Y Okamoto1, K Omichi, S Yamanaka, K Ikenaka, S Hase. 1. Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Okazaki National Research Institutes, Aichi, 444-8585, Japan.
Abstract
The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.
The N-linked n class="Chemical">sugar chains, n>n class="Chemical">GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.