Characterization of recombinantly expressed matrilin VWA domains.

VWA domains are the predominant independent folding units within matrilins and mediate protein-protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems.

Shark cartilage 14 kDa protein as a dendritic cells activator.

Low molecular weight components of shark cartilage are reported to have anti-tumor as well as immuno-stimulating effects. Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that have a key role in establishment of anti-cancer immune response. In this study, the effect of 14 kDa protein from shark cartilage was investigated on stimulation and maturation of dendritic cells. The isolated 14 kDa protein from shark cartilage extract was added to DCs medium during overnight culture and their maturation and T cells stimulation potential was investigated. The majority of shark-cartilage-treated DCs expressed higher levels of maturation markers and were more effective in stimulation of allogenic T cells compared with non-treated DCs (p < 0.05). Our results showed that shark cartilage 14 kDa protein can potentially be used in DC-mediated T-cells stimulation and induction of desirable immune responses in clinical trials such as cancer immunotherapy. However, further studies are required to examine this proposal.

Matrilins.

Marilins mediate interactions between macromolecular components of the extracellular matrix, e.g., collagens and proteoglycans. They are composed of von Willebrand factor type A and epidermal growth factor-like domains and the subunits oligomerize via coiled-coil domains. Matrilin-1 and -3 are abundant in hyaline cartilage, whereas matrilin-2 and -4 are widespread but less abundant. Mutations in matrilin genes have been linked to chondrodysplasias and osteoarthritis and recently characterization of matrilin-deficient mice revealed novel functions in mechanotransduction, regeneration, or inflammation. Due to their intrinsic adhesiveness and partially also low abundance, the study of matrilins is cumbersome. In this chapter, we describe methods for purification of matrilins from tissue, analysis of matrilins in tissue extracts, recombinant expression, and generation of matrilin-specific antibodies.

Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution.

The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease.