}D) and the negative frequency shift (-{Delta}f) measured by QCM-D upon the formation of HA films. Using a series of size-defined HA preparations, ranging in size from [~]2 kDa tetrasaccharides to [~]1 MDa polysaccharides, we establish a monotonic yet non-linear standard curve of the {Delta}D/-{Delta}f ratio as a function of HA size, which reflects the distinct conformations adopted by grafted HA chains depending on their size and surface coverage. We demonstrate that the standard curve can be used to determine the mean size of HA, as well as other GAGs, such as chondroitin sulfate and heparan sulfate, of preparations of previously unknown size in the range from 1 to 500 kDa, with a resolution of better than 10%. For polydisperse samples, our analysis shows that the process of surface-grafting preferentially selects smaller GAG chains, and thus reduces the average size of GAGs that are immobilised on surfaces comparative to the original solution sample. Our results establish a quantitative method to size HA and other GAGs grafted on surfaces, and also highlight the importance of sizing GAGs directly on surfaces. The method should be useful for the development and quality control of GAG-based surface coatings in a wide range of research areas, from molecular interaction analysis to biomaterials coatings.

biophysics↗

bioRxiv2022

An in vitro neuronal model replicating the in vivo maturation and heterogeneity of perineuronal nets

The perineuronal net (PNN) is a condensed form of extracellular matrix (ECM) that enwraps specific populations of neurons and regulates plasticity. To create a PNN, only three classes of components are needed: membrane bound hyaluronan by its synthetic enzyme hyaluronan synthases (HASs), a link protein and a CSPG. However, there is redundancy within the classes as multiple HAS isoforms, link proteins and CSPGs have been found in the PNN in vivo. The effect of this heterogeneity has on PNN function is unresolved. Currently, the most common way to address this question is through the creation and study of PNN component in knockout animals. Here, we reported the development of a primary neuronal culture model which reproduces the in vivo maturation and heterogeneity of PNNs. This model accurately replicated mature cortical PNNs, both in terms of the heterogeneity in PNN composition and its maturation. PNNs transitioned from an immature punctate morphology to the reticular morphology as observed in the mature CNS. We also observed a small population of PNNs that were mature at an earlier time point and a distinct composition, highlighting further heterogeneity. This model will provide a valuable tool for the study of PNN biology, their roles in diseases and the development of PNN focused plasticity treatment.

neuroscience↗

bioRxiv2019

Semaphorin 3A binding to chondroitin sulfate E enhances the biological activity of the protein, and cross-links and rigidifies glycosaminoglycan matrices

Semaphorin 3A (Sema3A) is a secreted protein that signals to cells through binding to neuropilin and plexin receptors and provides neurons with guidance cues key for axon pathfinding, and also controls cell migration in several other biological systems. Sema3A interacts with glycosaminoglycans (GAGs), an interaction that could localize the protein within tissues and involves the C-terminal domain of the protein. This domain comprises several furin cleavage sites that are processed during secretion and in previous works have hampered recombinant production of full-length wild type Sema3A, and the biochemical analysis of Sema3A interaction with GAGs. In this work, we have developed a strategy to purify the full-length protein in high yield and identified two sequences in the C-terminal domain, KRDRKQRRQR and KKGRNRR, which confer to the protein sub nM affinity for chondroitin sulfate and heparan sulfate polysaccharides. Using chemically defined oligosaccharides and solid phase binding assays, we report that Sema3A recognizes a (GlcA-GalNAc4S6S)2 motif but not a (GlcA2S-GalNAc6S)2 motif and is thus highly specific for type E chondroitin sulfate. Functionally, we found that Sema3A rigidified CS-E films that mimic the GAG presentation within extracellular matrices (ECMs), suggesting that Sema3A may have a previously unidentified function to cross-link and thus stabilize GAG-rich ECMs. Finally, we demonstrated that the full-length Sema3A is more potent at inhibiting neurite outgrowth than the truncated or mutant forms that were previously purified and that the GAG binding sites are required to achieve full activity. The results suggest that Sema3A can rigidify and cross-link GAG matrices, implicating Sema3A could function as an extracellular matrix organizer in addition to binding to and signaling through its cognate cell surface receptors.

biochemistry↗