Mutation Report for: F440V_human-CTSA

Human protective protein/cathepsin A (PPCA), a serine carboxypeptidase, forms a multienzyme complex with beta-galactosidase and neuraminidase and is required for the intralysosomal activity and stability of these two glycosidases. Genetic lesions in PPCA lead to a deficiency of beta-galactosidase and neuraminidase that is manifest as the autosomal recessive lysosomal storage disorder galactosialidosis. Eleven amino acid substitutions identified in mutant PPCAs from clinically different galactosialidosis patients have now been modeled in the three-dimensional structure of the wild-type enzyme. Of these substitutions, 9 are located in positions likely to alter drastically the folding and stability of the variant protein. In contrast, the other 2 mutations that are associated with a more moderate clinical outcome and are characterized by residual mature protein appeared to have a milder effect on protein structure. Remarkably, none of the mutations occurred in the active site or at the protein surface, which would have disrupted the catalytic activity or protective function. Instead, analysis of the 11 mutations revealed a substantive correlation between the effect of the amino acid substitution on the integrity of protein structure and the general severity of the clinical phenotype. The high incidence of PPCA folding mutants in galactosialidosis reflects the fact that a single point mutation is unlikely to affect both the beta-galactosidase and the neuraminidase binding sites of PPCA at the same time to produce the double glycosidase deficiency. Mutations in PPCA that result in defective folding, however, disrupt every function of PPCA simultaneously.

Mutations in the gene encoding lysosomal protective protein/cathepsin A (PPCA) are the cause of the lysosomal disorder galactosialidosis (GS). Depending on age of onset and severity of the symptoms, patients present with either an early infantile (EI), a late infantile (LI), or a juvenile/adult (J/A) form of the disease. To study genotype-phenotype correlation in this disorder, we have analyzed the mutations in the PPCA gene of eight clinically different patients. In two EI and one J/A patient, we have identified four novel point mutations (Val104Met, Leu208Pro, Gly411Ser and Ser23Tyr), that prevent phosphorylation and, hence, lysosomal localization and maturation of the mutant precursors. Two amino acid substitutions (Phe412Val and Tyr221Asn) are shared by five LI patients. These mutations appear to be pathognomonic for this phenotype, and determine the clinical outcome depending on whether they are present together or in combination with other mutations. The latter include a single base deletion and a novel amino acid change (Met378Thr), which generates an additional glycosylation site. Within the LI group, patients carrying the Phe412Val mutation are clinically more severe than those with the Tyr221Asn substitution. This is in agreement with the biochemical behavior of the Asn221-mutant protein, that is, like the Phe412Val protein, phosphorylated, routed to lysosomes and proteolytically processed, but its intralysosomal stability is intermediate between that of wild-type PPCA and Val412-PPCA. Overall, these results may explain the clinical heterogeneity observed in GS patients and may help to correlate mutant allelic combinations with specific clinical phenotypes.

The lysosomal disorder galactosialidosis is caused by deficiency of the protective protein in the absence of which the activities of the enzymes beta-galactosidase and neuraminidase are reduced. Aside from its protective function towards the two glycosidases, this protein has cathepsin A-like activity. A point mutation in the protective protein gene, resulting in the substitution of Phe412 with Val in the gene product, was identified in two unrelated patients with the late infantile form of the disease. Expression in COS-1 cells of a protective protein cDNA with the base substitution resulted in the synthesis of a mutant protein that lacks cathepsin A-like activity. The newly made mutant precursor was shown to be partially retained in the endoplasmic reticulum. Only a fraction is transported to the lysosomes where it is degraded soon after proteolytic processing into the mature two-chain form. Since the mutant precursor, contrary to the wild type protein, does not form homodimers, the dimerization process might be a condition for the proper targeting and stable conformation of the protective protein. These results clarify the mechanism underlying the combined deficiency in these patients, and give new insight into the structure-function relationship of the wild type protein.

We describe the clinical findings over the first 18 years of a patient with a novel phenotype for galactosialidosis, the storage disease produced by the combined deficiency of beta-galactosidase and neuraminidase. Clinical findings in the first few months included somewhat unusual appearance and hepatosplenomegaly. Dysostosis multiplex was evident by age 2 1/2 years. Mitral and aortic valvular disease appeared over the next few years and cardiac disease has become the most important clinical problem. Foam cells were present in the bone marrow, and vacuolated lymphocytes were present in the peripheral blood smear. The patient had no neurological symptoms, cherry red spots, or intellectual deterioration during the first 18 years. Evidence presented elsewhere indicates that the basic defect in this late infantile form of galactosialidosis (as is thought to be true for the other forms of galactosialidosis) is a reduced amount of the 32 kDa phosphoglycoprotein which associates with beta-galactosidase and alpha-neuraminidase in lysosomes.

We describe a 19-year-old white male with juvenile galactosialidosis. He presented with hip arthralgia and was found to have facial "coarseness," corneal clouding, mitral and aortic insufficiency, and hepatosplenomegaly. Ultrastructural studies of skin biopsy and peripheral blood lymphocytes showed membrane-bound inclusions containing sparse fibrillogranular material. Biochemical analysis showed elevated urinary sialyloligosaccharides and no free sialic acid. Fibroblast enzyme analysis showed low activities of both alpha-neuraminidase and beta-galactosidase. To date, most patients with juvenile galactosialidosis have been Japanese. However, unlike those patients, our patient did not have macular cherry-red spots, neurologic abnormalities, or mental retardation. We speculate that this young man represents a new subtype of juvenile galactosialidosis with a potentially different molecular defect from that of the Japanese variant.