Identification of HE1 as the second gene of Niemann-Pick C disease.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles.

The ribonucleocapsids of many plant viruses are extremely stable. The protein coat protects the RNA genome against degradation during the accumulation and spread of progeny virions. Chimeric single-stranded RNA molecules were transcribed in vitro from recombinant plasmids and later encapsidated, in vitro, into ribonucleoprotein particles (pseudoviruses) 60 nanometers long that resembled tobacco mosaic virus. Transcripts encoding an assayable enzyme, chloramphenicol acetyltransferase (CAT), were packaged into pseudovirus particles to assess the utility of this single-stranded RNA delivery system in a wide range of cell types. In all cases, packaged CAT messenger RNA was uncoated and transiently expressed. Significantly higher levels of CAT activity were detected with packaged than with naked CAT messenger RNA after inoculation of plant protoplasts in the presence of polyethylene glycol or abrasive inoculation of intact leaf surfaces. Structural events that lead to the uncoating and expression of CAT messenger RNA showed no cell specificity. This observation may support the view that the comparatively restricted host range of a true plant virus results from events that occur later during the infection cycle.

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.

Increased levels of glycoproteins containing mannose 6-phosphate in human breast carcinomas.

Newly synthesized enzymes destined for lysosomal localization contain mannose 6-phosphate (Man6-P) residues, allowing interaction with Man6-P receptors (MPRs) and subsequent intracellular targeting to the lysosome. In most cultured cells, lysosomal enzymes are rapidly dephosphorylated after targeting, but in some transformed cell lines, these proteins retain the Man6-P marker. To investigate the significance of this in human malignancy, we examined the persistence of the Man6-P marker in human breast biopsy specimens using MPR derivatives as affinity probes. In one approach, extracts of frozen tissue were standardized to protein content, fractionated by SDS-PAGE, immobilized on nitrocellulose, and probed with iodinated MPR. On average, carcinomas contained 4-fold higher levels of Man6-P glycoproteins than did benign tumors or normal breast samples. In about 15% of the carcinomas, levels of Man6-P glycoproteins were highly elevated (7-10-fold). Multiple Man6-P glycoproteins were detected, suggesting a general alteration in the synthesis or processing of many lysosomal enzymes in carcinomas. In a second approach, sections of formalin-fixed breast biopsy specimens were probed with biotinylated MPR. Malignant cells in 25 of 75 carcinomas exhibited granular cytoplasmic staining in what appears to be intracellular vesicles. Staining was specifically inhibited by Man6-P and was not observed in stromal components or lymphocytes. In addition, Man6-phosphorylated proteins were not detected in the 14 normal or benign biopsy samples examined. Staining appeared to be independent of most prognostic factors examined, including p53, cathepsin D, DNA ploidy, and hormone (estrogen and progesterone) receptor status. However, positive staining was significantly associated with high histological and nuclear grades (P < 0.05) and potentially with c-erbB-2 (P < 0.10), suggesting that elevated levels of Man6-P glycoproteins are associated with the more aggressive tumors.

Mapping determinants within cucumber mosaic virus and its satellite RNA for the induction of necrosis in tomato plants.

A mutant of the WL47 satellite (WL47-sat) RNA of cucumber mosaic virus (CMV), constructed in vitro, induces lethal necrosis in tomato plants when associated with either of two subgroup II strains of CMV: LS- or WL-CMV (D. E. Sleat and P. Palukaitis, Proc. Natl. Acad. Sci. USA 87:2946-2950, 1990). The phenotype of this mutant (WLM2-sat RNA) has been examined on tomato plants, after co-inoculation with a wider range of CMV strains. Necrosis was observed when WLM2-sat RNA was associated with any of the subgroup II CMV strains tested. However, WLM2-sat RNA ameliorated the symptoms induced by subgroup I CMV strains without inducing necrosis. Inoculation with WLM2-sat RNA with pseudorecombinants formed between subgroup I and subgroup II CMV strains showed an association of the necrosis induction phenotype with RNA 2 of the subgroup II strains. In contrast to WLM2-sat RNA, infectious transcripts of the naturally necrogenic D4-sat RNA induced lethal necrosis in tomato with all CMV strains tested. Experiments involving the exchange of sequences between WLM2- and D4-sat RNAs indicated that nucleotides sequences in either of two separate regions could influence the necrosis phenotype. Thus, the subgroup-specific necrosis-inducing phenotype may be due to subtle alterations in secondary and/or tertiary structure in the satellite RNA, as well as the presence or absence of particular nucleotide sequences.

Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro.

Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA. Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E. coli (EC). The omega' sequence enhanced expression of each mRNA in almost every translation system. While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega'. The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence. These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

Aminoglycoside-mediated suppression of nonsense mutations in late infantile neuronal ceroid lipofuscinosis.

The ability of aminoglycoside antibiotics to promote readthrough of eukaryotic stop codons has attracted interest in these drugs as potential therapeutic agents in human disorders caused by nonsense mutations. One disease for which such a therapeutic strategy may be viable is classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disorder with currently no effective treatment. Premature stop codon mutations in the gene CLN2 encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with disease in approximately half of children diagnosed with LINCL. The aim of this study was to examine the ability of the aminoglycoside gentamicin to restore TPP-I activity in LINCL cell lines. In one patient-derived cell line that was compound heterozygous for a commonly seen nonsense mutation, Arg208Stop and a different rare nonsense mutation, approximately 7% of normal levels of TPP-I were maximally restored with gentamicin treatment. In other cell lines from patients that were compound heterozygous for Arg208Stop and a splice junction mutation, approximately 0.5% of maximal activity was restored. These results suggest that pharmacological suppression of nonsense mutations by aminoglycosides or functionally similar pharmaceuticals may have therapeutic potential in LINCL.

Congenital ovine neuronal ceroid lipofuscinosis--a cathepsin D deficiency with increased levels of the inactive enzyme.

We recently showed that a form of neuronal ceroid lipofuscinosis (NCL) in white Swedish landrace sheep is caused by a missense mutation in the cathepsin D gene resulting in complete inactivation of the enzyme. Despite the lack of cathepsin D activity, the brains of the cathepsin D deficient sheep showed strongly increased staining for cathepsin D in immunohistochemistry. By Western blotting, a 5-10 fold increase in the level of cathepsin D was confirmed. These results indicate that the missense mutation in congenital NCL sheep results in the synthesis of an inactive yet stable cathepsin D.

Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase.

The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) is the multifunctional catalytic component of the viral RNA polymerase. A protocol has been developed for the synthesis of recombinant L protein that will support viral mRNA synthesis in vitro. COS cells were transfected with a transient expression vector (pSV-VSL1 [M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985]) which contains the simian virus 40 late promoter for the transcription of a cDNA copy of the L protein of the Indiana serotype of VSV. Cytoplasmic extracts of these cells efficiently transcribed VSV mRNAs in vitro in conjunction with N protein-RNA template purified from virus and recombinant phosphoprotein synthesized in Escherichia coli. mRNA synthesis was completely dependent upon addition of both bacterial phosphoprotein and extracts from cells transfected with the L gene. Extracts from mock-transfected cells or from cells transfected with the expression vector alone did not support VSV RNA synthesis. RNA synthesis was proportional to the concentration of cell extract used, with an optimum of 0.2 mg/ml. Rhabdoviruses and paramyxoviruses contain a highly conserved GDNQ motif which was mutated in the transfected L gene. All constructs with mutations within the core GDN abrogated transcriptional activity except for the mutant containing GDD, which retained 25% activity. Conserved amino acid changes outside of the core GDN and changes corresponding to other paromyxovirus and rhabdovirus L proteins retained variable transcriptional activity. These findings provide experimental evidence that the GDN of negative-strand, nonsegmented RNA viruses is a variant of the GDD motif of plus-strand RNA viruses and of the XDD motif of DNA viruses and reverse transcriptases.

A single nucleotide change within a plant virus satellite RNA alters the host specificity of disease induction.

Some RNA plant viruses contain satellite RNAs which are dependent upon their associated virus for replication and encapsidation. Some cucumber mosaic virus satellite RNAs induce chlorosis on any of several host plants, including either tobacco or tomato. The exchange of sequence domains between cDNA clones of chlorosis-inducing and non-pathogenic satellite RNAs delimited the chlorosis domain for both tobacco and tomato plants to the same region. Site-directed mutagenesis of one nucleotide (149) within this domain changed the host plant specificity for a chlorotic response to satellite RNA infection from tomato to tobacco. Within the chlorosis domain, three conserved nucleotides are either deleted or altered in all satellite RNAs that do not induce chlorosis. Deletion of one of these nucleotides (153) did not affect satellite RNA replication but rendered it non-pathogenic. Thus, two single nucleotides have been identified which play central roles in those interactions between the virus, its satellite RNA and the host plant, and together result in a specific disease state.

Ligand binding specificities of the two mannose 6-phosphate receptors.

Two mannose 6-phosphate (Man-6-P) receptors (MPRs) direct the vesicular transport of newly synthesized lysosomal enzymes that contain Man-6-P from the Golgi to a prelysosomal compartment. In order to understand the respective roles of the Mr = 46,000 cation-dependent (CD-) MPR and the Mr = 300,000 cation-independent (CI-) MPR in lysosomal targeting, an assay has been developed that simultaneously measures the relative affinity of each MPR for multiple ligands. Glycoproteins containing Man-6-P were affinity-purified from the metabolically labeled secretions of mutant mouse fibroblasts lacking both MPRs. They were incubated with purified MPRs, and the resulting receptor-ligand complexes were immunoprecipitated by anti-MPR monoclonal antibodies coupled to agarose beads. Ligands were eluted with Man-6-P and then quantified following SDS-polyacrylamide gel electrophoresis. Saturating concentrations of CI-MPR resulted in the complete recovery of each Man-6-P glycoprotein in receptor-ligand complexes. Apparent affinity constants ranged between 1 and 5 nM for the individual species. Ligands precipitated by the CD-MPR appeared identical to those bound by the CI-MPR, with apparent affinity constants ranging between 7 and 28 nM. The binding affinities of the two receptors for different ligands were not correlated, indicating that the two MPRs preferentially recognize different subsets of lysosomal enzymes. In addition, saturating levels of CD-MPR resulted in the precipitation of only 50% of the total input ligands, suggesting that the CD-MPR binds a subpopulation of the Man-6-P glycoproteins bound by the CI-MPR. These results provide a biochemical mechanism, which, in part, may explain the interaction of the two MPRs with overlapping yet distinct subsets of ligands in vivo.

Induction of tobacco chlorosis by certain cucumber mosaic virus satellite RNAs is specific to subgroup II helper strains.

Two satellite (sat) RNAs of cucumber mosaic virus (CMV), B2- and WL3-sat RNAs, which induce systemic chlorosis on tobacco, were inoculated onto tobacco with a number of CMV strains. Systemic chlorosis was observed only when these satellite RNAs were associated with subgroup II CMV strains. Infection of tobacco with various pseudorecombinants of subgroup I and II CMV strains, together with WL3- or B2-sat RNA, suggests that chlorosis is associated with RNA 2 of subgroup II CMV strains. Chlorosis was not induced when B2- or WL3-sat RNAs were inoculated onto tobacco with tomato aspermy virus. In contrast, the induction of chlorosis on tomato by B1-sat RNA did not show any clear dependence on the subgroup of its CMV helper strain although chlorosis did tend to be more severe in association with subgroup II CMV strains.

Site-directed mutagenesis of a plant viral satellite RNA changes its phenotype from ameliorative to necrogenic.

Comparison of the nucleotide sequences of cucumber mosaic virus (CMV) satellite RNAs, which induce necrosis on tomatoes, reveals a highly conserved region within their 3' halves. The sequence of WL1 satellite (WL1-sat) RNA, which attenuates CMV symptoms on tomatoes, differs from all necrogenic satellite RNAs at three nucleotide positions within this conserved region. These nucleotides were progressively mutated to determine what sequence is required for the induction of necrosis in tomatoes. Infectious transcripts from a cDNA clone of WL1-sat RNA, and its mutated derivatives, were assayed on tomato. Three of the four mutant satellite RNAs, in which two of the three nucleotides correspond to those present in necrogenic satellite RNAs, and the rest of the molecule corresponded to WL1-sat RNA, attenuated CMV symptoms on tomatoes, and were phenotypically identical to the parental WL1-sat RNA. One other mutant satellite RNA, in which all three mutated nucleotides corresponded to the sequence of necrogenic satellite RNAs and the rest of the molecule corresponded to WL1-sat RNA, induced a lethal necrosis on tomatoes. Necrosis was also observed when the same mutant satellite RNA was associated with a second CMV strain as helper virus. These results indicate that a single nucleotide change at any of the three nucleotides examined distinguishes necrogenic from nonnecrogenic satellite RNAs. This necrosis was similar to that induced by a naturally necrogenic satellite RNA. The various mutations did not modify the effect of WL1-sat RNA on the symptoms induced by CMV on tobacco or squash.

Selective encapsidation of CAT gene transcripts in TMV-infected transgenic tobacco inhibits CAT synthesis.

Young tobacco seedlings (F1-progeny), transformed to express chloramphenicol acetyltransferase (CAT) mRNA with or without a 3'-proximal copy of the origin-of-assembly sequence (OAS) from tobacco mosaic virus (TMV) RNA (residues 5118-5550), were inoculated with TMV. After 21 days, virus symptoms were observed and systemic TMV infections were confirmed by Western blotting for viral coat protein and by electron microscopy of leaf saps. CAT activities were measured in extracts of leaf discs taken before, and 21 days after, virus inoculation. On average, the systemic leaves from TMV-infected CAT-transgenic plants containing the OAS exhibited 3.2-fold less CAT activity than the equivalent leaves from CAT-transgenic control plants lacking the OAS. Hence selective, OAS-dependent encapsidation of nuclear DNA transcripts into TMV-like (pseudovirus) particles can reduce expression of a particular mRNA, post-transcriptionally, in vivo. Furthermore, these data indicate that TMV self-assembly is not restricted to an exclusive subcellular compartment in vivo, and that formation of natural pseudovirions (A. Siegel, Virology 46, 50-59 (1971)) may shut off specific host RNA functions.

Biochemical characterization of a lysosomal protease deficient in classical late infantile neuronal ceroid lipofuscinosis (LINCL) and development of an enzyme-based assay for diagnosis and exclusion of LINCL in human specimens and animal models.

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.

Studies on the mechanism of translational enhancement by the 5'-leader sequence of tobacco mosaic virus RNA.

Translation of foreign mRNAs is enhanced by a cis-acting derivative (omega') of the 5'-leader sequence (omega) of tobacco mosaic virus RNA (vulgare strain). To explain this effect we have conducted several experiments in vitro. 1. The presence of various 5'-terminal sequences, including omega', did not significantly increase the half-lives of chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII) mRNAs in wheat-germ extract. Also, a long leader sequence, unrelated to omega', did not enhance expression of NPTII mRNA in vitro. 2. The ability of several leader sequences, including omega', to form multiple initiation complexes with 80S (wheat germ) ribosomes was examined using CAT or NPTII mRNAs incubated in the presence of sparsomycin. Formation of disome complexes was unrelated to the capacity of a 5'-leader sequence to enhance translation. 3. Expression of CAT mRNA in both wheat germ extract and messenger-dependent rabbit reticulocyte lysate was less susceptible to inhibition by increasing salt concentration when a 5'-proximal omega' sequence was present. This effect was less marked when the CAT mRNA was capped. Conversely at high salt concentrations, capping was less stimulatory for mRNA with a 5'-proximal omega' sequence. These data suggest that omega' and the cap enhance translation, at least in part, by a similar mechanism. We propose that both features reduce RNA secondary structure, thereby rendering the 5' terminus more accessible to scanning by 40S ribosomal subunits and/or interaction with associated initiation factors. This conclusion was supported by computer-based secondary-structure analyses of our SP6 RNA polymerase transcript sequences. The ability of 5' leader sequences from brome mosaic virus RNA 3, alfalfa mosaic virus RNA 4, and the genomic RNAs of turnip yellow mosaic virus, Rous sarcoma virus or tobacco mosaic virus (tomato strain) to enhance mRNA translation in eukaryotic systems may also be correlated with their respective secondary structures. A different mechanism probably accounts for the omega'-dependent enhancement of mRNA expression in Escherichia coli or in E. coli cell-free systems.

Restricted replication of vesicular stomatitis virus in T lymphocytes is coincident with a deficiency in a cellular protein kinase required for viral transcription.

Vesicular stomatitis virus (VSV) fails to replicate in mouse T lymphocytes unless the cells have been mitogenically stimulated with concanavalin A (Con A). We have examined the possibility that the failure of VSV to replicate in unstimulated T lymphocytes can be attributed to a deficiency in a host protein kinase which activates the viral P protein by phosphorylation, thus rendering it transcriptionally competent. Soluble extracts were prepared from purified mouse T lymphocytes, with or without prior treatment with Con A. The ability of these extracts to phosphorylate bacterially synthesized P protein of two VSV serotypes was measured in vitro. Activity of the protein kinase on the P proteins of the Indiana or New Jersey serotypes of VSV increased, on average 2.4- and 2.1-fold respectively, after treatment of the cells with 3 micrograms/ml Con A. Higher concentrations of Con A induced proportional increases (up to 10-fold) in the activity of the host protein kinase. Activities of the kinase phosphorylating the P protein in separate populations of CD4- and CD8-containing murine T lymphocytes increased similarly on mitogenic activation. No biochemical or immunological differences were observed between the T cell protein kinase and the previously characterized protein kinase (casein kinase II) from BHK-21 cells. The activity of the kinase that phosphorylates the P protein did not vary in CV-1 cells on treatment with alpha- or gamma-interferon, both of which inhibited VSV replication. Similarly, casein kinase II activities in Raji and SIRC cells, which do not normally support VSV growth, were the same as in BHK-21 cells. Thus restriction of VSV replication in these cells, in contrast to T lymphocytes, was not associated with a deficiency in the host casein kinase II activity.

alpha-Glucosidase and N-acetylglucosamine-6-sulphatase are the major mannose-6-phosphate glycoproteins in human urine.

Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and alpha-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were beta-mannosidase (82%), hexosaminidase (27%) and alpha-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.

Prenatal testing for late infantile neuronal ceroid lipofuscinosis.

Classic late infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative disease in which autofluorescent "curvilinear" storage bodies accumulate in tissues from affected patients. Recently, the LINCL gene (CLN2) has been found to code for a pepstatin-insensitive lysosomal protease whose activity is deficient in LINCL specimens. We report the first 2 cases of successful prenatal testing for LINCL by using DNA and enzyme-based methods on amniocytes, and describe a new private mutation in one of the families analyzed. These approaches allow definitive prenatal diagnosis and represent a significant improvement over previous pathological methods.

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.