IVYA

The human 'protective protein' (HPP) forms a multi-enzyme complex with -galactosidase and neuraminidase in the lysosomes, protecting these two glycosidases from degradation. In humans, deficiency of HPP leads to the lysosomal storage disease galactosialidosis: a lysosomal storage disease inherited as an autosomal recessive trait. Patients with this disorder are biochemically diagnosed as having drastically reduced -galactosidase and neuraminidase activities in the lysosomes. Slightly different spectra of symptoms are observed : skeletal dysplasia, dysmorphism, progressive neurological deterioration, reduced life expectancy, and in some cases mental retardation, as well as impairment of cardiac and kidney function.

The activation mechanism of HPP is unique among proteases with known structure. It differs from the serine proteases in that the active site is preformed in the zymogen, but is blocked by a maturation subdomain. In contrast to the zinc metalloproteases and aspartic proteases, the chain segment physically rendering the catalytic triad solvent inaccessible in HPP is not cleaved off to form the active enzyme. The activation must be a multi-step process involving removal of the excision peptide and major conformational changes of the maturation subdomain, whereas the conformation of the enzymatic machinery is probably almost, or completely, unaffected.

HPP is synthesized as a 542 amino acid precursor with a molecular weight of 54 kDa and dimerizes soon after synthesis in the endoplasmic reticulum. The sequence contains two glycosylation sites (Asn117 and Asn305) and nine cysteines. After transport to the acidic endosomal/lysosomal compartments, the precursor undergoes a protease-mediated maturation process. A polypeptide of approximately 2 kDa, called the 'excision' peptide is removed from the protein yielding a 32 kDa and a 20 kDa chain held together by disulfide bridges

The crystal structure of the 108 kDa dimer of the precursor HPP has been elucidated by making extensive use of twofold density averaging. The monomer consists of a 'core' domain and a 'cap' domain.

HPP appears to be a multi-functional lysosomal enzyme. Although the exact physiological substrate(s) of HPP are not known, there is evidence that HPP may be secreted to participate extracellularly in the deactivation of selected bio-active peptides such as endothelin I. While loss of the protective capacity of HPP is directly linked to galactosialidosis, it is unclear to what extent loss of the enzymatic activity contributes to the clinical phenotype. Given its pleiotropic and distinct functions, as well as the capacity to be secreted and taken up by different cell types, HPP may have a function outside the multi-enzyme complex and possibly outside the lysosomes as well.

The crystal structure determination of the precursor form of HPP shows that the monomer contains approximately 20% structure and 30% helical structure. The protein fold can be divided into two domains: a 'core' domain, as commonly found in members of the hydrolase fold family and a 'cap' domain. The core domain, comprising residues 1–182 as well as 303–452, contains a central ten-stranded sheet. An additional ten helices and two small strands occur on both sides of the central sheet. The cap domain can be divided into a 'helical' subdomain consisting of three helices (residues 183–253) and a 'maturation' subdomain consisting of a three-stranded mixed sheet (residues 254–302).

Fig. 1.Schematic ribbon diagram of the HPP monomer (monomer 1). The 'core' domain is shown in yellow. The 'cap' domain consists of a 'helical' subdomain (red) and a 'maturation' subdomain (orange). The 'excision' peptide, located in the maturation subdomain is shown in light blue. The side chains of the catalytic triad Ser150, His429 and Asp372 (from right to left) are in green. Some secondary structure elements (assigned according to DSSP) are labeled.

There appear to be four disulfide bridges per monomer: Cys60–Cys334, Cys253–Cys303, Cys212–Cys228 and Cys213–Cys218.

HPP (only its mature form) possesses serine carboxypeptidase activity at acidic pH. Based on sequence alignments with serine carboxypeptidases, the catalytic triad in HPP has been proposed to be formed by the residues Ser150, His429 and Asp372 members of the hydrolase fold family, which includes enzymes with different catalytic functions such as the serine carboxypeptidases, dehalogenase, various lipases and acetylcholinesterase. Although the central core is the same (a central sheet flanked by helices on both sides), the cap domains in this protein family are quite diverse, both with respect to their folds as well as their sizes. HPP has one of the largest cap domains, with 121 residues forming the three-helix bundle of the helical subdomain and the three-stranded sheet of the maturation subdomain.

The oxyanion hole proposed to stabilize the negatively charged tetrahedral intermediate in serine carboxypeptidases is formed in HPP by the backbone amides of Gly57 and Tyr151.

In HPP, a pair of glutamic acid residues (Glu69 and Glu149) is positioned near the catalytic triad, with their carboxylate groups interacting with each other. In addition, an asparagin (Asn55) is oriented such that it forms a hydrogen bond to each of the two carboxylate groups of the glutamic acid pair. These three residues are conserved between HPP, CPW and CPY and have been implicated in regulating the low pH optimum for the carboxypeptidase activity found in the serine carboxypeptidases

HPP has a substrate preference for hydrophobic residues in the P1 and/or P1' binding pockets. The P1' pocket was identified as consisting of Tyr247 and Asp64, with Met430 and Thr304 at the far end.

The active-site cleft in the precursor is blocked by numerous residues from the maturation subdomain. The catalytic triad is rendered solvent inaccessible by residues Asn275, IIe276 and Phe277. These residues are part of the polypeptide Asp272–Phe277 which we call the 'blocking' peptide. This peptide is held down predominantly by hydrophobic contacts. Residue Asn275 of the blocking peptide appears to fill what might be part of the P1 binding pocket in the mature form. The blocking peptide does not assume a conformation that a productive peptide substrate would adopt. It is carefully positioned to avoid being cleaved by the nearby catalytic residues. Thus, substrate binding is prevented in the precursor form by the inaccessibility of the substrate-binding pockets. Our structure reveals that the inactivation mechanism of HPP is based on blocking of the active site, not upon conformational changes of the residues involved in catalysis or transition-state stabilization.But maturation must be accompanied by conformational changes.

In HPP a fourth mechanism for protease zymogen activation is revealed. In this case, the catalytic triad in the precursor form is in a catalytically competent conformation. Enzymatic activity is prevented by the blocking peptide. The blocking peptide is, however, not the same as the excision peptide. This leads to a distinct difference from other maturation mechanisms. After disappearance of the excision peptide, up to 35 residues filling the active-site cleft in the HPP precursor must rearrange to render the catalytic triad solvent accessible, but these residues do not get cleaved off. Removal of the excision peptide, and possibly a shift to lower pH in the endosome/lysosome, appears to be a trigger for this event.