title: Physiological substrates for human lysosomal beta -hexosaminidase S.
abstract: Human lysosomal beta-hexosaminidases remove terminal beta-glycosidically bound N-acetylhexosamine residues from a number of glycoconjugates. Three different isozymes composed of two noncovalently linked subunits alpha and beta exist: Hex A (alphabeta), Hex B (betabeta), and Hex S (alphaalpha). While the role of Hex A and B for the degradation of several anionic and neutral glycoconjugates has been well established, the physiological significance of labile Hex S has remained unclear. However, the striking accumulation of anionic oligosaccharides in double knockout mice totally deficient in hexosaminidase activity but not in mice expressing Hex S (Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C.J., Skop, E., Starr, C. M., Hoffmann, A., Sandhoff, K., Suzuki, K., and Proia, R. L., (1996) Nat. Genet. 14, 348-352) prompted us to reinvestigate the substrate specificity of Hex S. To identify physiological substrates of Hex S, anionic and neutral oligosaccharides excreted in the urine of the double knockout mice were isolated and analyzed. Using ESI-MS/MS and glycosidase digestion the anionic glycans were identified as products of incomplete dermatan sulfate degradation whereas the neutral storage oligosaccharides were found to be fragments of N-glycan degradation. In vitro, recombinant Hex S was highly active on water-soluble and amphiphilic glycoconjugates including artificial substrates, sulfated GAG fragments, and the sulfated glycosphingolipid SM2. Hydrolysis of membrane-bound SM2 by the recombinant Hex S was synergistically stimulated by the GM2 activator protein and the lysosomal anionic phospholipid bis(monoacylglycero)phosphate.
authors.affiliation: Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany.
authors.forename: Stefan T
authors.initials: ST
authors.lastname: Hepbildikler
authors.forename: Roger
authors.initials: R
authors.lastname: Sandhoff
authors.forename: Melanie
authors.initials: M
authors.lastname: Kolzer
authors.forename: Richard L
authors.initials: RL
authors.lastname: Proia
authors.forename: Konrad
authors.initials: K
authors.lastname: Sandhoff
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002460:Cell Line; D002809:Chondroitin Sulfates / Q000737:chemistry; D002855:Chromatography, Thin Layer; D003871:Dermatan Sulfate / Q000378:metabolism; D049289:G(M2) Activator Protein; D006017:Glycolipids; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007313:Insecta; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008246:Lysophospholipids / Q000737:chemistry; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D018345:Mice, Knockout; D008956:Models, Chemical; D008969:Molecular Sequence Data; D050178:Monoglycerides; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors; D014867:Water / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry*
chemical_list: D049289:G(M2) Activator Protein; D006017:Glycolipids; D008246:Lysophospholipids; D050178:Monoglycerides; D011134:Polysaccharides; D011506:Proteins; D011994:Recombinant Proteins; C012786:bis(monoacylglyceryl)phosphate; D014867:Water; D003871:Dermatan Sulfate; D002809:Chondroitin Sulfates; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases