Characterization of α-l-Iduronidase (Aldurazyme®) and its complexes.

Alpha-l-Iduronidase(IDUA) was the first enzyme replacement therapy approved for mucopolysaccharidosis type I and the corresponding recombinant protein drug, Aldurazyme®, is commercially available. In the frame of gel-based mass spectrometrical characterization of protein drugs, we intended to identify protein sequence and possible protein modifications. Moreover, we were interested in which aggregation/complex form Aldurazyme® would exist, which complexes were enzymatically active and in which form the naturally occurring enzyme would be present in the brain. Aldurazyme® was run on 2DE gel electrophoresis, spots were excised, in-gel digested with several proteases and identified by nano-LC-ESI-MS/MS on an ion trap. IDUA-activity was determined by a fluorometric principle. Blue-native gel electrophoresis with subsequent immunoblotting was carried out to show the presence of protein complexes. The protein was unambiguously identified by 100% sequence coverage; several amino acid substitutions were detected and protein modifications were novel phosphorylations on S59 and S482, histidine methylation at H572 and provide evidence for already known N-glycosylations. Four Aldurazyme® complexes that all were enzymatically active, were observed while a single complex was observed for the physiologically occurring IDUA in the brain. The findings are relevant for understanding chemistry, physiology, pharmacology and medicine of IDUA, design of further and interpretation of previous work.