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Human lysosomal protective protein/cathepsin A (CathA) is a multifunctional protein that exhibits not only protective functions as to lysosomal glycosidases, i.e., neuraminidase 1 (NEU1) and beta-galactosidase (GLB), but also its own serine carboxypeptidase activity, and exhibits conserved structural similarity to yeast and wheat homologs (CPY and CPW). Our previous study revealed that the R344 (Arg344) residue in CathA could contribute to the binding and recognition of the serine peptidase inhibitor chymostatin. We examined here the effects of substitution of R344 with other amino acids, including A, D, E, G, I, K, M, N, P, Q, S, and V, denoted as R344X, including the wild-type CathA, on expression of CathA activity and intracellular processing. Among the mutant gene products, the 54-kDa precursor/zymogen with the R344D substitution was not processed to the 32/20-kDa mature form with CathA activity in a fibroblastic cell line derived from a galactosialidosis patient. Molecular dynamics (MD) simulations on the total twelve R344X mutants and the wild-type revealed that only R344D takes on a significantly different conformation of S293-D295 in the excision peptide (M285-R298) compared to the other R344X mutants; the side chains of S293 and D295 in R344D are exposed on the molecular surface, although those in the other twelve R344X mutants are buried inside the protein. The results of the current work strongly suggest that the distinct conformational change of the S293-D295 region in the R344D protein causes the processing defect of the 54-kDa precursor of the R344D mutant gene product in cultured cells.

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.

GM1 gangliosidosis and Morquio B disease are distinct disorders both clinically and biochemically yet they arise from the same beta-galactosidase enzyme deficiency. On the other hand, galactosialidosis and sialidosis share common clinical and biochemical features, yet they arise from two separate enzyme deficiencies, namely, protective protein/cathepsin A and neuraminidase, respectively. However distinct, in practice these disorders overlap both clinically and biochemically so that easy discrimination between them is sometimes difficult. The principle reason for this may be found in the fact that these three enzymes form a unique complex in lysosomes that is required for their stability and posttranslational processing. In this review, I focus mainly on the primary and secondary beta-galactosidase deficiency states and offer some hypotheses to account for differences between GM1 gangliosidosis and Morquio B disease.

Mucolipidosis (ML) II and III are rare autosomal recessively inherited diseases characterized by deficiency of multiple lysosomal enzymes and, as a result, a generalized storage of macromolecules in lysosomes of cells of mesenchymal origin. In ML II and ML III fibroblasts, most, but not all, newly synthesized lysosomal enzymes are secreted into the medium instead of being targeted correctly to lysosomes. Defects in the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase underlie this effect. It is unknown how lysosomal phospholipases are targeted to the lysosomes of fibroblasts. In the present study lysosomal phospholipase activity was determined in delipidated fibroblast homogenates and plasma from ML II and ML III patients and controls using a [3H]choline-labeled phosphatidylcholine. After incubation, residual phosphatidylcholine and its labeled degradation products (lysophosphatidylcholine, glycerophosphorylcholine and choline phosphate) were quantified. We found that ML II and ML III fibroblasts are deficient in lysosomal phospholipase A and C activity. These enzymes were present in elevated amounts in plasma of ML II and ML III patients. These data indicate that phospholipases, like most other lysosomal enzymes in these diseases, are secreted into the blood instead of being targeted specifically to lysosomes. Thus, the mannose-6-phosphate receptor pathway is needed for proper delivery of lysosomal phospholipases to lysosomes. We also found that production of labeled choline phosphate was mainly due to the activity of acid sphingomyelinase instead of phospholipase C under the assay conditions used. Other active lipolytic enzymes were phospholipase A and lysophospholipase. No evidence for phospholipase D activity was found.

Inhibition of protein kinase C [PK-C] activity by sphingosine and its derivatives has been suggested to play a role in the pathogenesis of sphingolipidoses. In the present study, PK-C activity and PK-C-mediated phosphorylation of endogenous substrates were studied in skin fibroblasts from patients with mucolipidosis type 4 [ML-4], in which there is accumulation of the phospholipids phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine as well as gangliosides. Cytosolic PK-C activity in 5 ML-4 cell lines was comparable to that in control cells. PK-C activity in the particulate fraction of these cells was 84 +/- 14 pmol 32P/mg protein per min compared with 267 +/- 26 in control cells. Increasing the concentrations of the activating lipids in the reaction mixture did not enhance PK-C activity in ML-4 cells, suggesting a non-competitive inhibition of the kinase. Following partial purification of the enzyme from the particulate fraction PK-C activity increased to 288 +/- 14 and 339 +/- 12 pmol 32P/mg protein per min in ML-4 and control cells, respectively. The phosphorylation pattern of endogenous substrates in the particulate fraction of ML-4 cells differed from that in control cells both in the absence and in the presence of calcium and activating lipids. We suggest that PK-C may be involved in the pathogenesis of sphingolipidoses and that this may represent an example for a new type of pathogenetic mechanisms in inborn errors of metabolism.

Cultured fibroblasts from patients with the lysosomal storage disease, mucolipidosis II, produce complex glycosylated lysosomal enzymes which are preferentially excreted presumably due to the absence of specific phosphomannosyl recognition residues needed for intracellular retention. Complex glycosylated hydrolases are also produced by fibroblasts from patients with mucolipidosis I but an abnormal excretion is not apparent in this disorder. Intra- and extracellular distribution, lectin binding, and specific endocytosis were criteria used to compared the properties of intra- and extracellular beta-hexosaminidase derived from mucolipidosis I and normal fibroblast cultures. Mucolipidosis I fibroblasts did not hyperexcrete beta-hexosaminidase when maintained in serum-free medium. Using the specificity of ricin binding to terminal galactosyl residues, the most galactosylated forms of the enzyme derived from mucolipidosis I cell extracts and culture fluids were found in the mucolipidosis I cell extracts (50% of total enzyme). Mucolipidosis I-excreted beta-hexosaminidase which was eluted from ricin-120-Sepharose, was a high-uptake form in endocytosis experiments while unbound enzyme was a low-uptake form. These data suggest that beta-hexosaminidase molecules contained phosphomannosyl residues necessary for receptor-mediated endocytosis as well as galactosyl residues on the same molecule. The co-existence of complex chains with high-mannose chains did not interfere with the phosphomannose-mediated endocytosis of beta-hexosaminidase nor with the retention of endogenous enzyme. We can speculate that since complex oligosaccharide chains in the mucolipidosis I cellular enzyme persist due to a sialidase deficiency, more extensive sialylation of cellular enzyme in normal fibroblasts probably occurs at some point during post-translational processing. However, the presence of sialidase in normal cells initiates complex chain trimming in the lysosomes resulting in a less glycosylated end product.

Labelling of cultured human skin fibroblasts from either control subjects or patients with mucolipidosis II (I-cell disease) with [32P]phosphate resulted in tight association of phosphate with immunoprecipitated glucocerebrosidase, a membrane-associated lysosomal enzyme. Endoglycosidase F digestion of the immunoprecipitated glucocerebrosidase did not release labelled phosphate, suggesting that the phosphate was not associated with the oligosaccharide moiety of this glycoprotein. Purification of the enzyme from cells labelled with [32P]phosphate and [35S]methionine by an immunoaffinity chromatography procedure, which included a washing step with detergent, resulted in complete separation of the phosphate label from the peak of glucocerebrosidase activity and methionine labelling. We conclude that oligosaccharide phosphorylation, which is essential for transport of soluble lysosomal enzymes to the lysosomes in fibroblasts, does not occur in glucocerebrosidase.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.