Probing the biofunctionality of biotinylated hyaluronan and chondroitin sulfate by hyaluronidase degradation and aggrecan interaction
Artikel i vetenskaplig tidskrift, 2013

Molecular interactions involving glycosaminoglycans (GAGs) are important for biological processes in the extracellular matrix (ECM) and at cell surfaces, and also in biotechnological applications. Enzymes in the ECM constantly modulate the molecular structure and the amount of GAGs in our tissues. Specifically, the changeable sulfation patterns of many GAGs are expected to be important in interactions with proteins. Biotinylation is a convenient method for immobilizing molecules to surfaces. When studying interactions at the molecular, cell and tissue level, the native properties of the immobilized molecule, i.e. its biofunctionality, need to be retained upon immobilization. Here, the GAGs hyaluronan (HA) and chondroitin sulfate (CS), and synthetically sulfated derivatives of the two, were immobilized using biotin-streptavidin binding. The degree of biotinylation and the placement of biotin groups (end-on/side-on) were varied. The introduction of biotin groups could have unwanted effects on the studied molecule, but this aspect that is not always straightforward to evaluate. Hyaluronidase, an enzyme that degrades HA and CS in the ECM, was investigated as a probe to evaluate the biofunctionality of the immobilized GAGs, using both quartz crystal microbalance and high-performance liquid chromatography. Our results showed that end-on biotinylated HA was efficiently degraded by hyaluronidase, whereas already a low degree of side-on biotinylation destroyed the degrading ability of the enzyme. Synthetically introduced sulfate groups also had this effect. Hence hyaluronidase degradation is a cheap and easy way to investigate how molecular function is influenced by the introduced functional groups. Binding experiments with the proteoglycan aggrecan emphasized the influence of protein size and surface orientation of the GAGs for in-depth studies of GAG behavior.

Hyaluronidase

Surface-sensitive techniques

Glycosaminoglycan

Biotin

Aggrecan

Författare

Noomi Altgärde

Chalmers, Teknisk fysik, Biologisk fysik

Erik Nilebäck

Chalmers, Teknisk fysik, Biologisk fysik

Laura De Battice

Chalmers, Teknisk fysik, Biologisk fysik

I. Pashkuleva

ICVS/3B's - PT Government Associate Laboratory

Universidade do Minho

R. L. Reis

Universidade do Minho

ICVS/3B's - PT Government Associate Laboratory

Jana Becher

Biomaterials Department

Stephanie Möller

Biomaterials Department

Matthias Schnabelrauch

Biomaterials Department

Sofia Svedhem

Chalmers, Teknisk fysik, Biologisk fysik

Acta Biomaterialia

1742-7061 (ISSN)

Vol. 9 9 8158-8166

Ämneskategorier

Materialteknik

Den kondenserade materiens fysik

DOI

10.1016/j.actbio.2013.05.031

Mer information

Senast uppdaterat

2018-02-28