Specific substrate for CLN2 protease/tripeptidyl-peptidase I assay.

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.

Biochemistry of neuronal ceroid lipofuscinoses.

This chapter summarizes the recent advances that have been made with respect to biochemical characterization of the neurodegenerative diseases collectively known as neuronal ceroid lipofuscinoses (NCL) or Batten disease. Genomic and proteomic approaches have presently identified eight different forms of NCL (namely, CLN1 through CLN8) based on mutations in specific genes. CLN1 and CLN2 are caused by mutations in genes that encodes lysosomal enzymes,palmitoyl protein thioesterase and pepstatin-insensitive proteinase, respectively. The protein involved in the etiology of CLN3 is a highly hydrophobic, presumably transmembrane protein. NCL are considered as lysosomal storage diseases because of the accumulation of autofluorescent inclusion bodies. The composition of inclusion bodies varies in different forms of the NCL. The major storage component in CLN2 is the subunit c of mitochondrial ATP synthase complex and its accumulation is the direct result of lack of CLN2p in this disease. Mannose-6-phosphorylated glycoproteins accumulate in CLN3 and most likely their accumulation is the result of an intrinsic activity of the CLN3 protein. Significant levels of oligosaccharyl diphosphodolichol also accumulate in CLN3 and CLN2, whereas lysosomal sphingolipid activator proteins (saposins A and D) constitute major component of the storage material in CLN 1. The issue of selective loss of neuronal and retinal cells in NCL still remains to be addressed. Identification of natural substrates for the various enzymes involved in NCL may help in the characterization of the cytotoxic factor(s) and also in designing rationale therapeutic interventions for these group of devastating diseases.

Proteomic approach for the elucidation of biological defects in autism.

Proteomic-based approaches, which examine expressed proteins in tissues or cells, have great potential in the elucidation of biological defects in heterogeneous neurodevelopmental disorders such as autism. In this approach, tissue or cellular proteins from control and affected subjects are separated on two-dimensional (2-D) polyacrylamide gel electrophoresis, and those proteins that show marked changes in the concentration between control and affected subjects are identified by mass spectroscopy. This method has been successfully applied in the elucidation of the molecular biological defect in classic late-infantile neuronal ceroid lipofuscinosis (Sleat et al., 1997). Unlike the classical methods of genome-wide screening for chromosomal localization followed by positional cloning, the proteomic approach requires limited number of tissue samples and the study can be completed in a relatively short time. Currently, these methods are available for relatively abundant proteins and generally are not applicable for hydrophobic proteins because 2-D gel electrophoresis is not very effective in the analysis of hydrophobic proteins. The genetic defect results in either total loss of proteins or changes in molecular weight and/or isoelectric point will be detectable by the proteomic method. Because autism is a neurogenetic disorder, brain is the tissue of choice for proteomic study. For an oligogenic disorder such as autism, at least some of the aberrant (genes) proteins may be identified by this technology.

Retinoic acid reduces the yield of herpes simplex virus in Vero cells and alters the N-glycosylation of viral envelope proteins.

Treatment of Vero cells with all-trans-retinoic acid (RA) decreased the production of infectious herpes simplex virus (HSV) by 1000-10000-fold when compared with control cultures. Levels of total HSV envelope glycoproteins gB, gC and gD produced following RA treatment, were comparable with those found in control cultures. Following 24 h of RA treatment, lower molecular weight variants of gB, gC and gD were produced in addition to the typical molecular mass of each protein found in control samples. Between 24 and 48 h of RA treatment, the proportion of the lower molecular mass variants increased. When control and RA treated samples were incubated with peptide N-glycosidase F (PNGase F), which removes N-glycosylated sugars, the molecular weights of the respective gB, gC and gD proteins produced were comparable in both the groups, indicating that RA did not alter the primary sequence of viral proteins during protein synthesis or increase viral protein proteolysis. RA treatment increased [3H]mannose incorporation into glycoproteins in HSV infected cells but did not change [3H]glucosamine incorporation. We conclude that RA treatment does not reduce the synthesis of three major viral envelope glycoproteins but alters their N-glycosylation and postulate that the inhibitory effect of RA is related to its action on N-glycosylation.

A lysosomal pepstatin-insensitive proteinase as a novel biomarker for breast carcinoma.

Lysosomal proteinases play an important role in the turnover of intracellular proteins, and acidic proteinases such as cathepsin D are known to be increased in breast carcinoma. In the present study the activity of a newly discovered acidic lysosomal pepstatin-insensitive proteinase (CLN2p) was measured in breast tissues by the most sensitive and highly specific assay that we had developed for the diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) (2). Samples from eight normal subjects undergoing reductive mammoplasty and 200 patients with primary breast carcinoma were analyzed. The results suggest a two- to seventeen-fold higher CLN2p activity in tumors, which was significantly and positively correlated with already known breast cancer biomarkers such as levels of cathepsin D, estrogen receptor and progesterone receptor. These results suggest a diagnostic and prognostic potential for this novel acid proteinase in breast cancer.

Purification and characterization of bovine brain lysosomal pepstatin-insensitive proteinase, the gene product deficient in the human late-infantile neuronal ceroid lipofuscinosis.

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 beta-amyloid were substrates for the CLN2p, whereas angiotensin I, Leu-enkephalin, and gamma-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.

Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected].

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease.

Increased brain lysosomal pepstatin-insensitive proteinase activity in patients with neurodegenerative diseases.

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.

Mutational analysis of the defective protease in classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder.

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 novel assay for lysosomal pepstatin-insensitive proteinase and its application for the diagnosis of late-infantile neuronal ceroid lipofuscinosis.

A highly sensitive assay for mammalian lysosomal pepstatin-insensitive proteinase (LPIP) is described using a synthetic peptide substrate coupled to aminotrifluoromethyl coumarin (AFC). LPIP is an endocarboxyl proteinase which has specific sequence requirements of Phe-Phe around the carboxyl terminal. This HPLC based assay can detect patients suffering from late-infantile neuronal ceroid lipofuscinosis (LINCL) and also heterozygote carriers in cultured lymphoid cells and skin fibroblasts. None of the patients analyzed had detectable enzyme activity confirming the defective gene product, while carriers had about 50% activity when compared with the normal controls. Neurological controls comprised of patients with other neurodegenerative disorders have LPIP activities similar to normal controls. LPIP activity is also detectable in amniocytes and chorionic villi. Thus the assay reported can also be used for prenatal diagnosis of LINCL.

Clinical, pathologic, and neurochemical studies of an unusual case of neuronal storage disease with lamellar cytoplasmic inclusions: a new genetic disorder?

A child of first-cousin Puerto Rican parents had global developmental delay, failure to thrive, and hypotonia since early infancy. At 1 1/2 years of age, she developed clinical and electrophysiologic evidence of progressive motor and sensory neuropathy. At 2 1/2 years, she developed visual impairment and optic atrophy followed by gradual involvement of the 7th, 9th, 10th, and 12th cranial nerves. Uncontrollable myoclonic seizures began at 4 years and she died at 6 years of age. Motor nerve conduction velocities were initially normal and later became markedly slowed. Sensory distal latency responses were absent. Lysosomal enzyme activities in leukocytes and fibroblasts were normal. Sural nerve and two muscle biopsies showed only nondiagnostic abnormalities. Electron microscopy of lymphocytes, skin, and fibroblasts showed cytoplasmic inclusions. Light microscopy of frontal cortex biopsy showed neuronal storage material staining positively with Luxol fast blue, and electron microscopy showed cytoplasmic membranous bodies in neurons, suggesting an accumulation of a ganglioside. At autopsy, all organs were small but otherwise normal and without abnormal storage cells in the liver, spleen, or bone marrow. Anterior spinal nerve roots showed loss of large myelinated axons. The brain was small and atrophic; cortical neurons showed widespread accumulation of storage material, most marked in the pyramidal cell layer of the hippocampus. Subcortical white matter was gliotic with loss of axons and myelin sheaths. In cortical gray matter there was a 35% elevation of total gangliosides, with a 16-fold increase in GM3, a three- to four-fold increase in GM2 gangliosides, and a 15-fold elevation of lactosyl ceramide. GM3 sialidase activity was normal in gray matter at 3.1 nmols/mg protein per hour and lactosyl ceraminidase I and II activities were 70% to 80% of normal. In white matter, total myelin was reduced by 50% but its composition was normal. Phospholipid distribution and sphingomyelin content were normal in gray matter, white matter, and in the liver. These biochemical findings were interpreted as nonspecific abnormalities. The nature of the neuronal storage substance remains to be determined.

Glycosylation and palmitoylation are not required for the formation of the X-linked cone opsin visual pigments.

PURPOSE: This study was designed to test whether palmitoylation and glycosylation are required for the formation of the green opsin visual pigment. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a pMEP4ss recombinant plasmid containing wild-type bovine rhodopsin or wild-type or mutant (N32S) green opsin cDNA molecules that included a tag for the eight amino acid residues located at the C-terminus of rhodopsin. The opsins were induced by addition of CdCl2 into the medium and then reconstituted with 11-cis-retinal. The reconstituted opsins were purified by immunoaffinity chromatography, then analyzed by difference spectra, and by binding 35S-GTP in the presence of bovine transducin. Non-reconstituted opsins were analyzed by Western blotting and by pulse-labeling with 3H-palmitic acid followed by immunoprecipitation. RESULTS: Elimination of glycosylation by mutagenesis of the N-linked glycosylation site did not impair the ability of the resulting cone opsin to absorb light at the appropriate wavelength nor to activate transducin. Furthermore, as judged by pulse-labeling with 3H-palmitic acid and immunoprecipitation and by gas chromatography-mass spectroscopy, the wild type green opsin differs from rhodopsin by not being palmitoylated. CONCLUSIONS: Glycosylation and palmitoylation are not required for the formation of cone opsin visual pigments. For the previously described green opsin C203R mutation, disruption of folding and transport, rather than altered glycosylation is sufficient to explain the associated color vision deficiency.

Specific alterations in levels of mannose 6-phosphorylated glycoproteins in different neuronal ceroid lipofuscinoses.

Mannose 6-phosphate (Man-6-P) is a carbohydrate modification that is generated on newly synthesized lysosomal proteins. This modification is specifically recognized by two Man-6-P receptors that direct the vesicular transport of the lysosomal enzymes from the Golgi to a prelysosomal compartment. The Man-6-P is rapidly removed in the lysosome of most cell types; however, in neurons the Man-6-P modification persists. In this study we have examined the spectrum of Man-6-P-containing glycoproteins in brain specimens from patients with different neuronal ceroid lipofuscinoses (NCLs), which are progressive neurodegenerative disorders with established links to defects in lysosomal catabolism. We find characteristic alterations in the Man-6-P glycoproteins in specimens from late-infantile (LINCL), juvenile (JNCL) and adult (ANCL) patients. Man-6-P glycoproteins in LINCL patients were similar to controls, with the exception that the band corresponding to CLN2, a recently identified lysosomal enzyme whose deficiency results in this disease, was absent. In an ANCL patient, two Man-6-P glycoproteins were elevated in comparison with normal controls, suggesting that this disease also results from a perturbation in lysosomal hydrolysis. In JNCL, total levels of Man-6-P glycoproteins were 7-fold those of controls. In general this was reflected by increased lysosomal enzyme activities in JNCL but three Man-6-P glycoproteins were elevated to an even greater degree. These are CLN2 and the unidentified proteins that are also highly elevated in the ANCL.

Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

Farnesylation of Batten disease CLN3 protein.

The carboxyl terminal of the predicted amino acid sequence of the Batten disease CLN3 gene protein is CQLS. This motif is expected to be a site for farnesylation at the cysteine residue. In order to determine whether this is indeed farnesylated we have carried out the in-vitro prenylation of tetrapeptides CVLS, CAIL and CQLS using a farnesyl transferase preparation from bovine brain. The data shows that the CQLS is a good acceptor of a farnesyl group similar to CVLS while it is a poor acceptor of a geranylgeranyl group unlike CAIL, which is a good acceptor of a geranylgeranyl group. This suggests that the CLN3 gene product may be a farnesylated protein.

Inhibition of herpes simplex virus replication by retinoic acid.

The retinoic acid (RA) isomers all-trans-RA, 9-cis-RA and 13-cis-RA as well as other retinoids were tested for their ability to reduce the yield of herpes simplex virus-1 (HSV-1). RA isomers reduced HSV-1 replication whereas the other retinoids, retinol, retinal, beta-carotene and amide derivatives of RA were not inhibitory. All-trans-RA reduced the yield of HSV-1 by 100-fold at 5 micrograms/ml but 9-cis-RA and 13-cis-RA reduced viral replication by 10-fold. At a concentration of 10 micrograms/ml all-trans-RA and 9-cis-RA reduced virus yield by 1000-fold while 13-cis-RA decreased HSV-1 production by 100-fold. RA isomers at a concentration of 10 micrograms/ml were not cytotoxic for the Vero cells used in these studies. Immunofluorescence studies showed that all-trans-RA treated cell cultures exhibited small foci of virus specific immunostaining while untreated cultures displayed intense HSV-1 immunoreactivity in virtually the entire cell population. RA-dependent inhibition of HSV-1 replication required the presence of RA with the virus. HSV-1 replication proceeded when RA was removed from infected cells. Treatment of cell cultures with RA did not induce gene expression for type-1 interferon (IFN) or for the type-1 IFN inducible genes studied suggesting that RA inhibition of HSV-1 replication is not mediated by IFN. These studies have established the ability of RA to reduce the replication of HSV-1 in vitro.

Brain lysosomal hydrolases in neuronal ceroid-lipofuscinoses.

Although the neuronal ceroid-lipofuscinoses (NCLs) are often referred to as lysosomal storage disorders, information on brain lysosomal hydrolases in NCLs is not available. We have determined the specific activities of several acid hydrolases in postmortem brain gray matter of infantile (INCL), late infantile (LINCL), juvenile (JNCL), and adult (ANCL) forms of NCL, patients affected with other neurological disorders (ON), and normal controls. The specific activities of beta-hexosaminidase A and B were significantly high in JNCL gray matter, whereas in LINCL, the increase is significant only in beta-hexosaminidase compared to the controls. A significant increase in the activities of alpha-mannosidase, beta-glucuronidase, and acid phosphatase was also observed in LINCL and JNCL patients compared to the control values. beta-galactosidase activity was also found to be elevated in JNCL brains over the controls. In contrast, activities of beta-glucosidase and sialidase appeared to be lowered in INCL and LINCL. On the other hand, alpha-fucosidase, beta-mannosidase, and sulfatase were unaffected in NCLs brains. Thus, the present data indicate NCLs related abnormalities in some of the acid hydrolases in brain gray matter, which are primarily glycoproteins of lysosomal origin. These data in conjuction with the reported association of sphingolipid activator proteins (SAP) A and D and lysosomal glycoproteins with NCL storage bodies imply abberations in the glycoconjugate metabolism and lysosomal function.

Abnormal acid phosphatases in neuronal ceroid-lipofuscinoses.

Acid phosphatases in brain and cultured lymphoblasts from patients affected with neuronal ceroid-lipofuscinoses (NCL) were studied by starch gel electrophoresis. After electrophoresis the gel was incubated with 4-methyl umbelliferyl phosphate at pH 4.5 and the fluorescent reaction product was visualized under ultraviolet light. Control brain showed a single band with mobility of about 1 cm while NCL patients showed two additional fast moving bands. In the late-infantile, and in the adult form (Kufs disease), the middle band was prominent while the fast moving band was predominant in juvenile NCL. In long-term lymphoblasts, controls showed a single band of acid phosphatase activity while both juvenile and late-infantile NCL showed two additional fast moving bands. Obligate heterozygotes showed reduced levels of the fast moving bands. Fluorometric assay of acid phosphatase using 4-methylumbelliferyl phosphate as substrate showed a 2-fold increase in activity in the patients. The increased acid phosphatase activity is completely inhibited by tartrate. Lymphocyte hexosamnidase activities were unchanged in NCL patients lymphoblasts. Studies on brains of NCL patients and on cultured lymphoblasts from families with late-infantile and juvenile form of NCL showed that abnormal acid phosphatase is characteristic of NCL.