Proteomic Analysis of Brain and Cerebrospinal Fluid from the Three Major Forms of Neuronal Ceroid Lipofuscinosis Reveals Potential Biomarkers.

Clinical trials have been conducted for the neuronal ceroid lipofuscinoses (NCLs), a group of neurodegenerative lysosomal diseases that primarily affect children. Whereas clinical rating systems will evaluate long-term efficacy, biomarkers to measure short-term response to treatment would be extremely valuable. To identify candidate biomarkers, we analyzed autopsy brain and matching CSF samples from controls and three genetically distinct NCLs due to deficiencies in palmitoyl protein thioesterase 1 (CLN1 disease), tripeptidyl peptidase 1 (CLN2 disease), and CLN3 protein (CLN3 disease). Proteomic and biochemical methods were used to analyze lysosomal proteins, and, in general, we find that changes in protein expression compared with control were most similar between CLN2 disease and CLN3 disease. This is consistent with previous observations of biochemical similarities between these diseases. We also conducted unbiased proteomic analyses of CSF and brain using isobaric labeling/quantitative mass spectrometry. Significant alterations in protein expression were identified in each NCL, including reduced STXBP1 in CLN1 disease brain. Given the confounding variable of post-mortem changes, additional validation is required, but this study provides a useful starting set of candidate NCL biomarkers for further evaluation.

Effective intravenous therapy for neurodegenerative disease with a therapeutic enzyme and a peptide that mediates delivery to the brain.

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.

Proteomic analysis of mouse models of Niemann-Pick C disease reveals alterations in the steady-state levels of lysosomal proteins within the brain.

Niemann-Pick C disease (NPC) is a neurodegenerative lysosomal disorder characterized by storage of cholesterol and other lipids caused by defects in NPC1, a transmembrane protein involved in cholesterol export from the lysosome, or NPC2, an intralysosomal cholesterol transport protein. Alterations in lysosomal activities have been implicated in NPC pathogenesis therefore the aim of this study was to conduct a proteomic analysis of lysosomal proteins in mice deficient in either NPC1 or NPC2 to identify secondary changes that might be associated with disease. Lysosomal proteins containing the specific mannose 6-phosphate modification were purified from wild-type and Npc1(-/-) and Npc2(-/-) mutant mouse brains at different stages of disease progression and identified by bottom-up LC-MS/MS and quantified by spectral counting. Levels of a number of lysosomal proteins involved in lipid catabolism including prosaposin and the two subunits of ß-hexosaminidase were increased in both forms of NPC, possibly representing a compensatory cellular response to the accumulation of glycosphingolipids. Several other lysosomal proteins were significantly altered, including proteases and glycosidases. Changes in lysosomal protein levels corresponded with similar alterations in activities and transcript levels. Understanding the rationale for such changes may provide insights into the pathophysiology of NPC.

Systemic administration of tripeptidyl peptidase I in a mouse model of late infantile neuronal ceroid lipofuscinosis: effect of glycan modification.

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.

Loss of Niemann-Pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.

Niemann-Pick Type C (NPC) disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

Large-volume intrathecal enzyme delivery increases survival of a mouse model of late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a progressive neurodegenerative lysosomal storage disorder caused by mutations in TPP1, the gene encoding the lysosomal protease tripeptidyl-peptidase (TPP1). LINCL primarily affects children, is fatal and there is no effective treatment. Administration of recombinant protein has proved effective in treatment of visceral manifestations of other lysosomal storage disorders but to date, only marginal improvement in survival has been obtained for neurological diseases. In this study, we have developed and optimized a large-volume intrathecal administration strategy to deliver therapeutic amounts of TPP1 to the central nervous system (CNS) of a mouse model of LINCL. To determine the efficacy of treatment, we have monitored survival as the primary endpoint and demonstrate that an acute treatment regimen (three consecutive daily doses started at 4 weeks of age) increases median lifespan of the LINCL mice from 16 (vehicle treated) to 23 weeks (enzyme treated). Consistent with the increase in life-span, we also observed significant reversal of pathology and improvement in neurological phenotype. These results provide a strong basis for both clinical investigation of large-volume/high-dose delivery of TPP1 to the brain via the cerebrospinal fluid (CSF) and extension of this approach towards other neurological lysosomal storage diseases.

Intrathecal tripeptidyl-peptidase 1 reduces lysosomal storage in a canine model of late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by mutations in the gene encoding tripeptidyl-peptidase 1 (TPP1). LINCL patients accumulate lysosomal storage materials in the CNS accompanied by neurodegeneration, blindness, and functional decline. Dachshunds homozygous for a null mutation in the TPP1 gene recapitulate many symptoms of the human disease. The objectives of this study were to determine whether intrathecal (IT) TPP1 treatment attenuates storage accumulation and functional decline in TPP1-/- Dachshunds and to characterize the CNS distribution of TPP1 activity. TPP1 was administered to one TPP1-/- and one homozygous wild-type (WT) dog. An additional TPP1-/- and WT dog received vehicle. Four IT administrations of 32 mg TPP1 formulated in 2.3 mL of artificial cerebrospinal fluid (aCSF) or vehicle were administered monthly via the cerebellomedullary cistern from four to seven months of age. Functional decline was assessed by physical and neurological examinations, electrophysiology, and T-maze performance. Neural tissues were collected 48 h after the fourth administration and analyzed for TPP1 activity and autofluorescent storage material. TPP1 was distributed at greater than WT levels in many areas of the CNS of the TPP1-/- dog administered TPP1. The amount of autofluorescent storage was decreased in this dog relative to the vehicle-treated affected control. No improvement in overall function was observed in this dog compared to the vehicle-treated TPP1-/- littermate control. These results demonstrate for the first time in a large animal model of LINCL widespread delivery of biochemically active TPP1 to the brain after IT administration along with a decrease in lysosomal storage material. Further studies with this model will be necessary to optimize the dosing route and regimen to attenuate functional decline.

Crystal structure and autoactivation pathway of the precursor form of human tripeptidyl-peptidase 1, the enzyme deficient in late infantile ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis is a fatal childhood neurological disorder caused by a deficiency in the lysosomal protease tripeptidyl-peptidase 1 (TPP1). TPP1 represents the only known mammalian member of the S53 family of serine proteases, a group characterized by a subtilisin-like fold, a Ser-Glu-Asp catalytic triad, and an acidic pH optimum. TPP1 is synthesized as an inactive proenzyme (pro-TPP1) that is proteolytically processed into the active enzyme after exposure to low pH in vitro or targeting to the lysosome in vivo. In this study, we describe an endoglycosidase H-deglycosylated form of TPP1 containing four Asn-linked N-acetylglucosamines that is indistinguishable from fully glycosylated TPP1 in terms of autocatalytic processing of the proform and enzymatic properties of the mature protease. The crystal structure of deglycosylated pro-TPP1 was determined at 1.85 angstroms resolution. A large 151-residue C-shaped prodomain makes extensive contacts as it wraps around the surface of the catalytic domain with the two domains connected by a 24-residue flexible linker that passes through the substrate-binding groove. The proenzyme structure reveals suboptimal catalytic triad geometry with its propiece linker partially blocking the substrate-binding site, which together serve to prevent premature activation of the protease. Finally, we have identified numerous processing intermediates and propose a structural model that explains the pathway for TPP1 activation in vitro. These data provide new insights into TPP1 function and represent a valuable resource for constructing improved TPP1 variants for treatment of late infantile neuronal ceroid lipofuscinosis.

Residual levels of tripeptidyl-peptidase I activity dramatically ameliorate disease in late-infantile neuronal ceroid lipofuscinosis.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a hereditary neurodegenerative disease of childhood that is caused by mutations in the gene (CLN2) encoding the lysosomal protease tripeptidyl-peptidase I (TPPI). LINCL is fatal and there is no treatment of demonstrated efficacy in affected children but preclinical studies with AAV-mediated gene therapy have demonstrated promise in a mouse model. Here, we have generated mouse CLN2-mutants that express different amounts of TPPI activity to benchmark levels required for therapeutic benefits. Approximately 3% of normal TPPI activity in brain delayed disease onset and doubled lifespan to a median of approximately 9 months compared to mice expressing approximately 0.2% of normal levels. Expression of 6% of normal TPPI activity dramatically attenuated disease, with a median lifespan of approximately 20 months which approaches that of unaffected mice. While the lifespan of this hypomorph is shortened, disease is late-onset, less severe and progresses slowly compared to mice expressing lower TPPI levels. For gene therapy and other approaches that restore enzyme activity, these results suggest that 6% of normal TPPI activity throughout the CNS of affected individuals will provide a significant therapeutic benefit but higher levels will be required to cure this disease.

Timing of therapeutic intervention determines functional and survival outcomes in a mouse model of late infantile batten disease.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice. Intracranial injection of AAV1-hCLN2 resulted in widespread human TPP1 (hTPP1) activity in the brain that was 10-100-fold above wild-type levels. Injections before disease onset prevented storage and spared neurons from axonal degeneration, reflected by the preservation of motor function. Furthermore, the majority of CLN2 mutant mice treated pre-symptomatically lived for at least 330 days, compared with a median survival of 151 days in untreated CLN2 mutant controls. In contrast, although injection after disease onset ameliorated lysosomal storage, there was evidence of axonal degeneration, motor function showed limited recovery, and the animals had a median lifespan of 216 days. These data illustrate the importance of early intervention for enhanced therapeutic benefit, which may provide guidance in designing novel treatment strategies for cLINCL patients.

Activation of microglia acidifies lysosomes and leads to degradation of Alzheimer amyloid fibrils.

Microglia are the main immune cells of the brain, and under some circumstances they can play an important role in removal of fibrillar Alzheimer amyloid beta peptide (fAbeta). Primary mouse microglia can internalize fAbeta, but they do not degrade it efficiently. We compared the level of lysosomal proteases in microglia and J774 macrophages, which can degrade fAbeta efficiently, and we found that microglia actually contain higher levels of many lysosomal proteases than macrophages. However, the microglial lysosomes are less acidic (average pH of approximately 6), reducing the activity of lysosomal enzymes in the cells. Proinflammatory treatments with macrophage colony-stimulating factor (MCSF) or interleukin-6 acidify the lysosomes of microglia and enable them to degrade fAbeta. After treatment with MCSF, the pH of microglial lysosomes is similar to J774 macrophages (pH of approximately 5), and the MCSF-induced acidification can be partially reversed upon treatment with an inhibitor of protein kinase A or with an anion transport inhibitor. Microglia also degrade fAbeta if lysosomes are acidified by an ammonia pulse-wash or by treatment with forskolin, which activates protein kinase A. Our results indicate that regulated lysosomal acidification can potentiate fAbeta degradation by microglia.

Demonstration of lysosomal localization for the mammalian ependymin-related protein using classical approaches combined with a novel density shift method.

Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.

Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins.

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.

A frame shift mutation in canine TPP1 (the ortholog of human CLN2) in a juvenile Dachshund with neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage diseases characterized by progressive neuropathy and the accumulation of autofluorescent cytoplasmic granules. Clinical signs of a new canine NCL began in a 9-month-old male Dachshund with vomiting, mental dullness, and loss of previously learned commands and rapidly progressed to include disorientation, ataxia, visual deficits, generalized myoclonic seizures, and death at 12 months of age. Neurons throughout the CNS contained autofluorescent storage granules that stained with periodic acid-Schiff and Luxol fast blue stains. Electron microscopy revealed that the storage granule contents consisted of curvilinear-appearing material characteristic of human late infantile NCL caused by CLN2 mutations. Nucleotide sequence analysis of canine TPP1, the ortholog of human CLN2, revealed a single nucleotide deletion in exon 4 which predicted a frame shift with a premature stop codon. Brain tissue from the affected dog lacked detectable activity of the tripeptidyl-peptidase enzyme encoded by TPP1, whereas the specific activities of 15 other lysosomal enzymes were higher than those in the brains of three control dogs. The affected Dachshund was homozygous for the mutant c.325delC allele, his sire and dam were heterozygotes, and 181 unrelated dogs, including 77 Dachshunds, were all homozygous for the wild-type allele. A DNA assay that detects the mutant allele will help Dachshund breeders avoid producing affected puppies in future generations. Furthermore, this Dachshund NCL may prove to be a useful model for studying the pathogenesis of neurodegeneration in human late infantile NCL and for evaluating novel therapeutic interventions for this disease.

A mutation in the cathepsin D gene (CTSD) in American Bulldogs with neuronal ceroid lipofuscinosis.

We obtained DNA, brains, and eyes from American Bulldogs with neurodegeneration due to neuronal ceroid lipofuscinosis (NCL). The diagnosis of NCL was confirmed by detection of autofluorescent cytoplasmic inclusions within neurons throughout the brains, in retinal ganglion cells, and along outer limiting membranes of the retinas. Electron microscopy revealed that the inclusions had coarsely granular matrices surrounding well-delineated spherical structures and that the inclusions near the retinal outer limiting membranes were within photoreceptor cells, mostly cones. Affected American Bulldogs were homozygous for the A allele of a G to A transition in the cathepsin D gene (CTSD), which predicts the conversion of methionine-199 to an isoleucine. Only the G allele was detected in DNA samples from 131 randomly selected dogs representing 108 breeds other than American Bulldog; however, the A allele had a frequency of 0.28 among 123 genotyped American Bulldogs. Transmission analysis in a 99 dog pedigree of American Bulldogs indicated a probability of less than 10(-7) that alleles from any mutation unlinked to CTSD would be concordant with the pedigree and phenotypes of the dogs. Brain samples from affected dogs had 36% of the cathepsin D-specific enzymatic activity found in control dog brains; whereas, specific enzymatic activities of 15 other lysosomal enzymes were unchanged or increased. Compared to previously described NCLs in mice and sheep that completely lack cathepsin D activity, the clinical course of NCL in the American Bulldogs was less severe and more closely resembled that of many human NCLs.

Determination of the substrate specificity of tripeptidyl-peptidase I using combinatorial peptide libraries and development of improved fluorogenic substrates.

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.

Significance and prognostic value of lysosomal enzyme activities measured in surgically operated adenocarcinomas of the gastroesophageal junction and squamous cell carcinomas of the lower third of esophagus.

AIM: To establish whether there are fundamental differences in the biochemistries of adenocarcinomas of the gastroesophageal junction (GEJ) and the squamous cell carcinomas of the lower third of the esophagus (LTE). METHODS: Between February 1, 1997 and February 1, 2000, we obtained tissue samples at the moment of resection from 54 patients for biochemical analysis. The full set of data could be comprehensively analyzed in 47 of 54 patients samples (81%). Of these, 29 were adenocarcinomas of the GEJ Siewert type I (n = 8), type II (n = 12), type III (n = 9), and 18 presented as squamous cell carcinomas of the LTE. We evaluated the mean values of 11-lysosomal enzyme and 1-cytosol protease activities of the tumorous and surrounding mucosae as well as their relative activities, measured as the ratio of activity in tumor and normal tissues from the same patient. These data were further analyzed to establish the correlation with tumor localization, TNM stage (lymph-node involvement), histological type (papillary, signet-ring cell, tubular), state of differentiation (good, moderate, poor), and survival (or=24 mo). RESULTS: In adenocarcinomas, the activity of alpha-mannosidase (AMAN), cathepsin B (CB) and dipeptidyl-peptidase I (DPP I) increased significantly as compared to the normal gastric mucosa. In squamous cell carcinomas of the esophagus, we also found a significant difference in the activity of cathepsin L and tripeptidyl-peptidase I in addition to these three. There was a statistical correlation of AMAN, CB, and DPP I activity between the level of differentiation of adenocarcinomas of the GEJ and lymph node involvement, because tumors with no lymph node metastases histologically confirmed as well-differentiated, showed a significantly lower activity. The differences in CB and DPP I activity correlated well with the differences in survival rates, since the CB and DPP I values of those who died within 24 mo following surgical intervention were significantly higher than of those who survived for 2 years or more. CONCLUSION: Adenocarcinomas of the GEJ form a homogenous group from a tumor-biochemical aspect, and differ from the biochemical characteristics of squamous cell carcinomas of the LTE on many points. When adenocarcinomas of the GEJs are examined at the preoperative phase, the ratio of the performed AMAN, CB, and DPP I enzymatic activity of the tissue sample from the tumor and adjacent intact mucosa within 2 cm of the tumor may have a prognostic value even in the preoperative examination period, and may indicate that ranking of these patients into the neo-adjuvant treatment group should be considered.

Lysosomal enzyme activities: new potential markers for Sjögren's syndrome.

OBJECTIVE: To evaluate the changes in lysosomal enzyme activities in leukocytes of patients with Sjögren's syndrome. METHODS: Leukocytes were obtained from 38 patients with Sjögren's syndrome and 36 healthy subjects. The activities of the following glycosidases were measured: alpha-glucosidase (AGU), beta-galactosidase (BGA), alpha-mannosidase (AMAN), beta-glucuronidase (GCU), beta-hexosaminidase (HEX), and the following proteases: cathepsin B (CATH B), dipeptidyl peptidase I (DPP I), cathepsin H (CATH H), dipeptidyl peptidase II (DPP II), tripeptidyl peptidase I (TPP I), and cathepsin D (CATH D) activity. RESULTS: Activity of the glycosidases beta-galactosidase, alpha-mannosidase, beta-glucuronidase and beta-hexosaminidase, as well as of the peptidases cathepsin B, cathepsin D, dipeptidyl peptidase I, and tripeptidyl peptidase I, was elevated during the first 5 years of SS, and it increased further between 5 and 10 years after diagnosis. CONCLUSIONS: The elevated activities of the lysosomal enzymes in Sjögren's syndrome patients may play a role in tissue damage by accelerated breakdown of glycoproteins in lysosomes.

The human brain mannose 6-phosphate glycoproteome: a complex mixture composed of multiple isoforms of many soluble lysosomal proteins.

The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes.

Metabolic changes in the lower esophageal sphincter influencing the result of anti-reflux surgical interventions in chronic gastroesophageal reflux disease.

AIM: With the availability of a minimally invasive approach, anti-reflux surgery has recently experienced a renaissance as a cost-effective alternative to life-long medical treatment in patients with gastroesophageal reflux disease (GERD). We are not aware of the fact whether reflux episodes causing complaints for a long time i.e., at least for one year are associated with metabolic changes in the lower esophageal sphincter, and if so, whether these may influence functional results achieved after anti-reflux surgery. METHODS: Between 1 January 2001 and 31 December 2002 we performed anti-reflux surgery on 79 patients. Muscle samples were taken from the lower esophageal sphincter (LES) in 33 patients during anti-reflux surgery. Inclusion criteria were: LES resting pressure below 10 mmHg and a marked, pH proven acid exposure to the esophagus of at least one year's duration, causing subjective complaints and requiring continuous proton pump inhibitor treatment. Control samples were obtained from muscle tissue in the gastroesophageal junction that had been removed from 17 patients undergoing gastric or esophageal resection. Metabolic and lysosomal enzyme activities and special protein concentrations 16 parameters in total were evaluated in tissue taken from control specimens and tissue taken from patients with GERD. The biochemical parameters of these intra-operative biopsies were used to correlate the results of anti-reflux operations (Visick I and II-III). RESULTS: In the reflux-type muscle, we found a significant increase of the energy-enzyme activities e.g., creatine kinase, lactate dehydrogenase, beta-hydroxybutyrate dehydrogenase, and aspartate aminotransaminase-. The concentration of the structural protein S-100 and the myofibrillar protein troponin I were also significantly increased. Among lysosomal enzymes, we found that the activities of cathepsin B, tripeptidyl-peptidase I, dipeptidyl-peptidase II, beta-hexosaminidase B, beta-mannosidase and beta-galactosidase were significantly decreased as compared to the control LES muscles. By analyzing the activity values of the 9 patients in Visick groups II and III at two months post-surgery, we found a significant increase in the activity of the so-called energy-enzyme values and in the concentration of structural and myofibrillar proteins as compared to the rest of the reflux patients. CONCLUSION: Our results call attention to the metabolic changes that occurred in the LES muscles of reflux patients. The developing hypertrophy-like changes of LES muscles may be a reason for complaints after anti-reflux surgery, which consisted mainly of reports of persisting dysphagia.