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Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is an autosomal recessive condition caused by variants in the TPP1 gene, leading to deficient activity of the lysosomal enzyme tripeptidyl peptidase I (TPP1). We update on the spectrum of TPP1 variants associated with CLN2 disease, comprising 131 unique variants from 389 individuals (717 alleles) collected from the literature review, public databases, and laboratory communications. Previously unrecorded individuals were added to the UCL TPP1‐specific database. Two known pathogenic variants, c.509–1 G>C and c.622 C>T (p.(Arg208*)), collectively occur in 60% of affected individuals in the sample, and account for 50% of disease‐associated alleles. At least 86 variants (66%) are private to single families. Homozygosity occurs in 45% of individuals where both alleles are known (87% of reported individuals). Atypical CLN2 disease, TPP1 enzyme deficiency with disease onset and/or progression distinct from classic late‐infantile CLN2, represents 13% of individuals recorded with associated phenotype. NCBI ClinVar currently holds records for 37% of variants collected here. Effective CLN2 disease management requires early diagnosis; however, irreversible neurodegeneration occurs before a diagnosis is typically reached at age 5. Timely classification and public reporting of TPP1 variants is essential as molecular testing increases in use as a first‐line diagnostic test for pediatric‐onset neurological disease., Neuronal ceroid lipofuscinosis type 2 (CLN2 disease) is an autosomal recessive condition caused by variants in the TPP1 gene, leading to deficient activity of the lysosomal enzyme tripeptidyl peptidase I (TPP1). We update on the spectrum of TPP1 variants associated with CLN2 disease, comprising 131 unique variants from 389 individuals (717 alleles) collected from the literature review, public databases, and laboratory communications.

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3–5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.

Introduction Neuronal Ceroid Lipofuscinosis type 2 (CLN2) is a neurodegenerative lysosomal disease which leads to early dementia and death without treatment. The recently available therapy consists of intracerebroventricular enzyme substitution: cerliponase alfa. In this report, we describe the evolution of the first French children treated with cerliponase alfa. Method CLN2 Clinical Rating Scale Motor-Language (CLN2 ML) assesses the motor and language evolution of CLN2 patients. We retrospectively studied patients' medical records: clinical symptoms, MRI conclusions, gene mutation, side effects of infusions, patient's age and CLN2 ML scores at diagnosis, at the beginning of enzyme replacement therapy (ERT) and at the last evaluation. Seven patients were included. Results Average age at diagnosis was 50 months ( ±10) with CLN2 ML score equal to 3.6 [1.5–5]. Average age at the beginning of ERT was 56 months ( ±13) with CLN2 ML score equal to 3.1 [1–5]. At the last available evaluation, average age was 82 months ( ±20) with CLN2 ML score equal to 2.8 [0–5]. Thus, in 26 months, the mean CLN2 ML score only decreased by 0.3 points. However, patients with a CLN2 ML score greater than three at the onset of ERT experienced a stabilisation or improvement of clinical signs, whereas patients with a CLN2 ML score less than three at baseline continue to deteriorate. Conclusion For patients starting ERT at an early stage of the disease, cerliponase alfa changes the natural history of the disease with a halt in disease progression or even a slight improvement in clinical symptoms.

Hypoxic events are known to cause substantial damage to the hippocampus, cerebellum and striatum. The impact of hypoxic shock on other brain parts is not sufficiently studied. Recent studies show that tripeptidyl peptidase I (TPPI) activity in fish is altered after a hypoxic stress pointing out at a possible enzyme involvement in response to hypoxia. Similar studies are not performed in mammals. In this work, the effect of sodium nitrite-induced acute hypoxic shock on the rat brain was studied at different post-treatment periods. Morphological changes in cerebral cortex, cerebellum, medulla oblongata, thalamus, mesencephalon and pons were assessed using silver-copper impregnation for neurodegeneration. TPPI activity was biochemically assayed and localized by enzyme histochemistry. Although less vulnerable to oxidative stress, the studied brain areas showed different histopathological changes, such as neuronal loss and tissue vacuolization, dilatation of the smallest capillaries and impairment of neuronal processes. TPPI activity was strictly regulated following the hypoxic stress. It was found to increase 12-24h post-treatment, then decreased followed by a slow process of recovery. The enzyme histochemistry revealed a temporary enzyme deficiency in all types of neurons. These findings indicate a possible involvement of the enzyme in rat brain response to hypoxic stress.

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca(2+) and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K(M) compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys.

The blood–brain barrier (BBB) presents a major challenge to effective treatment of neurological disorders, including lysosomal storage diseases (LSDs), which frequently present with life-shortening and untreatable neurodegeneration. There is considerable interest in methods for intravenous delivery of lysosomal proteins across the BBB but for the most part, levels achievable in the brain of mouse models are modest and increased lifespan remains to be demonstrated. In this study, we have investigated delivery across the BBB using a mouse model of late-infantile neuronal ceroid lipofuscinosis (LINCL), a neurodegenerative LSD caused by loss of tripeptidyl peptidase I (TPP1). We have achieved supraphysiological levels of TPP1 throughout the brain of LINCL mice by intravenous (IV) coadministration of recombinant TPP1 with a 36-residue peptide that contains polylysine and a low-density lipoprotein receptor binding sequence from apolipoprotein E. Importantly, IV administration of TPP1 with the peptide significantly reduces brain lysosomal storage, increases lifespan and improves neurological function. This simple “mix and inject” method is immediately applicable towards evaluation of enzyme replacement therapy to the brain in preclinical models and further exploration of its clinical potential is warranted.

Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2(-/-) mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin-proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2(-/-) neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a lysosomal storage disorder caused by mutations in the CLN2 gene and a deficiency of tripeptidyl peptidase I (TPP-I). Prior studies with adeno-associated virus (AAV) serotype 2 or 5 mediated transfer of the CLN2 complementary DNA to the central nervous system (CNS) of CLN2(-/-) mice cleared CNS storage granules, but provided no improvement in the phenotype or survival of this model of LINCL. In this study, AAV serotypes (AAV2, AAV5, AAV8, and AAVrh.10) were compared for the delivery of the same CLN2 expression cassette. AAVrh.10, derived from rhesus macaque, provided the highest TPP-I level and maximum spread beyond the site of injection. The AAVrh.10-based vector functioned equally well in naive rats and in rats previously immunized against human serotypes of AAV. When administered to the CNS of CLN2(-/-) mice, the AAVrh.10CLN2 vector provided widespread TPP-I activity comparable to that in the wild-type mice. Importantly, the AAVrh.10CLN2-treated CLN2(-/-) mice had significant reduction in CNS storage granules and demonstrated improvement in gait, nest-making abilities, seizures, balance beam function, and grip strength, as well as having a survival advantage.

Tripeptidyl peptidase I (TPP-I) is a lysosomal peptidase with unclear physiological function. TPP-I deficiency is associated with late-infantile neuronal ceroid lipofuscinosis (NCL), a fatal neurodegenerative disease of childhood that is characterized by loss of neurons and photoreceptor cells. We have developed two novel fluorogenic substrates, [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110, that are cleaved by TPP-I in living cells. Fluorescence of liberated rhodamine 110 was detected by flow cytometry and was dependent on the level of TPP-I expression. Rhodamine-related fluorescence could be suppressed by preincubation with a specific inhibitor of TPP-I. When investigated by fluorescent confocal microscopy, rhodamine signals colocalized with lysosomal markers. Thus, cleavage of these rhodamide-derived substrates is a marker for mature enzymatically active TPP-I. In addition, TPP-I-induced cleavage of [Ala-Ala-Phe]2-rhodamine 110 could be visualized in primary neurons. We conclude that [Ala-Ala-Phe]2-rhodamine 110 and [Arg-Nle-Nle]2-rhodamine 110 are specific substrates for determining TPP-I activity and intracellular localization in living cells. Further, these substrates could be a valuable tool for studying the neuronal pathology underlying classical late-infantile NCL. This article contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations inCLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains ofCLN2−/−mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that ofCLN2+/+control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection sites. Together, these data support the development of direct CNS gene transfer using an AAV2 vector expressing the CLN2 cDNA for the CNS manifestations of LINCL.

Late-infantile neuronal ceroid lipofuscinosis (CLN2), previously known as the late-infantile form of Batten disease, is a lysosomal storage disease which results from mutations in the gene that codes for tripeptidyl peptidase-I (TPP-I). This disease is characterised by progressive neurodegeneration in young children although the molecular mechanisms responsible for neuronal cell death are unclear. TPP-I is an exopeptidase which removes N-terminal tripeptides from small peptides, including several peptide hormones. We report that the degradation of the neuropeptide, neuromedin B, by mouse brain cells is restricted to lysosomes and that the pattern of degradation products is consistent with a predominant role for TPP-I. Neuromedin B is degraded by a similar pathway in a mouse neuronal cell line and also in cultured human fibroblasts. A specific inhibitor of TPP-I is able to abolish neuromedin B degradation in a variety of cell types. Fibroblasts from CLN2 patients, which are deficient in TPP-I activity, are unable to degrade neuromedin B. These observations suggest that TPP-I is the predominant proteolytic enzyme responsible for the intracellular degradation of neuromedin B. The inability of cells from CLN2 patients to degrade neuromedin B and other neuropeptides may contribute to the pathogenesis of the disease.

The neuronal ceroid lipofuscinoses (NCL), also known as Batten disease, are a group of inherited severe neurodegenerative disorders primarily affecting children. They are characterised by the accumulation of autofluorescent storage material in many cells. Children suffer from visual failure, seizures, progressive physical and mental decline and premature death, associated with the loss of cortical neurones. Six genes have been identified that cause human NCL (CLN1, CLN2, CLN3, CLN5, CLN6, CLN8), and approximately 150 mutations have been described. The majority of mutations result in a characteristic disease course for each gene. However, mutations associated with later disease onset or a more protracted disease course have also been described. At least seven common mutations exist, either with a world‐wide distribution or associated with families from specific countries. All mutations are described in the NCL Mutation Database (http://www.ucl.ac.uk/ncl http://www.ucl.ac.uk/ncl).

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.

Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small proteins. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, late infantile neuronal ceroid lipofuscinosis. The pathological consequences of loss of activity are only manifested in neuronal cells suggesting that TPP-I may be involved in the lysosomal degradation of neuropeptides. We have investigated the degradation of the C-terminal octapeptide of sulphated cholecystokinin (CCK-8S) by a lysosomal fraction purified from mouse brain. Degradation products were characterised by reversed phase HPLC and mass spectrometry. Incubation of CCK-8S with brain lysosomes results in the sequential removal of the tripeptides DY(SO(3)H)M and Glycl-Tryptophanyl-Methionine from the N-terminus of CCK-8S. Degradation of CCK-8S in the isolated lysosomal fraction is completely prevented by Ala-Ala-Phe-chloromethyl ketone, an inhibitor of TPP-I. Butabindide, a specific inhibitor of TPP-II, a cell surface peptidase which also cleaves CCK-8S, inhibits TPP-I but kinetic studies indicate that the Ki for inhibition of TPP-I is 1000-fold higher than the Ki for the inhibition of TPP-II. Consequently, higher concentrations of butabindide are required for the inhibition of CCK-8S degradation by TPP-I than by TPP-II. These results indicate that whereas cell surface TPP-II is responsible for regulating extracellular CCK-8S levels, lysosomal TPP-I is largely responsible for the degradation of CCK-8S which enters the cell by receptor-mediated endocytosis.

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.

To understand the precise distribution of tripeptidyl peptidase I (TPP-I), a defect of which has been shown to induce late infantile neuronal ceroid lipofuscinosis, various tissues from rats and mice were analyzed using biochemical and immunohistochemical techniques. Western blot analyses showed that a protein band immunoreactive to anti-TPP-I appeared in tissue extracts of both animals at a molecular weight of approximately 47 kD. Protein levels of TPP-I differed among tissues; they were high in the rat brain, liver, stomach, kidney, thyroid and adrenal glands and in the mouse brain, stomach, kidney, and testis. The proteolytic activity of TPP-I was detectable; it differed in the tissues examined and did not always reflect the expression levels of the protein in the tissues. In particular, the TPP-I activity was low in the brains of both animals and high in the rat testis, although its protein levels were high in the former tissue and low in the latter. Double immunostaining showed the immunoreactivity for TPP-I to be well localized in granular structures of epithelial cells in renal tubules and the cerebral choroid plexus, both of which were also stained with lamp2, a lysosomal membrane protein marker, indicating that TPP-I is a lysosomal enzyme. The immunoreactivity was intense in F4/80-immunopositive macrophages/microglial cells located in various tissues including the thymus, spleen, liver, alimentary tract, and central nervous system. Although the immunoreactivity differed depending on the tissues and even within the same tissues between the species, it was detected in all tissues examined, especially in nerve cells, some types of endocrine cells, and oxyntic cells such as gastric parietal cells and bone osteoclasts. However, the immunoreactivity was faint and week in rat thyroid gland, although its protein level was high in the tissue. These lines of evidence suggest that TPP-I, a lysosomal serine proteinase, is widely distributed in rat and mouse tissues, although its expression levels vary among them.

We examined 26 individuals with clinical and electron microscopic signs of late infantile neuronal ceroid lipofuscinosis (LINCL). In 22 cases, we found both pathogenic alleles. Sixteen patients exclusively carried either one or a combination of the two common mutations R208X and IVS5-1G > C. In the remaining cases, four missense mutations could be detected, of which R127Q, N286S, and T353P represent novel, previously not described alleles. A clinical performance score was developed by rating motor, visual, and verbal functions and the incidence of cerebral seizures in 3-month intervals during the course of the disease. A Total Disability Score was derived by summing up the single scores for motor, visual, and verbal functions. The 16 individuals with the two common mutations were grouped together (referred to as standard patients), and the 5th, 50th, and 95th centiles were calculated and graphically depicted over time. The scores for motor function and language ability dropped earliest and progressed very similarly in the standard patients. The performance curves of two children with the N286S mutation slightly diverged from the 95th centile. However, the performance curves of one patient with atypical LINCL carrying the R127Q mutation fell far beyond the 95th centile. The presented performance rating clearly and quantitatively delineates the disease course of the LINCL patients and hence offers a useful tool for clinical evaluation of future therapeutic interventions. In addition, the described performance score system can be applied to other types of neuronal ceroid lipofuscinoses and could be adapted to various other neurodegenerative diseases of childhood.

Tripeptidyl aminopeptidase I (TPP I) is a lysosomal exopeptidase that is widely distributed throughout the central nervous system (CNS) and internal organs in many mammalian species. The enzyme is involved in the breakdown of collagen and different peptides. The carotid body (CB) is the main peripheral arterial chemoreceptor playing an important role in the control of breathing and the autonomic control of cardiovascular function. In response to hypoxia its neuron-like glomus cells release a variety of peptide transmitters that trigger an action potential through the afferent fibers, thus conveying the chemosensory information to the CNS. In the present study we investigated the histochemical localization of TPP I in the CB of rats. Enzyme histochemistry showed high activity of TPP I in CB glomeruli. In particular, the glomus cells contained many TPP I-positive granules, while the glial-like sustentacular cells displayed a slightly fainter reaction. The interglomerular connective tissue was also weakly stained. The results show that both the parenchymal cells of the rat CB express, albeit with different intensity, TPP I. Taken together with our previous enzyme histochemical investigations on the rat CB, it seems likely that the glomus cells possess enzymatic equipment necessary for the neuropeptide intracellular and collagen extracellular initial degradation. These findings also suggest that TPP I is involved in the general turnover of chemotransmitters between glomus cells and sensory nerve endings which emphasizes its importance for chemoreception under hypoxic conditions.

Use of fusion protein tags to investigate lysosomal proteins can be complicated by the acidic, protease-rich environment of the lysosome. Potential artifacts include degradation or release of the tag and acid quenching of fluorescence. Tagging can also affect protein folding, glycosylation and/or trafficking. To specifically investigate the use of fluorescent tags to reveal lysosomal localization, we tested mCherry derivatives as C-terminal tags for Niemann-Pick disease type C protein 2 (NPC2), a luminal lysosomal protein. Full-length mCherry was released from the NPC2 chimera while deletion of the 11 N-terminal residues of mCherry generated a cleavage-resistant (cr) fluorescent variant. Insertion of proline linkers between NPC2 and crmCherry had little effect while Gly-Ser linkers promoted cleavage. The NPC2-crmCherry fusion was targeted to the lysosome and restored function in NPC2-deficient cells. Fusion of crmCherry to known and candidate lysosomal proteins revealed that the linkers had different effects on lysosomal localization. Direct fusion of crmCherry impaired mannose 6-phosphorylation and lysosomal targeting of the lysosomal protease tripeptidyl peptidase I (TPP1), while insertion of linkers corrected the defects. Molecular modeling suggested structural bases for the effects of different linkers on NPC2 and TPP1 fusion proteins. While mCherry fusion proteins can be useful tools for studying the lysosome and related organelles, our findings underscore the potential artifacts associated with such applications.

A lysosomal pepstatin-insensitive proteinase (CLN2p) deficiency is the underlying defect in the classical late-infantile neuronal ceroid lipofuscinosis (LINCL, CLN2). The natural substrates for CLN2p and the causative factors for the neurodegeneration in this disorder are still not understood. We have now purified the CLN2p from bovine brain to apparent homogeneity. The proteinase has a molecular mass of 46 kDa and an aminoterminal sequence, L-H-L-G-V-T-P-S-V-I-R-K, that is identical to the human enzyme. Peptide:N-glycosidase F and endoglycosidase H treatment of the CLN2p reduced its molecular mass to 39.5 and 40.5 kDa, respectively, suggesting the presence of as many as five N-glycosylated residues. The CLN2p activity was not affected by common protease inhibitors, and thiol reagents, metal chelators, and divalent metal ions had no significant effect on the proteolytic activity of the CLN2p. Among the naturally occurring neuropeptides, angiotensin II, substance P, and β-amyloid were substrates for the CLN2p, whereas angiotensin I, Leu-enkephalin, and γ-endorphin were not. Peptide cleavage sites indicated that the CLN2p is a tripeptidyl peptidase that cleaves peptides having free amino-termini. Synthetic amino- and carboxyl-terminal peptides from the subunit c sequence, which is the major storage material in LINCL, are hydrolyzed by the CLN2p, suggesting that the subunit c may be one of the natural substrates for this proteinase and its accumulation in LINCL is the direct result of the proteinase deficiency.

Tripeptidyl peptidase-I (TPP-I) is a lysosomal peptidase which cleaves tripeptides from the N-terminus of peptides. The function of the enzyme is unclear but its importance is demonstrated by the fact that mutations in TPP-I are responsible for late infantile neuronal ceroid lipofuscinosis, a lethal lysosomal storage disease. As a step towards identifying its natural substrates, we have used a series of synthetic peptides, based on angiotensin-II, to explore the effects of peptide chain length and the effects of amino acid substitutions at the P1 and P1' positions on the rate of catalysis. With the exception of angiotensin-(1-8) (angiotensin-II), which is a relatively poor substrate for TPP-I, the rate of catalysis increases with increasing chain length. K(cat)/K(m) values increase 50-fold between angiotensin-(1-5) and angiotensin-(1-14). TPP-I shows little specificity for the nature of the amino acids in the P1 and P1' positions, K(cat)/K(m) values varying only 5-fold for a range of substitutions. However, Pro or Lys in the P1 position and Pro in the P1' positions are incompatible with TPP-I activity. These observations suggest that TPP-I is a non-specific, but essential, peptidase involved in the latter stages of lysosomal protein degradation.

Batten disease, or human late-infantile neuronal ceroid lipofuscinosis (LINCL) is a familiar progressive degenerative disease affecting children, caused by a deficiency of a lysosomal proteinase (tripeptidyl peptidase I, TPP-I) and characterized by the accumulation of autofluorescent storage bodies in the brain and other tissues of the body. Current methodology used to diagnose this disease needs to be improved in order to have less invasive techniques with higher resolution and shorter assay time. In this report, we discuss the potential merits of micellar electrokinetic chromatography as an excellent tool that requires minute samples but offers high resolution and a short running time for monitoring TPP-I activity in human and animal specimens.

Tripeptidyl peptidase I (TPP I) is a lysosomal exopeptidase that cleaves tripeptides from the free N-termini of oligopeptides. Mutations in this enzyme are associated with the classic late-infantile form of neuronal ceroid lipofuscinosis (CLN2), an autosomal recessive disorder leading to severe brain damage. To gain more insight into CLN2 pathogenesis and the role of TPP I in human tissues in general, we analyzed the temporal and spatial distribution of TPP I in the brain and its localization in internal organs under normal and pathological conditions. We report that TPP I immunoreactivity appears in neurons late in gestation and increases gradually in the postnatal period, matching significantly the final differentiation and maturation of neural tissue. Endothelial cells, choroid plexus, microglial cells, and ependyma showed TPP I immunostaining distinctly earlier than neurons. Acquisition of the adult pattern of TPP I distribution in the brain at around the age of 2 years correlates with the onset of clinical signs in CLN2 subjects. In adults, TPP I was found in all types of cells in the brain and internal organs we studied, although the intensity of TPP I labeling varied among several types of cells and showed a noticeable predilection for cells and/or organs associated with peptide hormone and neuropeptide production. In addition, TPP I immunoreactivity was increased in aging brain, neurodegenerative and lysosomal storage disorders, and some differentiated neoplasms and was reduced in ischemic/anoxic areas and undifferentiated tumors. These findings suggest that TPP I is involved in general protein turnover and that its expression may be controlled by various regulatory mechanisms, which highlights the importance of this enzyme for normal function of cells and organs in humans.

The classic late infantile neuronal ceroid lipofuscinosis (LINCL, CLN2) is a fatal neurodegenerative disorder that results from mutations in a gene encoding a lysosomal proteinase, known as CLN2 protease (CLN2p) or tripeptidyl peptidase I (TPP-I). Three different substrates, fluorescein isothiocyanate-labelled haemoglobin, A-F-F-7-amino-4-methylcoumarin (AAF-AMC) and G-F-F-L-7-amino-4-trifluoromethylcoumarin (GFFL-AFC) have been used for the CLN2p/TPP-I assay with varying degrees of residual activities in patients with LINCL. Further, conclusive identification of carriers are not possible with the first two substrates. An assay for the CLN2p/TPP-I based on the cleavage of amino terminal tripeptide from G-F-F-L-AFC was applied to prenatal and postnatal diagnosis of LINCL patients and heterozygote carriers. In leukocytes, the CLN2p/TPP-I activities in controls and heterozygote carriers were 1995 +/- 154 (n = 15) and 918 +/- 253 (n = 15) nmol/h/mg protein respectively. No CLN2p/TPP-I activity was detectable in all but two patients. These two patients had less than 2% residual activity, and had delayed clinical symptoms for LINCL. This shows that the G-F-F-L-AFC is a highly specific substrate for the CLN2p/TPP-I assay. The fact that with this substrate the enzyme cleaves a peptide bond between the two amino acids may be the reason for the high level of specificity.

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative diseases that include infantile NCL (INCL; OMIM 256730, where the defective gene is CLN1 ), classical late infantile NCL (LINCL; OMIM 204500, where the defective gene is CLN2 ), two variant late infantile NCLs (OMIM 256731, where the defective gene is CLN5 ; and OMIM 601780, where the defective gene is CLN6 ), juvenile NCL (JNCL; OMIM 204200; defective gene, CLN3 ), a juvenile onset epilepsy with progressive mental retardation (EPMR; OMIM 600143; defective gene, CLN8 ), and adult NCL (Kufs disease; OMIM 204300; defective gene, CLN4 ) [reviewed in Ref. (1)]. Patients experience a progressive and profound loss of neurons in the central nervous system that generally is associated with increasingly severe epileptic seizures, visual failure, ataxia, mental deterioration, and early death. At the cellular level, the NCLs are characterized by accumulation of autofluorescent storage material in lysosome-like organelles in neurons and other cell types. Different forms of NCLs have traditionally been classified on the basis of age of onset of symptoms and cellular morphology and, more recently, by molecular criteria. Molecular analysis has revealed that different mutations in a given NCL gene can produce distinct clinical presentations [e.g., CLN2 deficiencies can lead to late infantile as well as juvenile onset disease (2), CLN1 deficiencies to infantile as well as late infantile and juvenile onset disease (3), and CLN3 deficiencies to juvenile as well as delayed onset disease (4)(5)]. Because of the existence of multiple NCL disease genes that can have overlapping clinical manifestations, simple clinical assays for NCL classification are useful before proceeding to DNA-based analysis. Enzyme-based assays are now available for diagnosis of CLN2 deficiencies. The CLN2 gene encodes a lysosomal protein (6) that has sequence similarity to bacterial pepstatin-insensitive endoproteinases (7). We …

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progressive and fatal neurodegenerative disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant sequence similarity to prokaryotic pepstatin-insensitive acid proteases. We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological samples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fibroblasts, and amniocytes). The enzyme has a pH optimum of 3.5 and is rapidly inactivated at neutral pH. A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease). A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indicates that the assay can distinguish homozygotes, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing. Analysis of archival specimens indicates that several specimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2. Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2. In addition, several animals with NCL-like neurodegenerative symptoms [mutant strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate. Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity.

Summary The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G→C transversion in the invariant AG of a 3′ splice junction, found in 38 of 115 alleles, and a C→T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg→His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

A tripeptidyl peptidase I from Dictyostelium discoideum was purified 744-fold to near homogeneity. The enzyme is 214 kDa in size and is composed of two monomers with a M(r) of 107 kDa. It has two pH optima at pH 4.5 and 5.9 and is a serine peptidase with no aminopeptidase or dipeptidyl peptidase activity. The enzyme was relatively specific showing activity on ala-ala-phe-p-nitroaniline but also acted on substrates with proline in the P1 position in contrast to mammalian TPP I. The K(m) values of the enzyme at pH 4.5 for ala-ala-phe-, ala-phe-pro- and ala-ala-pro-p-nitroanilines were 27 microM, 437 microM and 888 microM, respectively. The enzyme is most abundant during the amoeba stage of the life cycle but is present in the early stages of development and may therefore have a dual role in the organism in mobilizing amino acids or in processing specific peptides or proteins.

A recent study has shown mutations in CLN2 gene, that encodes a novel lysosomal pepstatin-insensitive proteinase (LPIP), in the pathophysiology of late-infantile neuronal ceroid lipofuscinosis (LINCL). We have measured the LPIP activities in brains from various forms of human neuronal ceroid lipofuscinoses (NCL), canine ceroid lipofuscinosis and other neurodegenerative disorders with a highly sensitive assay using a tetrapeptide Gly-Phe-Phe-Leu-amino-trifluoromethyl coumarin (AFC) as substrate. Brain LPIP has a pH optimum of 3.5 and an apparent km of 100 microM for the crude enzyme. The enzyme activity is totally absent in LINCL patients. Pronounced increase in the LPIP activity was seen in patients suffering from infantile (INCL), juvenile (JNCL) and adult (ANCL) forms of neuronal ceroid lipofuscinoses. LPIP activity was also found to be increased about two-fold in Alzheimer's disease when compared with normal or age-matched controls, while in globoidal-cell leukodystrophy (Krabbe's disease) it was similar to the normal controls. Although mannose-6-phosphorylated LPIP is increased 13-fold in brains of patients with JNCL, this form of LPIP did not have any enzyme activity. The mechanism by which LPIP activities are increased in a wide range of neurodegenerative diseases is unknown, although neuronal loss, followed by gliosis are common characteristics of these diseases.

Mutations in the CLN2 gene result in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disease. In this report, we present the complete sequence of the human CLN2 gene and define its physical relationship with two other genes that have been previously mapped to chromosome 11p15. The CLN2 gene consists of 13 exons and 12 introns and spans 6.65 kb. By S1 mapping and primer extension, the 5'-terminus of the CLN2 mRNA was mapped to 32 nucleotides upstream of the proposed initiation codon. A number of other elements were found to be located in close proximity to CLN2, including the gene encoding transcription factor TAFII30, the gene encoding intregrin-linked kinase, and an approximately 914-bp fragment that is 82% identical to antithrombin III. In addition, an EST cDNA clone that is transcribed on the strand opposite to CLN2 and that overlaps a portion of the CLN2 gene was identified. Finally, a set of primer pairs are presented for the amplification of the coding sequences, putative promoter, and splice junctions of the CLN2 gene. Taken together, this information will facilitate the molecular analysis of and genetic testing for classical LINCL.

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a recessive genetic disease of childhood caused by deficiencies in the lysosomal protease tripeptidyl peptidase I (TPP1). Disease is characterized by progressive and extensive neuronal death. One hurdle towards development of enzyme replacement therapy is delivery of TPP1 to the brain. In this study, we evaluated the effect of modifying N-linked glycans on recombinant human TPP1 on its pharmacokinetic properties after administration via tail vein injection to a mouse model of LINCL. Unmodified TPP1 exhibited a dose-dependent serum half-life of 12 min (0.12 mg) to 45 min (2 mg). Deglycosylation or modification using sodium metaperiodate oxidation and reduction with sodium borohydride increased the circulatory half-life but did not improve targeting to the brain compared to unmodified TPP1. Analysis of liver, brain, spleen, kidney and lung demonstrated that for all preparations, >95% of the recovered activity was in the liver. Interestingly, administration of a single 2 mg dose (80 mg/kg) of unmodified TPP1 resulted in ∼10% of wild-type activity in brain. This suggests that systemic administration of unmodified recombinant enzyme merits further exploration as a potential therapy for LINCL.

Tripeptidyl peptidase I (TPPI) - a lysosomal serine protease - is encoded by the CLN2 gene, mutations that cause late-infantile neuronal ceroid lipofuscinosis (LINCL) connected with profound neuronal loss, severe clinical symptoms and early death at puberty. Developmental studies of TPPI activity levels and distribution have been done in the human and rat central nervous systems (CNS) and visceral organs. Similar studies have not been performed in mouse. In this paper, we follow up on the developmental changes in the enzyme activity and localization pattern in the CNS and visceral organs of mouse over the main periods of life - embryonic, neonate, suckling, infantile, juvenile, adult and aged - using biochemical assays and enzyme histochemistry. In the studied peripheral organs (liver, kidney, spleen, pancreas and lung) TPPI is present at birth but further its pattern is not consistent in different organs over different life periods. TPPI activity starts to be expressed in the brain at the 10th embryonic day but in most neuronal types it appears at the early infantile period, increases during infancy, reaches high activity levels in the juvenile period and is highest in adult and aged animals. Thus, in mice TPPI activity becomes crucial for the neuronal functions later in development (juvenile period) than in humans and does not decrease with aging. These results are essential as a basis for comparison between normal and pathological TPPI patterns in mice. They can be valuable in view of the use of animal models for studying LINCL and other neurodegenerative disorders.

There are 35 missense mutations among 68 different mutations in the TPP1 gene, which encodes tripeptidyl peptidase I (TPPI), a lysosomal aminopeptidase associated with classic late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). To elucidate the molecular mechanisms underlying TPPI deficiency in patients carrying missense mutations and to test the amenability of mutant proteins to chemical chaperones and permissive temperature treatment, we introduced individually 14 disease-associated missense mutations into human TPP1 cDNA and analyzed the cell biology of these TPPI variants expressed in Chinese hamster ovary cells. Most TPPI variants displayed obstructed transport to the lysosomes, prolonged half-life of the proenzyme, and residual or no enzymatic activity, indicating folding abnormalities. Protein misfolding was produced by mutations located in both the prosegment (p.Gly77Arg) and throughout the length of the mature enzyme. However, the routes of removal of misfolded proteins by the cells varied, ranging from their efficient degradation by the ubiquitin/proteasome system to abundant secretion. Two TPPI variants demonstrated enhanced processing in response to folding improvement treatment, and the activity of one of them, p.Arg447His, showed a fivefold increase under permissive temperature conditions, which suggests that folding improvement strategies may ameliorate the function of some misfolding TPPI mutant proteins. Hum Mutat 31:1–12, 2010. © 2010 Wiley-Liss, Inc.

Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 angstroms resolution reveals a globular structure with a subtilisin-like fold, a Ser475-Glu272-Asp360 catalytic triad, and an octahedrally coordinated Ca2+-binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

LINCL (late-infantile neuronal ceroid lipofuscinosis) is a fatal neurodegenerative disease resulting from mutations in the gene encoding the lysosomal protease TPPI (tripeptidyl-peptidase I). TPPI is expressed ubiquitously throughout the body but disease appears restricted to the brain. One explanation for the absence of peripheral pathology is that in tissues other than brain, other proteases may compensate for the loss of TPPI. One such candidate is another lysosomal aminopeptidase, DPPI (dipeptidyl-peptidase I), which appears to have overlapping substrate specificity with TPPI and is expressed at relatively low levels in brain. Compensation for the loss of TPPI by DPPI may have therapeutic implications for LINCL and, in the present study, we have investigated this possibility using mouse genetic models. Our rationale was that if DPPI could compensate for the loss of TPPI in peripheral tissues, then its absence should exacerbate disease in an LINCL mouse model but, conversely, increased CNS (central nervous system) expression of DPPI should ameliorate disease. By comparing TPPI and DPPI single mutants with a double mutant lacking both proteases, we found that the loss of DPPI had no effect on accumulation of storage material, disease severity or lifespan of the LINCL mouse. Transgenic expression of DPPI resulted in a ∼2-fold increase in DPPI activity in the brain, but this had no significant effect on survival of the LINCL mouse. These results together indicate that DPPI cannot functionally compensate for the loss of TPPI. Therapeutic approaches to increase neuronal expression of DPPI are therefore unlikely to be effective for treatment of LINCL.

Tripeptidyl peptidase I (TPP I) is the first mammalian representative of a family of pepstatin-insensitive serine-carboxyl proteases, or sedolisins. The enzyme acts in lysosomes, where it sequentially removes tripeptides from the unmodified N terminus of small, unstructured polypeptides. Naturally occurring mutations in TPP I underlie a neurodegenerative disorder of childhood, classic late infantile neuronal ceroid lipofuscinosis (CLN2). Generation of mature TPP I is associated with removal of a long prosegment of 176 amino acid residues from the zymogen. Here we investigated the inhibitory properties of TPP I prosegment expressed and isolated from Escherichia coli toward its cognate protease. We show that the TPP I prosegment is a potent, slow-binding inhibitor of its parent enzyme, with an overall inhibition constant in the low nanomolar range. We also demonstrate the protective effect of the prosegment on alkaline pH-induced inactivation of the enzyme. Interestingly, the inhibitory properties of TPP I prosegment with the introduced classic late infantile neuronal ceroid lipofuscinosis disease-associated mutation, G77R, significantly differed from those revealed by wild-type prosegment in both the mechanism of interaction and the inhibitory rate. This is the first characterization of the inhibitory action of the sedolisin prosegment.

Gly-Pro-, Gly-Pro-Met- and Ala-Ala-Phe- N ′-(2-hexyl-1,3-dioxo-2,3-dihydro-1H-benzo[ de ]isoquinolin-6-yl)-hydrazides are synthesized by guanidinium/uronium type condensing reagent and used as fluorogenic substrates to localize dipeptidyl peptidase IV and tripeptidyl peptidase I activities in mammalian tissue sections. Enzyme hydrolysis releases 2-hexyl-6-hydrazino-1H-benzo[ de ]isoquinoline-1,3(2H)-dione, which couples with piperonal to form insoluble fluorescent hydrazone, precipitating on the enzyme locations and marking them. The fluorescent technique reveals precisely the enzymes locations at the lack of background noise in a single incubation step. It avoids most of the drawbacks of the previously proposed fluorescent histochemical techniques and can be valuable for the in situ studies of these enzymes in norm and pathology.

The lysosomal lumen contains numerous acidic hydrolases involved in the degradation of carbohydrates, lipids, proteins, and nucleic acids, which are basic cell components that turn over continuously within the cell and/or are ingested from outside of the cell. Deficiency in almost any of these hydrolases causes accumulation of the undigested material in secondary lysosomes, which manifests itself as a form of lysosomal storage disorder (LSD). Mutations in tripeptidyl-peptidase I (TPP I) underlie the classic late-infantile form of neuronal ceroid lipofuscinoses (CLN2), the most common neurodegenerative disorders of childhood. TPP I is an aminopeptidase with minor endopeptidase activity and Ser475 serving as an active-site nucleophile. The enzyme is synthesized as a highly glycosylated precursor transported by mannose-6-phosphate receptors to lysosomes, where it undergoes proteolytic maturation. This review summarizes recent progress in understanding of TPP I biology and molecular pathology of the CLN2 disease process, including distribution of the enzyme, its biosynthesis, glycosylation, transport and activation, as well as catalytic mechanisms and their potential implications for pathogenesis and treatment of the underlying disease. Promising data from gene and stem cell therapy in laboratory animals raise hope that CLN2 will be the first neurodegenerative LSD for which causative treatment will become available for humans.

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1′ and P2′ residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.