Molecular pathology of galactosialidosis in a patient affected with two new frameshift mutations in the cathepsin A/protective protein gene.

Galactosialidosis is a recessively inherited lysosomal storage disease characterized by the combined deficiency of neuraminidase and beta-galactosidase secondary to the genetic deficiency of cathepsin A/protective protein. In lysosomes, cathepsin A forms a high-molecular-weight complex with beta-galactosidase and neuraminidase that protects these enzymes against intralysosomal proteolysis. In a patient affected with late infantile form of galactosialidosis, we found two new cathepsin A mutations, a two-nucleotide deletion, c517delTT and an intronic mutation, IVS8+9C-->G resulting in abnormal splicing and a five-nucleotide insertion in the cathepsin A cDNA. Both mutations cause frameshifts and result in the synthesis of truncated cathepsin A proteins, which, as suggested by structural modeling, are incapable of dimerization, complex formation, and catalysis. However, enzymatic assays, gel-filtration, and Western blot analysis of the patient's cultured skin fibroblast extracts showed the presence of a small amount of normal-size, catalytically active cathepsin A and cathepsin A-beta-galactosidase 680 kDa complex, suggesting that a low amount of cathepsin A mRNA is spliced normally and produces the wild-type protein. This may contribute to the relatively mild phenotype of the patient and illustrates the importance of critically comparing molecular results with clinical and biochemical phenotypes.

Ultrastructural and immunocytochemical study of skin fibroblasts from normal and sialidosis patients.

The objectives of this study were to analyze morphologically, morphometrically and immunocytochemically the lysosomal compartment of normal fibroblasts and of fibroblasts with neuraminidase deficiency. The immunocytochemical analyses consisted of quantifying the distribution of saposins and beta-galactosidase in the lysosomes of these cells to test the hypothesis that neuraminidase deficiency is associated with an impairment in the transport of these proteins to the lysosomal compartment. To test this idea, cultured skin fibroblasts of patients with or without sialidosis were prepared for electron microscopy and probed with antibodies against lysosomal beta-galactosidase and lysosomal saposins. The lysosomes of the affected cells had an abnormal accumulation of incompletely digested membranes which was associated with a significant lowering in the density of antigenic sites per lysosome. However, due to a significant increase in the number of lysosomes per affected cell, the total number of antigenic sites in control and neuraminidase deficient cells was similar. This presumably compensatory effect indicates that although the rate of production of beta-galactosidase and saposins remains unchanged, the transport of these molecules to the lysosomes is somehow affected. Our data also indicate that in the fibroblasts, lysosomes require a normal concentration of the three enzymes to maintain neuraminidase activity and sphingolipid degradation.

Ring chromosome 20 and possible assignment of the structural gene encoding human carboxypeptidase-L to the distal segment of the long arm of chromosome 20.

We report on a 14-year-old boy with ring chromosome 20. Clinical manifestations included postnatal growth retardation, epilepsy, microcephaly, behaviour disorder, minor facial anomalies, small sella turcica, possible partial growth hormone deficiency, and mental retardation. A decreased activity of enzyme carboxypeptidase-L/protective protein (CP/PP) in cultured fibroblasts was demonstrated in our patient and a patient with a karyotype 46,XY,-14, + der(14)t(14;20)(14pter----14q32.3::20q13.1----20qter)m at. This suggests possible assignment of the CP/PP gene to the distal segment of 20q.

Human neuraminidase is a 60 kDa-processing product of prosaposin.

Human neuraminidase was purified from placenta as part of a large molecular weight complex with lysosomal beta-galactosidase and carboxypeptidase. Passage of this purified complex through a sialic acid-affinity column (fetuin-agarose) retained a minor 60 kDa protein which was eluted with 100 mM N-acetylneuraminic acid. This 60 kDa protein is recognized in Western blots of the purified complex by an anti-prosaposin antibody which at the same time was able to inhibit neuraminidase activity in the preparation. Furthermore, probing of cultured skin fibroblasts of patients affected with neuraminidase deficiency using the antiprosaposin antibody revealed an abnormal 57 kDa protein. These results indicate that the 60 kDa protein is derived from prosaposin and has the characteristics of a neuraminidase.

Deficient lysosomal carboxypeptidase activity in galactosialidosis.

In the lysosome, the glycosidases neuraminidase (EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23) are associated to a 52 kDa "protective protein" to form a large multi-enzymatic complex. Deficient synthesis or inactivation of this protective protein causes galactosialidosis, a lysosomal storage disorder in man in which both neuraminidase and beta-galactosidase activities are deficient. Since the protective protein possesses extensive sequence homology with carboxypeptidase Y (carb Y) and the KEX 1 gene product from yeast, we have used the artificial substrate N-CBZ-Phe-Leu to detect and characterize the peptidase activity of the lysosomal carboxypeptidase (carb L). Using both a purified preparation of the lysosomal multi-enzymatic complex and cultured skin fibroblasts of patients affected with galactosialidosis, we demonstrate that the 52 kDa protective protein is responsible for carb L activity. The fibroblasts of three patients affected with late infantile and juvenile galactosialidosis were found to be deficient in carb L activity (1.4% of normal mean value).

Structure of the lysosomal neuraminidase-beta-galactosidase-carboxypeptidase multienzymic complex.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed