Analyse von Glykosaminoglykanen in humanen Zellen und Körperflüssigkeiten

Glycosaminoglycans (GAGs) are found mainly in connective tissue as constituents of proteoglycans, covalently linked to the core protein. They participate in and regulate several cellular events and physiological processes. The sequence of [Delta]-disaccharides in GAGs is crucial for their proper function. The human xylosyltransferases XT-I and XT-II catalyse the initial and rate-limiting step in the biosynthesis of GAGs by the transfer of xylose to selected serine residues in the core protein of proteoglycans (PGs). For the analysis of GAGs, a HPLC method facilitating the separation of 16 [Delta]-disaccharide standards derivatized with the fluorophore 2-aminoacridone was developed. This novel method allows the quantitative analysis of the [Delta]-disaccharide composition of hyaluronic acid (HA), chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and heparin (H). The method represents the first HPLC application ever to accomplish baseline separation of either seven [Delta]-disaccharides from HS/H or nine [Delta]-disaccharides from CS/DS/HA with one column and buffer system after fluorophore labeling in two runs. This is also the first HPLC method achieving the separation of all nine fluorophore-labeled GAG [Delta]-disaccharides from CS/DS/HA in one run. The run times are shorter compared to any existing HPLC separation of labeled GAG [Delta]-disaccharides from either CS/DS/HA or HS/H. Since only conventional HPLC apparatus is needed our method can be used for routine GAG disaccharide analysis in every laboratory. In order to evaluate the newly developed method, GAGs from different blood cells were characterized. Optimized methods with good recovery and high purity were developed for the purification of platelets and granulocytes using gradient centrifugation and were successfully applied. The average purities were 99.4 per cent for platelet samples and 98.3 per cent for granulocyte samples. For the first time, normal ranges of the GAG [Delta]-disaccharide compositions from platelets and granulocytes were determined. The composition of GAG [Delta]-disaccharides from granulocytes was characterized for the first time and [Delta]di-mono4S_CS und [Delta]di-0S_HA were identified as the main components. Furthermore, the [Delta]-disaccharide compositions of GAGs from 22 different human cell lines were analysed for differences in the total GAG amount between the investigated adherent and suspension cell lines. For the 5 suspension cell lines investigated (blastoma cell lines and hematological cancer cell lines), a significantly decreased total GAG amount was found compared to the 17 adherent cell lines (carcinoma cell lines, sarcoma cell lines and fibroblast cell lines) (p = 0.03). The amounts of HA, CS/DS and HS/H were decreased in the suspension cell lines with only the amount of HS/H being significantly decreased (p = 0.037) in suspension cell lines in comparison with adherent cell lines. The percentage of [Delta]di-mono0S_HS was found to be significantly decreased (p = 0.006) in suspension cell lines compared to adherent cell lines. The percentage of [Delta]di-di(6,N)S_HS was significantly increased (p = 0.0002) in suspension cell lines in comparison with adherent cell lines. [Delta]di-mono6S_HS and [Delta]di-di(2,N)S_HS were exclusively detected in the adherent cell lines. In order to examine changes in the serum GAGs concentration and composition caused by polymorphisms in the xylosyltransferase genes XYLT1 and XYLT2, 223 blood donors were screened for 3 missense polymorphisms in XYLT1 and XYLT2. 48 samples comprising 23 case samples in which one of the missense mutations had been found and 25 wildtype/control samples were analyzed for their serum GAGs and XT activity. Here, it could be shown that the SNP c.343G>T is associated with a significantly (p<0.01) reduced GAG amount compared to the wildtype group.