Development of novel enzyme activity assays for human sulfatases and related glycosidases to indicate lysosomal storage diseases in Newborn

Abstract

Proteoglycans and sulfated glycosaminoglycans build very important structures in eukaryotic organisms. They can be found inside the cell and on its surface, but they especially represent a major component of the extracellular matrix of animal and human cells. Their physiological function reaches from simple shock absorbers and lubricants in joints and cartilage to various important signaling processes during cell division or inflammation. During cell metabolism, the appearance of these complex frameworks changes constantly. Worn-out parts are transported to lysosomes within the cell were they are degraded and the harvested carbohydrates are recycled to form new glycosaminoglycan chains. To degrade such highly sulfated biopolymers, special enzymes are needed that cleave these functionalities before other hydrolases can process remaining glycosaminoglycan chains further. This highly ordered sequences of consecutively acting lysosomal enzymes can lead to metabolic imbalance, if certain enzymes show no or diminished activity. These rare, inherited, genetic conditions are called lysosomal storage diseases. Often no signs of the disease are present at birth, but their progressive nature produces first symptoms of irreversible damage usually in early childhood. The need to reliably identify these conditions at a most early stage is emphasized by the development of new promising treatment options for this group of metabolic disorders and to ensure most effective therapy. The use of dried blood spot cards allows the implementation of enzyme activity assays in routine new born screenings. Within this work the synthesis and screening evaluation of novel enzyme assay substrates for the detection of five individual sulfatase- and two related glycosidase-deficiencies is described. After incubation, samples are analyzed be UHPLC tandem MS analysis. To enable robust quantification via this method, corresponding degradation products and isotopically labeled internal standards were prepared utilizing the advantages of copper catalyzed alkyne azide click chemistry for this combinatorial synthetic task. Mass spectrometry offers a very sensitive and specific analytical method to measure enzymatic activity. Additionally enzyme assay sets that bare an unnatural glycosidic bond were prepared to investigate the influence of these modifications on assay performance. C-glycosidic compounds for the detection of MPS IV A and VI as well as N-linked compounds to screen for MPS II and IV A were prepared and analyzed. Chromatographic separation of analyte and remaining enzyme substrate is a necessary but time consuming hurdle. After meticulous method optimization one last possibility remains to enhance screening throughput further. The development of well-coordinated assay sets allows measuring several individual samples in one single injection thus reducing acquisition time substantially. The applicability of this multiplexing technique was demonstrated by probing for either the same disease in three samples or by analyzing one blood sample to screen for three (MPS II, MPS IV A and MPS VI) different enzyme deficiencies simultaneously.