The formation of surfaces decorated with biomacromolecules such as proteins, glycans, or nucleic acids with well-controlled orientations and densities is of critical importance for the design of in vitro models, e.g., synthetic cell membranes and interaction assays. To this effect, ligand molecules are often functionalized with an anchor that specifically binds to a surface with a high density of binding sites, providing control over the presentation of the molecules. Here, we present a method to robustly and quantitatively control the surface density of one or several types of anchor-bearing molecules by tuning the relative concentrations of target molecules and free anchors in the incubation solution. We provide a theoretical background that relates incubation concentrations to the final surface density of the molecules of interest and present effective guidelines toward optimizing incubation conditions for the quantitative control of surface densities. Focusing on the biotin anchor, a commonly used anchor for interaction studies, as a salient example, we experimentally demonstrate surface density control over a wide range of densities and target molecule sizes. Conversely, we show how the method can be adapted to quality control the purity of end-grafted biopolymers such as biotinylated glycosaminoglycans by quantifying the amount of residual free biotin reactant in the sample solution.

• MeSH
• biopolymery MeSH
• biotin * chemie   MeSH
• buněčná membrána MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biopolymery MeSH
• biotin * MeSH

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 (ΔD) and the negative frequency shift (- Δf) 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 ΔD/ - Δf 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.

• MeSH
• buněčná adheze MeSH
• chondroitinsulfáty MeSH
• glykosaminoglykany * chemie   MeSH
• kyselina hyaluronová * chemie   MeSH
• mikrorovnovážné techniky křemenného krystalu MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chondroitinsulfáty MeSH
• glykosaminoglykany * MeSH
• kyselina hyaluronová * MeSH

The perineuronal net (PNN) is a mesh-like proteoglycan structure on the neuronal surface which is involved in regulating plasticity. The PNN regulates plasticity via multiple pathways, one of which is direct regulation of synapses through the control of AMPA receptor mobility. Since neuronal pentraxin 2 (Nptx2) is a known regulator of AMPA receptor mobility and Nptx2 can be removed from the neuronal surface by PNN removal, we investigated whether Nptx2 has a function in the PNN. We found that Nptx2 binds to the glycosaminoglycans hyaluronan and chondroitin sulphate E in the PNN. Furthermore, in primary cortical neuron cultures, the addition of NPTX2 to the culture medium enhances PNN formation during PNN development. These findings suggest Nptx2 as a novel PNN binding protein with a role in the mechanism of PNN formation.

• MeSH
• C-reaktivní protein metabolismus   MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• nervová síť chemie   cytologie   metabolismus   MeSH
• neurony chemie   metabolismus   MeSH
• neuroplasticita fyziologie   MeSH
• perineuronální satelitní buňky chemie   metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• proteiny nervové tkáně metabolismus   MeSH
• vazba proteinů fyziologie   MeSH
• zrakové korové centrum chemie   cytologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-reaktivní protein MeSH
• neuronal pentraxin MeSH Prohlížeč
• proteiny nervové tkáně MeSH