Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 Å resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis.Late infantile neuronal ceroid lipofuscinosis (LINCL)³ (OMIM number 204500) is a neurodegenerative lysosomal storage disease of childhood that presents typically between the ages of 2 and 4 years with the onset of seizures. Disease progression is reflected by blindness, dementia, mental retardation, and an increase in the severity of seizures. LINCL is always fatal, and the life span of patients is typically 6-15 years. LINCL is caused by mutations in TPP1 (previously named CLN2, for ceroid lipofuscinosis neuronal type 2 gene) (1), which normally encodes a lysosomal protease, tripeptidyl-peptidase 1 (TPP1, EC 3.4.14.9) (2, 3).There is currently no treatment of demonstrated efficacy for LINCL, but promising progress is being made in some directions. Proof-of-principle for virus-mediated gene therapy has been established in a mouse model of LINCL, with a significant improvement in disease phenotype achieved with the use of adeno-associated virus vectors expressing TPP1 (4-7). Affected children have also been treated with adeno-associated virus vectors, although it is too soon to determine whether significant clinical benefits have been achieved in these early trials (8). Enzyme replacement therapy, an approach that has proven successful in a number of other lysosomal storage diseases, has also been investigated in LINCL. Purified recombinant human TPP1 that contains the mannose 6-phosphate lysosomal targeting modification can be taken up by LINCL fibroblasts where it degrades storage material (9), and the protein has been introduced into the cerebrospinal fluid of the LINCL mouse model via intraventricular injection, resulting in significant uptake into the brain and some correction of neuropathology (10).For therapeutic approaches that rely upon replacing a mutant gene product with a functional protein via recombinant methods, e.g. gene and enzyme replacement therapy, a thorough understanding of the biological and biophysical properties of the protein in question are essential for success. Thus, for LINCL, considerable effort has been directed toward the investigation of TPP1, and as a result, this is a well characterized enzyme at the functional and molecular levels (reviewed in Refs. 11, 12). TPP1 encodes a 563-residue preproprotein with a cleavable N-terminal 19-residue signal sequence. The proenzyme (residues 20-563) is a soluble monomer that undergoes proteolytic cleavage in the lysosome, converting the zymogen to an active, mature protease (residues 196-563) (1). Studies on purified pro-TPP1 demonstrate that maturation is autocatalytic in vitro (13, 14) but may involve other proteases in vivo (15). TPP1 is glycosylated, and its N-linked oligosaccharides have been implicated in maturation, activity, targeting, and stability of the processed enzyme (16, 17).TPP1 is a serine protease (14) that possesses two catalytic functions as follows: a primary tripeptidyl exopeptidase activity with a pH optimum of ∼5.0 that catalyzes the sequential release of tripeptides from the unsubstituted N termini of substrates (18), and a much weaker endoproteolytic activity with a pH optimum of ∼3.0 (19). TPP1 exhibits broad substrate specificity (20) and is the only mammalian member of the S53 sedolisin family (reviewed in Ref. 21), which includes a number of unusual bacterial serine peptidases (22). High resolution crystal structures of both free and inhibitor-bound complexes have been determined for three bacterial members of this family (sedolisin (23-26), kumamolisin (27, 28), and kumamolisin-As (29, 30)), and for one (kumamolisin), the structure of a mutant, inactive precursor form has also been obtained (28). These proteins share a common subtilisin-like fold, an octahedrally coordinated calcium-binding site, and an active site that contains an unusual Ser-Glu-Asp (SED) catalytic triad, rather than the Ser-His-Asp (SHD) triad of subtilisin (31, 32). Chemical modification studies of TPP1 have revealed that Ser⁴⁷⁵ is the active site nucleophile (14). Modeling studies suggest that Glu²⁷² and Asp²⁷⁶ complete the catalytic triad and that Asp³⁶⁰ is homologous to the conserved Asn in the subtilisin family in its role in stabilization of the oxyanion of the tetrahedral intermediate during catalysis (33). Site-directed mutagenesis studies are consistent with these conclusions (14, 34).A detailed understanding of the tertiary structure of TPP1 may have implications for developing or improving therapeutic strategies. First, a high resolution model would provide the basis for targeted protein engineering efforts to design TPP1 derivatives with increased half-life prior to and/or upon delivery to the lysosome. Successful creation of a longer lived TPP1 molecule could significantly enhance gene or enzyme replacement approaches to LINCL. Second, a structural model for TPP1 could be valuable in designing derivatives tagged with protein transduction domains to facilitate crossing of the blood-brain barrier for delivery to the central nervous system from the bloodstream. In this study, we present the crystal structure of the proform of human TPP1 at 1.85 Å resolution. This model provides novel insights into the structural basis for the pH-induced auto-activation of the proform of TPP1. A structure of glycosylated pro-TPP1 has been independently determined, displaying features similar to those of deglycosylated TPP1.⁴