Analysis of lysosomal Neu4 sialidase associated proteins by using mass spectrometry (MS/MS)

Abstract

Sialidases are glycohydrolytic enzymes which remove sialic acid residues from glycoproteins, oligosaccharides and glycolipids. There are 4 different sialidases known in mammalians. These are Neu1 (lysosomal), Neu2 (cytoplasmic), Neu3 (cell membrane) and Neu4 (lysosomal/mitochondrial) sialidase. Sialidases are involved in many metabolic and cellular processes interactioning with another proteins or work together in multiprotein complexes. For example, Neu1 is only active with betagalactosidase and cathepsin A enzyme in lysosome. Interactions of sialidases Neu2, Neu3 and Neu4 enzyme with other proteins remain unknown In our study, we aimed to identify proteins which have interaction with sialidase Neu4 as well as Neu1 by using mass spectrometry analysis to find new possible roles of sialidases. Our bait protein's cDNA was tagged with calmodulin binding protein as well as streptavidin binding protein. After transfection and expression of vectors to mammalian cells, proteins were purified using tandem affinity purification (TAP). We identified some associated proteins with sialidase Neu1 and Neu4 by using MS/MS analysis and bioinformatics.