TAILIEUCHUNG - Báo cáo khóa học: Photoaffinity labelling of the Human GM2-activator protein Mechanistic insight into ganglioside GM2 degradation

The GM2-activator protein (GM2AP) is an essential cofactor for the degradation of ganglioside GM2 by lyso-somal b-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membrane-bound substrate at the lipid–water interphase. Inherited defects in the gene encoding this glycoprotein result ina fatal neurological storage disorder, the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this gly-coprotein cofactor, we synthesized the two photoaffinity labels [ 14 C]C3 -TPD-GM2 and [ 14 C]C7 -TPD-GM2. . | Eur. J. Biochem. 271 614-627 2004 FEBS 2004 doi Photoaffinity labelling of the Human GM2-activator protein Mechanistic insight into ganglioside GM2 degradation Michaela Wendeler1 Joera Hoernschemever1 . Daniel Hoffmann2 Thomas Kolter1 Guenter Schwarzmann1 and Konrad Sandhoff1 1 Kekule-Institut fur Organische Chemie und Biochemie der Universitat Bonn D-53121 Bonn 2Stiftung caesar Bonn Germany The GM2-activator protein GM2AP is an essential cofactor for the degradation of ganglioside GM2 by lysosomal b-hexosaminidase A. It mediates the interaction between the water-soluble exohydrolase and its membranebound substrate at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein result in a fatal neurological storage disorder the AB variant of GM2-gangliosidosis. To elucidate the mode of action of this glycoprotein cofactor we synthesized the two photoaffinity labels 14C C3-TPD-GM2 and 14C C7-TPD-GM2. Incubation of GM2AP with these substrate analogues and subsequent irradiation led to covalent labelling of the protein. After separation of tryptic peptides by reverse-phase HPLC the labelled peptide fractions were analysed by MALDI-TOF and sequenced by ESI-Q-TOF mass spectrometry. Both labels were found to be specifically photoincorporated into a part of the surface loop comprising residues V153-L163 a stretch of amino acids that was previously identified as the most flexible region in the crystal structure of the activator. Our results provide strong evidence that this loop constitutes the part of the activator protein that directly interacts with the ganglioside substrate suggesting that the hydrophobicity and the great structural mobility of this element are crucial for the extraction of the membrane-embedded glycolipid its stabilization inside the spacious cavity and its guidance to the enzyme s active site. This study demonstrates that the approach of photoaffinity labelling in conjunction with accurate .

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