dc.description.abstract
The extracellular matrix (ECM) is a complex network of proteins and carbohydrates. Among others, it consists of structural proteins such as collagen, as well as proteoglycans (PGs), which have attached covalently bound and negatively charged carbohydrates called glycosaminoglycans (GAGs). GAGs play an important role in the development and maintenance of cellular functions and communication of cells. PGs, which carry the GAG keratan sulfate (KS), have important functions in the homeostasis, neuronal development, and regenerative processes. Changes in the structure and biochemical composition of KS, such as sulfation, are often associated with various pathologies. Therefore, the early detection of changes in GAG composition, expression, and distribution in the ECM is of great diagnostic interest. However, a reliable detection depends on the availability of specifically binding molecules such as carbohydrate-specific antibodies. In this work, the KS-binding monoclonal antibody (mAb) MZ15 was further characterized and established for imaging. Using commercially available hybridoma cells and protein A/G affinity chromatography, the mAb MZ15 could be produced in mg scale. Then, by utilizing synthetic and chemically defined oligomeric GAGs, the specificity of the mAb MZ15 for highly sulfated KS oligosaccharides was confirmed by glycan microarray assays. After the binding of the mAb MZ15 to KS was validated in a defined environment, binding was also demonstrated in a more complex environment of extracted PGs from rat cornea and salmon cupula by Western blotting. Enzymatic digestion using PNGase F and Niallia circulans keratanase II led to a strong reduction or complete loss of signal, confirming efficient enzymatic digestion of KS structures. Furthermore, the mAb MZ15 was used for the visualization of KS by immunofluorescence microscopy on tissue sections of rat, salmon, and human eyes. Thereby, KS was detected in the corneal stroma across all tested species. Additionally, it was shown that random fluorescent labeling of the mAb MZ15 has no influence on the visualization of KS. Binding of the mAb MZ15 towards KS was confirmed by enzymatic digestion of the tissue sections using keratanase II, resulting in abolished fluorescence in the stroma. Furthermore, visualization of KS was established using a second imaging technique, the laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Therefore, first a site-directed introduction of a metal label into the mAb MZ15 was performed. The labeled antibody was then used on rat and human eyes. Element-specific measurement using LA-ICP-MS allowed for a detection of KS with high spatial resolution in the cornea of the eyes. After the suitability of the mAb MZ15 for imaging of KS was validated, a single-chain variable fragment (ScFv) based on the full-length antibody was developed. Accordingly, genes of the variable regions of the mAb MZ15 heavy and light chain were specifically amplified. After sequencing and bioinformatic analysis, the KS-binding paratope was identified. Then, the ScFv expression vector was generated and the ScFv was expressed and purified from E. coli. For KS imaging in rat eyes, the ScFv was equally suitable like the full-length antibody MZ15. Next, an apparent binding affinity (KDapp) of the mAb MZ15 to KS of 80 nM was determined by surface plasmon resonance spectroscopy. However, a binding affinity for the ScFv could not be determined. Moreover, various GAG-degrading enzymes were produced to be utilized in the Collaborative Research Center 1340 (CRC1340). To cover a wide range of processible GAGs, the enzymes heparinase I, heparinase II and heparinase III from Pedobacter heparinus, keratan sulfate endo-1,4-β-galactosidase from Sphingobacterium multivorum, keratanase II from Niallia circulans, and chondroitinase ABC from Proteus vulgaris were heterologously expressed in E. coli. Thereby, keratanase II and chondroitinase ABC were produced as 6xHis tagged proteins. Additionally, the enzymes heparinase I, heparinase II, heparinase III, and keratanase II were produced and purified as triple-tagged fusion proteins, where the tags consisted of maltose-binding protein, 6xHis tag, and Strep tag II, respectively. By photometric measurement, the following activities of the triple-tagged heparinases were determined: heparinase I: 0.6 U/mg, heparinase II (substrate: heparin): 38.5 U/mg and heparinase III: 31.7 U/mg. Furthermore, the analysis of heparinase digestion products by HPLC using the respective substrates heparin and heparan sulfate revealed expected substrate specificities for the produced heparinases.
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