Compensatory expression of human N-acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma.

The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markersessential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (alpha2, beta2, gamma2). The alpha- and beta-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the gamma-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GNPTG cause mucolipidosis type III gamma, which is characterized by missorting and cellular loss of lysosomal enzymes leading to lysosomal accumulation of storage material. Using plasmon resonance spectrometry, we showed that recombinant gamma-subunit failed to bind the lysosomal enzyme arylsulfatase A. Additionally, the overexpression of the gamma-subunit in COS7 cells did not result in hypersecretion of newly synthesized lysosomal enzymes expected for competition for binding sites of the endogenous phosphotransferase complex. Analysis of fibroblasts exhibiting a novel mutation in GNPTG (c.619insT, p.K207IfsX7) revealed that the expression of GNPTAB was increased whereas in gamma-subunit overexpressing cells the GNPTAB mRNA was reduced. The data suggest that the gamma-subunit is important for the balance of phosphotransferase subunits rather for general binding of lysosomal enzymes.

Missense mutation in the N-acetylglucosamine-1-phosphotransferase gene (GNPTA) in a patient with mucolipidosis II induces changes in the size and cellular distribution of GNPTG.

Mucolipidosis type II (ML II; I-cell disease) and mucolipidosis III (ML III; pseudo Hurler polydystrophy) are autosomal recessively inherited disorders caused by a defective N-acetylglucosamine 1-phosphotransferase (phosphotransferase). The formation of mannose 6-phosphate markers in soluble lysosomal enzymes is impeded leading to their increased excretion into the serum, to cellular deficiency of multiple hydrolases, and lysosomal storage of non-digested material. Phosphotransferase deficiency is caused by mutations in GNPTA and GNPTG encoding phosphotransferase subunits. Here we report on an adolescent with progressive joint contractions and other signs of mucolipidosis II who survived to the age of 14 years. Impaired trafficking of lysosomal enzymes cathepsin D and -hexosaminidase in metabolically labeled fibroblasts was documented. Mutations in the GNPTG gene and alterations in the GNPTG mRNA level were not detected. A different electrophoretic mobility of the 97 kDa GNPTG dimer suggested posttranslational modification abrogating the compartmentalization of GNPTG in the Golgi apparatus. A nucleotide substitution in the GNPTA gene (c.3707A>T) was identified altering the predicted C-terminal transmembrane anchor of the phosphotransferase subunit. The data demonstrate that defective GNPTA not only impairs lysosomal enzyme targeting but also the availability of intact GNPTG required for phosphotransferase activity and assembly of subunits.

Missense mutations in N-acetylglucosamine-1-phosphotransferase alpha/beta subunit gene in a patient with mucolipidosis III and a mild clinical phenotype.

Mucolipidosis type III (ML III, pseudo-Hurler polydystrophy), an autosomal recessive inherited disorder of lysosomal enzyme targeting is due to a defective N-acetylglucosamine 1-phosphotransferase (phosphotransferase) activity and leads to the impaired formation of mannose 6-phosphate markers in soluble lysosomal enzymes followed by their increased excretion into the serum. Mutations in the phosphotransferase gamma subunit gene (GNPTAG) have been reported to be responsible for ML III. Here we report on a 14-year-old adolescent with a mild clinical phenotype of ML III. He presented with progressive joint stiffness and swelling. Urinary oligosaccharide and mucopolysaccharide excretion was normal. Lysosomal enzyme activities were significantly elevated in the serum and decreased in cultured fibroblasts. Impaired trafficking of the lysosomal protease cathepsin D (CtsD) was confirmed by metabolic labeling of the patient's fibroblasts. Neither mutations in the GNPTAG gene nor alterations in the GNPTAG mRNA level were detected whereas the steady state concentration of the 97 kDa GNPTAG dimer was reduced. Most importantly, the patient is homozygous for a pathogenic nucleotide substitution and a polymorphism in the phosphotransferase alpha/beta subunit gene (GNPTA). The data indicate that defects in genes other than GNPTAG can be linked to ML III contributing to the variability of the phenotype.

A novel mutation in UDP-N-acetylglucosamine-1-phosphotransferase gamma subunit (GNPTAG) in two siblings with mucolipidosis type III alters a used glycosylation site.

The N-acetylglucosaminyl-1-phosphotransferase (termed phosphotransferase) catalyzes the initial step in the formation of mannose 6-phosphate (M6P) residues required for the efficient transport of soluble lysosomal enzymes. The phosphotransferase is a multisubunit enzyme composed of three subunits (alpha2beta2gamma2) that are products of two genes. The gene encoding the gamma-subunit (GNPTAG) appears to be defective in patients with mucolipidosis type III (ML III). We have analyzed the GNPTAG gene in two siblings with ML III showing elevated activities of several lysosomal enzymes in cultured fibroblasts serum and diminished activities in cultured fibroblasts. Immunoprecipitation of metabolically labeled cathepsin D (CtsD) from fibroblasts revealed that the sorting/transport of this lysosomal protease was affected. Addition of ammonium chloride inhibiting pH-dependent processes, such as the CtsD-M6P receptor interaction, indicated that 15 to 20% of the newly synthesized CtsD is transported in ML III fibroblasts in an M6P-dependent manner. By direct sequencing a novel homozygous mutation, c.347_349delACA (p.Asn116del), was identified affecting a potential N-linked glycosylation site. Western blot analysis of extracts from control fibroblasts detect a 97 kDa glycosylated dimer whereas ML III cells contain a GNPTAG dimer of reduced molecular mass. These data suggest that the loss of the used glycosylation site in the gamma subunit may affect the intracellular localization of GNPTAG and the overall efficiency of M6P formation.

Homodimeric galectin-7 (p53-induced gene 1) is a negative growth regulator for human neuroblastoma cells.

The extracellular functions of galectin-7 (p53-induced gene 1) are largely unknown. On the surface of neuroblastoma cells (SK-N-MC), the increased GM1 density, a result of upregulated ganglioside sialidase activity, is a key factor for the switch from proliferation to differentiation. We show by solid-phase and cell assays that the sugar chain of this ganglioside is a ligand for galectin-7. In serum-supplemented proliferation assays, galectin-7 reduced neuroblastoma cell growth without the appearance of features characteristic for classical apoptosis. The presence of galectin-3 blocked this effect, which mechanistically resembles that of galectin-1. By virtue of carbohydrate binding, galectin-7 thus exerts neuroblastoma growth control similar to galectin-1 despite their structural differences. In addition to p53-linked proapoptotic activity intracellularly, galectin-7, acting as a lectin on the cell surface, appears to be capable of reducing cancer cell proliferation in susceptible systems.

Identification of a CTL4/Neu1 fusion transcript in a sialidosis patient.

The deficiency of the lysosomal neuraminidase (NEU1; sialidase) causes the storage disorder sialidosis with symptoms ranging from eye abnormalities and neurological disturbances to skeletal malformations, mental retardation and early death. Sialidosis patients encompassing a wide spectrum of clinical symptoms were screened for mutations in neu1. We identified the same homozygous interstitial deletion (11 kb) in two patients causing the fusion of exon 10 of CTL4 (New Gene 22; NG22) with the 3'-UTR of neu1. In one patient we found the resulting CTL4/Neu1 fusion transcript, in the other we detected an alternatively spliced CTL4 transcript (retention of intron 9).

Substrate specificity and inhibitor studies of a membrane-bound ganglioside sialidase isolated from human brain tissue.

A ganglioside-specific sialidase that controls cellular functions such as growth, differentiation, and adhesion has been observed in a variety of cells, but its characterization proved difficult due to firm membrane attachment and lability of the purified enzyme. Here we report on the specificity toward gangliosides and susceptibility to certain inhibitors of a ganglioside sialidase solubilized and purified 5100-fold from human brain. The sialidase removed terminal sialic acids from gangliosides GM3, GM4, GD3, GD2, GD1 a, GD1 b, GT1 b and GQ1 b, but was inactive toward gangliosides with sialic acid in a branching position (as in GM1 and GM2). Lyso-GM3 and -GD1a were good substrates, too, whereas O-acetylation of the sialic acid as in 9-O-acetyl-GD3 caused strongly reduced cleavage. The new influenza virus drug 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Zanamivir) exhibited an IC50 value of about 7 x 10(-5) M that was in the range of the 'classical' sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; the bacterial sialidase inhibitor 4-nitrophenyloxamic acid, however, was ineffective. The glycosaminoglycans heparan sulfate, heparin, chondroitin sulfates A and B, as well as dextran sulfate and suramin, were all strongly inhibitory, suggesting that glycosaminoglycans present on the cell surface or in the extracellular matrix may influence the ability of the sialidase to alter the ganglioside composition of the membrane.