Proc. Natl. Acad. Sci. USA Vol. 95, pp. 621–625, January 1998 Genetics The atomic model of the human protective proteinycathepsin A suggests a structural basis for galactosialidosis GABBY RUDENKO*†‡,ERIK BONTEN†,WIM.G.J.HOL*, AND ALESSANDRA D’AZZO†§ *Department of Biological Structure, Howard Hughes Medical Institute, University of Washington, Box 357742, Health Sciences Building Room K-428, Seattle, WA 98195-7742; and †Department of Genetics, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105 Communicated by Elizabeth F. Neufeld, University of California School of Medicine, Los Angeles, CA, November 26, 1997 (received for review October 6, 1997) ABSTRACT Human protective proteinycathepsin A 2-kDa peptide, generating the mature and active form (5, 6, 8). (PPCA), a serine carboxypeptidase, forms a multienzyme The enzyme is active at both acidic and neutral pH and functions complex with b-galactosidase and neuraminidase and is re- as cathepsin Aydeamidaseyesterase on a subset of neuropeptides quired for the intralysosomal activity and stability of these two (9–11). The dual and separable activities of the protein may glycosidases. Genetic lesions in PPCA lead to a deficiency of reflect its capacity to participate in a variety of cellular processes b-galactosidase and neuraminidase that is manifest as the not necessarily restricted to the lysosomal compartment, although autosomal recessive lysosomal storage disorder galactosia- its physiologic role is so far unknown. PPCA’s protective function lidosis. Eleven amino acid substitutions identified in mutant resides in its ability to form a multienzyme complex with b-ga- PPCAs from clinically different galactosialidosis patients lactosidase and neuraminidase, contributing to the stability and have now been modeled in the three-dimensional structure of lysosomal activity of both glycosidases (12, 13). Thus, the clinical the wild-type enzyme. Of these substitutions, 9 are located in phenotype of galactosialidosis is apparently dictated by the loss of positions likely to alter drastically the folding and stability of PPCA protective function, with so far no discernible contribution the variant protein. In contrast, the other 2 mutations that are from the cathepsin A deficiency (2). The interaction with PPCA associated with a more moderate clinical outcome and are is especially crucial for neuraminidase, which looses its enzymatic characterized by residual mature protein appeared to have a activity in absence of PPCA, whereas b-galactosidase maintains milder effect on protein structure. Remarkably, none of the at least 10–15% of the normal enzyme values (2). In turn, the mutations occurred in the active site or at the protein surface, severe early-infantile forms of galactosialidosis present with fea- which would have disrupted the catalytic activity or protective tures that are also observed in both the infantile and congenital function. Instead, analysis of the 11 mutations revealed a type II sialidosis, caused by a single neuraminidase deficiency, and substantive correlation between the effect of the amino acid b the GM1-gangliosidosis, caused by a primary defect in -galacto- substitution on the integrity of protein structure and the sidase (14, 15). This is consistent with PPCA deficiencies causing general severity of the clinical phenotype. The high incidence both glycosidases to malfunction. of PPCA folding mutants in galactosialidosis reflects the fact The majority of the mutations identified so far in galacto- that a single point mutation is unlikely to affect both the b sialidosis patients are missense mutations, and as seen in other -galactosidase and the neuraminidase binding sites of PPCA lysosomal storage disorders, the patient’s clinical severity often at the same time to produce the double glycosidase deficiency. depends on the combination of the two mutant alleles encod- Mutations in PPCA that result in defective folding, however, ing different PPCA variants (16–19). Many of these mutations disrupt every function of PPCA simultaneously. were characterized according to their expression levels and functional deficits (16–18). For a few of them, homology Galactosialidosis is a neurodegenerative lysosomal storage dis- modeling has been attempted by using the distantly related ease that is inherited as an autosomal recessive trait (1, 2). wheat serine carboxypeptidase structure (about 30% amino Patients are classified according to age of onset and the severity acid sequence identity) (20, 21). However, a precise structural of symptoms as severe early infantile or milder late infantile and understanding of how any of these mutations result in a y juvenile adult. The clinical features include cardiac and kidney dysfunctional protein can only come from the three- involvement, skeletal dysplasia, dysmorphism, angiokeratoma, dimensional structure of PPCA itself (7). Herein, we have progressive neurological deterioration, reduced life expectancy, modeled 11 amino acid substitutions in the three-dimensional and in some cases, mental retardation. At the biochemical level, b structure of the wild-type PPCA precursor. The results of this galactosialidosis is diagnosed as a combined deficiency of -ga- study give insights into the structural basis of this unusual lactosidase and neuraminidase activities in the lysosomes (3–5) lysosomal disease. that leads to storage of sialyloligosaccharides and glycopeptides in patient’s tissues and body fluids (ref. 2 and references therein). The primary defect, however, stems from the genetic alteration MATERIALS AND METHODS y of a third lysosomal protein, protective protein cathepsin A Modeling studies of the mutations were performed by using (PPCA) (1, 5). the macromolecular modeling package O (22) on a Silicon PPCA is a multifunctional enzyme with distinct protective and Graphics interactive computer graphics system, Indigo XZ catalytic activities (6). It is synthesized as a 452-amino acid (54 kDa) precursor (5), containing four disulfide bridges and two y Abbreviations: PPCA, protective proteinycathepsin A; ER, endoplas- glycosylation sites (5, 7). In the endosomal lysosomal compart- mic reticulum. ment, the precursor undergoes endoproteolytic removal of a Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, Biology Department, Brookhaven National Lab- The publication costs of this article were defrayed in part by page charge oratory, Upton, NY 11973 (reference 1IVY). ‡Present address: Howard Hughes Medical Institute, University of payment. This article must therefore be hereby marked ‘‘advertisement’’ in Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, accordance with 18 U.S.C. §1734 solely to indicate this fact. Room Y4–206, Dallas, TX 75235-9050. © 1998 by The National Academy of Sciences 0027-8424y98y95621-5$2.00y0 §To whom reprint requests should be addressed. e-mail: alessandra. PNAS is available online at http:yywww.pnas.org. [email protected]. 621 Downloaded by guest on September 27, 2021 622 Genetics: Rudenko et al. Proc. Natl. Acad. Sci. USA 95 (1998) R4000. Further analysis of the effects of each amino acid W37R, S62L, and L208P, hydrogen bond donors or acceptors substitution, including interatomic contacts and solvent- are buried in the protein interior with no possibility for a accessible surface calculations, was carried out by using pro- partner. Loss of a hydrogen bonding partner is probably grams from the CCP4 suite (23). The atomic structure used for particularly detrimental for L208P, where a proline residue in this analysis has been refined to 2.2 Å with a crystallographic the middle of helix Ha2 disrupts the hydrogen bonding pattern 5 Rfactor of 21.3% (Rfree 26.8%) (7). Figures were drawn with (Fig. 2f). The mutations Q21R and W37R introduce a posi- MOLSCRIPT (24) and RASTER3D (25). tively charged residue in the interior of the protein with no apparent possibility of charge compensation, thereby decreas- RESULTS ing the stability of these mutants. Internal cavities, formed by the introduction of smaller residues, might also contribute to We divided the 11 amino acid substitutions identified in PPCA protein destabilization for Y367C and L208P. For Y367C, variants from different galactosialidosis patients into two introducing an extra free cysteine could have promoted the groups according to the ability of the mutant proteins to reach formation of improper disulfide bonds and concomitant mis- the lysosomes. Nine of the variants (17, 18) do not compart- folded intermediates. Although it is difficult to predict the mentalize in lysosomes and have the following amino acid precise effects of these mutations on protein structure without substitutions: Q21R, S23Y, W37R, S62L, V104M, L208P, the crystal structure of the different mutant proteins, overall Y367C, M378T, and G411S (group 1). Mutations Y221N and the impact of the group 1 mutations on protein folding is F412V result in a diminished but significant amount of protein expected to be severe. still detectable in the lysosomes (group 2) (16, 18). A 12th Similarly to Y367C, mutation M378T was modeled in the mutation (SpDEx7) that generates an alternative splice site is native structure without causing any strain because the sub- leaky and allows for a small amount of correctly spliced mRNA stituted residue is smaller than the wild-type amino acid. resulting in low levels of wild-type protein (17). The mutations However, it was shown that no functional protein is produced S23Y, S62L, V104M, L208P, Y367C,