The atomic model of the human protective protein/cathepsin A suggests a structural basis for galactosialidosis

Abstract

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.

Figure 1

Schematic diagram of the PPCA monomer, which is present as a dimer in the crystal structure. The core domain contains the catalytic triad (shown in blue). The cap domain consists of a three-helical bundle and a small mixed β-sheet involved in enzyme inactivation. The PPCA mutations found in galactosialidosis patients are shown in red (group 1) or green (group 2).

Figure 2

Panel of PPCA mutations in group 1. Wild-type residues are shown in yellow, and the mutant side chains are shown in purple. Relevant side chains that interact with the residue of interest are depicted and labeled. β-strands, helices, and coils indicate the secondary structure elements that form the scaffold for the interacting residues. Color coding: carbon atoms, aqua spheres; nitrogen atoms, blue spheres; oxygen atoms, red spheres; sulfur atoms, green spheres; hydrogen bonds, green dotted lines. The impact of each mutation on the protein structure was assessed as follows. (a) Steric clashes due to incorporation of a larger side chain, positive charge introduced in hydrophobic interior, loss of hydrogen bond donor for N^ɛ1 Trp³⁷, no hydrogen bond acceptors for N^η1 Arg at position 21, and N^η2 Arg at position 21. (b) Steric clashes upon introduction of a larger side chain and loss of hydrogen bond to N^ɛ2 His³⁵. (c) Positive charge introduced in hydrophobic interior and no hydrogen bond acceptors for N^η1 Arg³⁷ and N^η2 Arg³⁷. (d) Steric clashes due to introduction of a larger side chain, forcing two β-strands (Cβ3 and Cβ6) in the central β-sheet apart, and loss of hydrogen bonds to main chain atoms: O Leu⁹⁸ and N Asn⁵⁵. (e) Steric clash with backbone carbonyl of Gly⁵⁹ due to incorporation of a larger side chain while presence of disulfide Cys⁶⁰–Cys³³⁴ limits possibilities for conformational changes accommodating the mutation. (f) Loss of hydrogen bond donor for main chain atom O Leu²⁰⁸, located in the middle of a helix, cavity created possibly disrupting packing. (g) Extra cysteine introduced, could promote formation of incorrect disulfides, smaller side chain incorporated introducing cavity. (h) New N-linked glycosylation site created at the dimer interface, and cavity introduced at the dimer interface due to incorporation of a smaller side chain. (i) Steric clashes due to incorporation of a larger side chain while backbone conformation at position 411 demands Ramachandran angles only favorable for Gly.

Figure 3

Panel of PPCA mutants in group 2. (a) The locations of Phe⁴¹² in the PPCA precusor dimer. (b) Substitution of Phe⁴¹² (in yellow) with Val (in magenta). (c) The predicted effects of the F412V mutation on protein structural integrity. (d) The environment of Tyr²²¹ in the PPCA dimer. (e) Substitution of Tyr²²¹ (in yellow) with Asn (in magenta). A lilac sphere has been modeled to show how a water molecule or ion could participate in the interactions between Asn²²¹ and the other side chains, allowing the formation of a more extensive hydrogen bonding network between the loop with Asn²²¹, the blocking peptide, and the helix containing Asp¹⁸⁷. (f) Proposed effects of the Y221N mutation on the local protein environment. Color coding: carbon atoms, aqua spheres; nitrogen atoms, blue spheres; oxygen atoms, red spheres; sulfur atoms, green spheres; blocking peptide (residues 272–277), orange; hydrogen bonds, green dotted lines.