The GM2 gangliosidoses, Tay-Sachs and Sandhoff diseases, are caused by mutations in the HEXA (alpha-subunit) and HEXB (beta-subunit) genes, respectively. Each gene encodes a subunit for the heterodimeric lysosomal enzyme, beta-hexosaminidase A (alpha beta), as well as for the homodimers beta-hexosaminidase B (beta beta) and S (alpha alpha). In this study, we have produced mice that have both Hexa and Hexb genes disrupted through interbreeding Tay-Sachs (Hexa-/-) and Sandhoff (Hexb-/-) disease model mice. Lacking both the alpha and beta-subunits these 'double knockout' mice displayed a total deficiency of all forms of lysosomal beta-hexosaminidase including the small amount of beta-hexosaminidase S present in the Sandhoff disease model mice. More surprisingly, these mice showed the phenotypic, pathologic and biochemical features of the mucopolysaccharidoses, lysosomal storage diseases caused by the accumulation of glycosaminoglycans. The mucopolysaccharidosis phenotype is not seen in the Tay-Sachs or Sandhoff disease model mice or in the corresponding human patients. This result demonstrates that glycosaminoglycans are crucial substrates for beta-hexosaminidase and that their lack of storage in Tay-Sachs and Sandhoff diseases is due to functional redundancy in the beta-hexosaminidase enzyme system.

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Abstract. The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey kidney COS-I cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-I cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-I cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.

Glycoprotein components of the nuclear pore are essential for nuclear transport and are modified by both glycosylation and phosphorylation. The function and control of these post-translational modifications are poorly understood. Glycosylation of the major rat nuclear pore glycoprotein, p62, was examined in vitro using recombinant p62 as a substrate. Rat p62 was expressed in Escherichia coli and purified to near homogeneity. Kinetic analysis using a partially purified mammalian transferase suggests that the recombinant protein is an excellent substrate (Km = 0.30 microM) for the transfer of GlcNAc from UDP-GlcNAc (Km = 1.8 microM). Localization of the sites of O-linked GlcNAc glycosylation of rat p62 was performed by a combination of deletion analysis of in vitro translation products and by immunoprecipitation of [14C]GlcNAc-labeled proteolytic fragments. The amino terminus of rat p62 is poorly glycosylated with no O-linked GlcNAc sites between Lys22 and Lys97; the carboxyl terminus has one known glycosylation site at Ser471. The majority of the glycosylation sites in rat p62 are likely to occur on the six clustered Ser residues in the central Ser/Thr-rich region from Ser270 to Thr294. A synthetic peptide derived from this region is a good substrate for O-GlcNAc addition (Km = 30 microM) and a potent competitive inhibitor of p62 glycosylation (Ki = 15 microM). It is proposed that this Ser/Thr-rich domain functions as a linker region between the amino-terminal beta-pleated sheet and the carboxyl terminal alpha-helical domains. O-Glycosylation and phosphorylation of this linker region could provide a dynamic means of altering the conformation of p62 during nuclear pore assembly and disassembly.