Figure 1.

Cat-315 epitopes are reduced in the brain of GnT-IX-deficient (KO) mice. A, Structures of the wild-type (WT) GnT-IX allele, targeting construct, and targeted GnT-IX allele. The dark boxes represent exons. The open box shows the pgk-neo cassette. B, BamHI restriction site. The expected sizes of the BamHI digestion products of the gene are shown for the WT allele (10 kb) and mutated allele (5.4 kb). B, The top shows a Southern blot analysis of BamHI-digested genomic DNA derived from WT, heterozygous, and homozygous mutant mice hybridized with the probe. The bottom shows a PCR analysis of genomic DNA extracted from the tails of these mice. The wild-type allele generates a PCR product of 380 bp, while the mutant allele generates a product of 550 bp. C, The GnT-IX enzyme activities in the brain microsomal fractions prepared from WT and GnT-IX-deficient mice (KO) are presented as means ± SD (n = 3). N.D., not detected. D, The upper structure shows an N-linked glycan in which GnT-IX catalyzes the transfer of GlcNAc to both mannose arms via β1,6-linkage in vitro (Inamori et al., 2003). The enclosed residues show the PHA-L4 epitope. The lower structure shows an O-mannosyl glycan in which GnT-IX catalyzes the branched structure (Inamori et al., 2004). The enclosed residues show possible Cat-315 epitopes (Dino et al., 2006). E, Brain lysates from WT, GnT-V-deficient, GnT-IX-deficient, or double KO mice were incubated with or without PNGase, and then analyzed by PHA-L4 lectin blotting or Western blotting with an anti-GAPDH antibody. F, Brain lysates from WT and GnT-IX-deficient KO mice at 1 week of age (1 Wk) or 8–12 months of age (Adult) were analyzed by Western blotting with Cat-315 and anti-GAPDH antibodies (left). In the right graph, the bars represent the relative Cat-315 immunoreactivities shown as means ± SEM (n = 5). *p < 0.00005.