Larry W. Hancock

Generalized N-Acetylneuraminic Acid Storage Disease: Quantitation and Identification of the Monosaccharide Accumulating in Brain and Other Tissues

Abstract: Brain and other tissues from a patient with extensive neonatal as‐cites and clinical symptoms suggestive of a severe neurovisceral storage disorder were examined following autopsy for the accumulation of oligosaccharides. This carbohydrate analysis revealed the presence of large amounts (3–21 μmol/g fresh weight) of sialic acid in brain, liver, and kidney tissue as the major abnormality. Exhaustive characterization of the accumulating material by gel filtration, gas‐liquid chromatography, thin‐layer chromatography, and GLC‐mass spectrometry positively identified the saccharide as free N‐acetylneuraminic acid. Based on the accumulation of only free N‐acetylneuraminic acid in the tissue of this patient, and normal activities of lysosomal enzymes involved in the catabolism of cellular glycoproteins, this storage disorder appears to result from a previously unreported defect in glycoconjugate metabolism.

N-Acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 mumol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [3H]glucosamine-HCl or N-[3H]acetylmannosamine showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.

Lysosomal Degradation of Glycoproteins and Glycosaminoglycans

The catabolism of brain glycoconjugates is a complex process involving the interaction of endoglycosidases, exoglycosidases, and proteinases, as summarized in Table 1. Lysosomal catabolism of these glycoconjugates further requires the delivery of both the catabolic enzymes and their substrates to this organelle, and the maintenance of a functional milieu having the proper acidic pH and complement of cofactors (e.g., cations) to facilitate efficient catabolism.

N-acetylneuraminic acid accumulation in a buoyant lysosomal fraction of cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease.

Cultured fibroblasts from control individuals and two patients affected with the infantile variant of generalized N-acetylneuraminic acid (NeuAc) storage disease were disrupted by nitrogen cavitation, and the post-nuclear supernatant fractions were subjected to subcellular fractionation on Percoll gradients. Accumulating NeuAc in affected fibroblasts (approx. 150 nmol/mg protein) co-localized with the lysosomal marker N-acetyl-beta-hexosaminidase (Hex), in a fraction with a mean density of 1.035 g/ml. In contrast, more than 70% of the Hex activity of control cells sedimented in comparable gradients with a density of more than 1.07 g/ml. The lysosomal localization of NeuAc accumulation in affected fibroblasts was confirmed by treatment of post-nuclear supernatant fractions with 0.5 mM Gly-Phe-beta-naphthylamide (20 min, 37 degrees C) prior to centrifugation, which resulted in the simultaneous loss of latency of Hex and free NeuAc, and their association with the soluble fraction on Percoll gradients. The results provide direct evidence for the accumulation of free NeuAc in a unique buoyant lysosomal fraction of affected fibroblasts.

N-Acetyl-β-hexosaminidase B deficiency in cultured fibroblasts from a patient with progressive motor neuron disease

A patient with a 20-year history of progressive motor neuron disease was previously found to have profoundly low levels of N-acetyl-beta-hexosaminidase (Hex) in serum and leukocytes; Hex activity in cultured skin fibroblasts was in the low normal range. By thermal inactivation and cellulose acetate electrophoresis, the residual activity appeared to be Hex A. In the present study, the residual activity in cultured skin fibroblasts was further characterized as Hex A by thermal inactivation at reduced temperatures and ion exchange chromatography; no evidence was obtained for a diffusible inhibitor of Hex activity. After labeling with [3H]leucine, immunoprecipitation with polyclonal antibody to Hex B, and SDS-polyacrylamide gel electrophoresis, both alpha and beta polypeptide chains were visualized, confirming the presence of Hex A. The results suggest that, in the patients fibroblasts, a defect in beta-chain synthesis or processing precludes the self-association of beta-chains to form Hex B, but does not prevent the association of alpha- and beta-chains to form Hex A.

Evidence for two catabolic endoglycosidase activities in β-mannosidase-deficient goat fibroblasts

Cultured skin fibroblasts derived from Nubian goats deficient in lysosomal beta-mannosidase, which had previously been shown to accumulate storage oligosaccharides with the structures Man beta 4GlcNAc beta 4GlcNAc and Man beta 4GlcNAc (in the ratio of 2.7:1) were evaluated for their ability to catabolize exogenous [3H]GlcN-labelled glycoproteins isolated from the secretions of cultured goat or human fibroblasts. Regardless of the source of exogenous labelled glycoprotein, affected goat fibroblasts took up the labelled glycoprotein from the culture medium and subsequently accumulated the same major labelled oligosaccharide, identified as Man beta 4GlcNAc beta 4GlcNAc; no such oligosaccharide accumulated in normal goat fibroblasts under the same conditions. Tunicamycin-treated affected fibroblasts also took up labelled exogenous glycoprotein and accumulated labelled storage trisaccharide, further suggesting the direct accumulation of storage trisaccharide from impaired glycoprotein-associated oligosaccharide catabolism. Treatment of metabolically labelled affected fibroblasts with leupeptin, an inhibitor of lysosomal cathepsins, resulted in the 2- to 6-fold inhibition of trisaccharide accumulation, while having little effect on the uptake of [3H]GlcN or the accumulation of labelled disaccharide. The results are most consistent with the presence of two endoglycosidases, an endo-beta-N-acetylglucosaminidase and an endo-aspartylglucosaminidase, in goat fibroblasts. These two activities, rather than heterogeneous core oligosaccharide structures, are responsible for the ultimate accumulation of storage oligosaccharides with one and two GlcNAc residues at their reducing terminus.

Regulation of GM2 ganglioside metabolism in cultured cells

GM2-ganglioside (II3NeuAcGgOse3Cer) is a minor component of adult nervous tissue, but is probably an oncofetal antigen. Its biological role is unknown, but several lines of evidence indicate its potential role in cell adhesion both in the retina and in oligodendrocytes. The biosynthesis of GM2-ganglioside appears to be tightly regulated, since it is a key intermediate in complex ganglioside synthesis. The specific GM3: hexosaminyl-transferase is activated under conditions which activate cyclic AMP-dependent protein kinase, and cell transformation with retroviruses inactivates it. Catabolism of GM2 requires the concerted action of three gene products (alpha-chain, beta-chain and activator protein in a thermolabile alpha beta 2 AP complex referred to as HexA). Defects in either three components results in the neuronal storage of GM2 ganglioside and the manifestations of Tay-Sachs Disease in children or motor neuron disease in adults.

Synthesis and processing of lysosomal α-fucosidase in cultured human fibroblasts

Abstract The lysosomal enzyme α- l -fucosidase from human skin fibroblasts is synthesized as a 53 kDa glycosylated precursor which is then proteolytically processed to a 50 kDa mature form. This was confirmed by pulse-chase labeling studies with chase times up to 72 h. In fibroblasts treated with 1-deoxymannojirimycin to prevent trimming of high mannose oligosaccharides, endoglycosidase H (endo H) treatment completely deglycosylated and reduced the size of immunoprecipitated α-fucosidase by 4–5 kDa, suggesting the presence of two oligosaccharide units. Endoglycosidase H and endo F studies on untreated α-fucosidase suggested the presence of one complex-type and one high mannose-type unit, and that the final processing from 53 to 50 kDa did not involve the removal of carbohydrate. Processing was inhibited by the thiol proteinase inhibitor Ep-459, but not by Ep-475 or leupeptin. Since Ep-459 treatment increased both α-fucosidase activity (3-fold) and the amount of immunoprecipitable α-fucosidase protein in normal human skin fibroblasts, this suggests a role for cysteine-like proteinases either directly or indirectly in lysosomal hydrolase processing and turnover. Subcellular fractionation studies revealed that the proteolytic processing of the 53 kDa precursor to the 50 kDa mature form occurred in the lysosome, or some other dense organelle.

Inborn Errors of Complex Carbohydrate Catabolism

Complex carbohydrates of the nervous system are degraded in lysosomes by the sequential action of a group of exoglycosidases known collectively as the lysosomal hydrolases. Inherited defects in the synthesis, assembly, or turnover of these hydrolases lead to storage diseases in humans (Spranger, 1987) and a variety of domestic animals. Those involving the nervous system result in spectacular neuropathology and provide the best evidence for the types of glycoconjugates synthesized by nervous tissue, as well as their rate of turnover. For example, in Tay—Sachs disease, storage material (GM2 ganglioside) predominates in nervous tissue, especially motor neurons, and is virtually absent from visceral tissue. The variable level of accumulation of GM2 in different brain regions (identified morphologically as multilamellar cytosomes) can be related to different levels of synthesis and degradation. This clearly manifests itself in patients with partial hexosaminidase (HexA) deficiencies, who exhibit symptoms of motor neuron disease, or spinocerebellar degeneration with other neuronal function (such as vision and intelligence) relatively intact. The absence of GM2 storage outside the CNS reflects the lack of GM2 synthesis in nonneural tissue. However, since lysosomal hydrolases are synthesized constitutively in all tissues, GM2 can be fed to fibroblasts from HexA-deficient patients, and its steady accumulation observed. Thus, storage patterns in patients with inherited enzyme defects can be used to give an accurate reflection of glycoconjugate content of the CNS versus nonneural tissue and this will be emphasized on an enzyme/disease, case-by-case basis.

Molecular heterogeneity in lysosomal storage diseases

The availability of specific antibodies and cDNA probes for lysosomal hydrolases has revealed unexpected heterogeneity among the human inherited lysosomal storage diseases. Using alpha-fucosidase and N-acetyl-beta-D-hexosaminidase deficiency variants as examples, it has been determined that a lysosomal hydrolase deficiency can result from DNA deletion mutations, failure to synthesize mRNA because of defective splicing, posttranslational defects in assembly, and synthesis of a precursor enzyme that is prematurely proteolytically degraded through lack of a protective protein. In some cases (fucosidosis), the different genotypes cannot be distinguished phenotypically, whereas in others (beta-hexosaminidoses) the phenotypes can range from infantile neurodegeneration through juvenile motor neuron disease to adult neurodysfunction. Biochemical studies on both diseases have revealed several distinct genotypes. We show that some forms of fucosidosis result from unstable enzyme that can be stabilized by protease inhibitors, whereas partial beta-hexosaminidase deficiencies cannot be corrected by these protease inhibitors.