The Batten disease gene product (CLN3p) is a Golgi integral membrane protein

Batten disease (juvenile neuronal ceroid lipofuscinosis) is a recessive neurodegenerative disorder of childhood. The gene, CLN3, was recently identified and found to encode a novel 438 amino acid protein of unknown function. In order to gain insight into the function of the Batten disease protein (CLN3p), we investigated its subcellular localization. Protein constructs incorporating CLN3p fused to the green fluorescence protein or an eight amino acid peptide tag were transiently expressed in fibroblasts, HeLa and COS-7 cells. A juxtanuclear, asymmetric localization pattern was observed that correlated with the Golgi apparatus in all three cell types. However, a proportion of transiently transfected cells exhibited a punctate vesicular distribution throughout the cytoplasm in addition to or without the Golgi localization. In order to account for localization patterns arising from intracellular protein transport disruption due to exaggerated overexpression in transiently transfected cells, we isolated a stably transfected cell line expressing only one copy of the CLN3 -GFP DNA construct. Fluorescence and biochemical analyses using this cell line demonstrated that CLN3p is an integral membrane protein that localizes primarily in the Golgi apparatus. The functional implications of this finding are discussed.     

Isolation and chromosomal mapping of a mouse homolog of the Batten disease gene CLN3

We describe the isolation and chromosomal mapping of a mouse homolog of the Batten disease gene, CLN3. Like its human counterpart, the mouse cDNA contains an open reading frame of 1314 bp encoding a predicted protein product of 438 amino acids. The mouse and human coding regions are 82 and 85% identical at the nucleic acid and amino acid levels, respectively. The mouse gene maps to distal Chromosome 7, in a region containing genes whose homologs are on human chromosome 16p12, where CLN3 maps. Isolation of a mouse CLN3 homolog will facilitate the creation of a mouse model of Batten disease.     

Refined localization of the Batten disease gene (CLN3) by haplotype and linkage disequilibrium mapping to D16S288-D16S383 and exclusion from this region of a variant form of Batten disease with granular osmiophilic deposits

Haplotype analysis in a collaborative collection of 143 families with juvenile-onset neuronal ceroid lipofuscinosis (JNCL) or Batten (Spielmeyer-Vogt-Sj?gren) disease has permitted refined localization of the disease gene, CLN3, which was assigned to chromosome 16 in 1989. Recombination events in four maternal meioses delimit new flanking genetic markers for CLN3 which localize the gene to the chromosome interval 16p12.1-11.2 between microsatellite markers D16S288 and D16S383. This narrows the position of CLN3 to a region of 2.1 cM, a significant reduction from the previous best interval. Using haplotypes, analysis of the strong linkage disequilibrium that exists between genetic markers within the D16S288-D16S383 interval and CLN3 shows that CLN3 is in closest proximity to loci D16S299 and D16S298. Analysis of markers across the D16S288-D16S383 region in four families with a variant form of JNCL characterized histologically by cytosomal granular osmiophilic deposits (GROD) has excluded linkage of the gene locus to the CLN3 region of chromosome 16, suggesting that JNCL with GROD is not an allelic form of JNCL.     

Phenol sulfotransferases: candidate genes for Batten disease

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis; JNCL) is an autosomal recessive neurodegenerative disorder, characterized by the cytosomal accumulation of autofluorescent proteolipopigments in neurons and other cell types. The Batten disease gene (CLN3) has not yet been identified, but has been mapped to a small region of human chromosome area 16p12.1-p11.2. We recently reported the fortuitous discovery that the cytosolic phenol sulfotransferase gene (STP) is located within this same interval of chromosome 16p. Since phenol sulfotransferase is expressed in neurons, can sulfate lipophilic phenolic compounds, and is mapped near CLN3, STP is considered as a candidate gene for Batten disease. YAC and cosmid cloning results have further substantiated the close proximity of STP and a highly related sulfotransferase (STM), encoding the catecholamine-preferring enzyme, to the CLN3 region of chromosome 16p. In this report, we summarize some of the recent progress in the identification of two phenol sulfotransferase genes (STP and STM) as positional candidate genes for Batten disease.     

A yeast model for the study of Batten disease

Although the CLN3 gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive. We previously cloned the Saccharomyces cerevisiae homologue to the human CLN3 gene, designated BTN1, which is not essential and whose product is 39% identical and 59% similar to Cln3p. We report that btn1-Delta deletion yeast strains are more resistant to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (denoted ANP), a phenotype that is complemented in yeast by the human CLN3 gene. Furthermore, the severity of Batten disease in humans and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease.     

Evidence for phosphorylation of CTP:phosphocholine cytidylyltransferase by multiple proline-directed protein kinases

Reversible phosphorylation of CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of phosphatidylcholine biosynthesis, is thought to play a role in regulating its activity. In the present study, the hypothesis that proline-directed kinases play a major role in phosphorylating cytidylyltransferase is substantiated using a c-Ha-ras-transfected clone of the human keratinocyte cell line HaCaT. Cellular extracts from epidermal growth factor-stimulated HaCaT cells and from ras-transfected HaCaT cells phosphorylated cytidylyltransferase much stronger as compared with extracts from quiescent HaCaT cells. The tryptic phosphopeptide pattern of cytidylyltransferase phosphorylated by cell-free extracts from ras-transfected HaCaT cells was similar compared with the patterns of cytidylyltransferase phosphorylated by p44mpkmitogen-activated protein kinase and p34cdc2 kinase in vitro, whereas in the case of casein kinase II the pattern was different. Furthermore, in c-Ha-ras-transfected HaCaT cells the in vivo phosphorylation state of cytidylyltransferase was 2-fold higher as compared with untransfected HaCaT cells. This higher phosphorylation of cytidylyltransferase in the ras-transfected clone was reduced to a level below the phosphorylation of cytidylyltransferase in untransfected cells, using olomoucine, a specific inhibitor of proline-directed kinases. The reduced phosphorylation of cytidylyltransferase in olomoucine-treated cells correlated with an enhanced stimulation of enzyme activity by oleic acid.     

Priming of platelet alphaIIbbeta3 by oxidants is associated with tyrosine phosphorylation of beta3

Reactive oxygen species play an important role at the site of vascular injuries and arterial thromboses. We studied the mechanism mediating platelet aggregation induced by H2O2, a major cellular oxidant. Exposure to H2O2 triggered platelet aggregation, but only when the platelets were stirred. Strong platelet aggregation induced99032416 required the presence of the tyrosine phosphatase inhibitor sodium orthovanadate (NaVO4) and was dependent on the participation of integrin alphaIIbbeta3 (glycoprotein IIb-IIIa). A specific inhibitor of alphaIIbbeta3 blocked platelet aggregation induced by H2O2 and NaVO4, thus confirming that aggregation requires this receptor. In the presence of H2O2 and NaVO4, multiple platelet substrates were phosphorylated on tyrosine. Such tyrosine kinase response was necessary but not sufficient to activate alphaIIbbeta3, as detected by binding of soluble fibrinogen to platelets. Stirring of the platelets exposed to H2O2 and NaVO4 was also needed to allow for binding of fibrinogen to alphaIIbbeta3. The tyrosine kinase inhibitor genistein was able to block platelet aggregation induced by H2O2 and NaVO4, thus confirming that tyrosine kinase activity was needed to trigger alphaIIbbeta3 activation on stirring. N-Acetyl-L-cysteine, a cell-permeant antioxidant, blocked the tyrosine phosphorylation of platelet substrates and also the platelet aggregation induced by H2O2 and NaVO4. We found that beta3 was phosphorylated on tyrosine in platelets exposed to H2O2 and NaVO4, even in the absence of aggregation. Hence, tyrosine phosphorylation of beta3 might contribute to the "priming" of alphaIIbbeta3 induced by H2O2 and NaVO4, whereby the receptor can become activated on stirring of the platelets.     

Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation

We have shown that a synthetic vitamin K analog, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or compound 5 (Cpd 5), potently inhibits cell growth and suggested that the analog exerts its effects mainly via sulfhydryl arylation rather than redox cycling. Since protein-tyrosine phosphatases (PTPases), which have pivotal roles in many cellular functions, have a critical cysteine in their active site, we have proposed PTPases as likely targets for Cpd 5. To test this hypothesis, we examined the effects of Cpd 5 on protein tyrosine phosphorylation of cellular proteins and on the activity of PTPases. We found that Cpd 5 rapidly induced protein tyrosine phosphorylation in a human hepatocellular carcinoma cell line (Hep3B) at growth inhibitory doses, and the effect was blocked by thiols but not by non-thiol antioxidants or tyrosine kinase inhibitors. Cpd 5 inhibited PTPase activity, which was also significantly antagonized by reduced glutathione. Furthermore, the well studied PTPase inhibitor orthovanadate also induced protein tyrosine phosphorylation and growth inhibition in Hep3B cells. These results suggest that inhibition of cellular PTPases by sulfhydryl arylation and subsequent perturbation of protein tyrosine phosphorylation may be involved in the mechanisms of Cpd 5-induced cell growth inhibition.     

Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.     

Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) was inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, was activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt were found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway.     

Complement receptor 3 of macrophages is associated with galectin-1-like protein

We have previously identified a 16-kDa protein with a pI of 5.1 (P16/5.1) that is associated with macrophage CR3. Microsequencing of P16/5.1 indicated exclusive homology to the beta-galactoside-binding lectin, galectin-1. Abs specific to a galectin-1 unique peptide reacted with P16/5.1. The association of P16/5.1 with CR3 was specifically inhibited by lactose, which binds with high affinity to galectin-1. These data together with similarities in molecular mass and pI suggest that P16/5.1 is galectin-1. Two-color immunofluorescence staining revealed the expression of galectin-1 on the macrophage surface and its colocalization with CR3. However, a surplus of CR3 was free of galectin-1, and some galectin-1 molecules were associated with cell surface receptors other than CR3. Based on these results we propose two models depicting the functional significance of CR3-galectin-1 association: 1) homodimeric galectin-1 possessing a divalent sugar binding site may act as an extracellular adapter molecule that cross-links CR3 with other receptors; and 2) association of galectin-1 with beta-galactosides on the extracellular domain of CR3 may modify the binding affinity of the receptor to its ligand. These possibilities are not mutually exclusive and can clarify the mode by which CR3 transmits signals in macrophages.     

Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.     

Control of juvenile hormone biosynthesis. Evidence for phosphorylation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase of insect corpus allatum

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme of juvenile hormone biosynthesis, have been measured in the supernatants of homogenates (10,000 x g) prepared from the corpora allata of the adult tobacco hornworm moth, Manduca sexta. Enzyme activity was inhibited 80% by 50 mM NaF, a known phosphoprotein phosphatase inhibitor, if present during extirpation of the glands and all subsequent workup of the tissue. Reductase activity was also significantly decreased (20-30%) in homogenates preincubated with 4 mM MgCl2 and 2 mM ATP. These results provide evidence that reductase in the insect undergoes phosphorylation and dephosphorylation similar to that occurring with reductase of mammalian liver. If so, this would provide a rapid method for modulating juvenile hormone synthesis.     

The Lyn-catalyzed Tyr phosphorylation of the transmembrane band-3 protein of human erythrocytes

Band-3 protein (approximately 95 kDa), the major and multifunctional transmembrane protein of human erythrocytes, has been shown to be phosphorylated by endogenous Tyr-protein kinases on different Tyr residues at its N and C cytoplasmic domains. Both the added p36syk (catalytic domain of p72syk) and Lyn kinases are able to phosphorylate the isolated cytoplasmic domain of band 3 (cdb3), yielded by chymotryptic digestion of band 3 in the isolated membranes (ghosts). However, the two Tyr-protein kinases exhibited different phosphorylation behaviours when added to the isolated erythrocyte membranes. More precisely, the added p36syk markedly Tyr phosphorylates the band-3 protein, whereas the added Lyn phosphorylates it very poorly. It is of interest that Lyn can associate with membranes and markedly phosphorylate band 3 when this latter protein has been previously phosphorylated by p36syk, i.e. the p36(syk)-catalyzed phosphorylation is proposed to be a prerequisite for the association of Lyn with the membrane (likely to band 3) and for the Lyn-catalyzed phosphorylation of different band-3 Tyr sites.     

Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.     

Progressive intracranial occlusive disease associated with deficiency of protein S. Report of two cases

BACKGROUND AND PURPOSE: Deficiency of the free fraction of protein S has been associated with arterial or venous stroke. The pathogenesis of vascular occlusion in patients with protein S deficiency is not known. We present two cases of cerebral infarction and deficiency of protein S in which the subjects had progressive intracranial occlusions. CASE DESCRIPTION: A 16-year-old girl was admitted because of left brain stem infarction and protein S deficiency. Cerebral angiography disclosed stenosis of the right intracranial vertebral artery and occlusion of the left posterior cerebral artery. A second angiogram performed 18 months later disclosed occlusion of the right vertebral intracranial artery. In the second case, a 17-year-old girl was admitted because of left hemispheric cerebral infarction and protein S deficiency. Cerebral angiography showed stenosis of the left anterior cerebral artery, left supraclinoid internal artery, and left middle cerebral artery. A second cerebral angiogram performed 5 months later disclosed occlusion of the left anterior cerebral artery and poor hemispheric perfusion through the left middle cerebral artery. CONCLUSIONS: Based on our cases, we postulate that some patients with prothrombotic states may develop progressive intracranial arterial occlusions, possibly secondary to a permanent thrombogenic stimulus. We suggest routinely searching for prothrombotic states in young patients with intracranial occlusion, especially if the occlusion is progressive and other causes are not obvious.     

Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix

COS-7 cells transfected with a DNA construct encoding the 133 amino acids in norrin plus six histidine residues at its carboxyl terminus were pulse-labeled with [35S]cysteine, and the labeled norrin was examined in cell lysates, the medium, and the extracellular matrix. SDS-gel electrophoresis under reducing conditions showed that the norrin expressed had an apparent Mr = 14,000 and was present only in cell lysates and the extracellular matrix. Under nonreducing conditions, most of the norrin in the extracellular matrix was oligomers that contained up to approximately 20 monomers. One of the major extracellular species of norrin under reducing conditions after cross-linking of norrin oligomers with bis(sulfosuccinimidyl)suberate had an apparent Mr = 28,000, consistent with covalent cross-linked dimers. Thus the covalently cross-linked dimers are key structural components of norrin oligomers. By site-directed mutagenesis, the codon for half-cystine 95 in norrin was changed to one encoding alanine. The norrin C95A found in the extracellular matrix of cells transfected with this mutant was the size of dimers, indicating that half-cystine 95 is involved in oligomer formation. The corresponding half-cystine residue in human prepro-von Willebrand factor is also involved in interchain disulfide bond formation, which is consistent with the sequence identity of the half-cystine residues in norrin and part of the half-cystine residues in a disulfide-rich domain of von Willebrand factor. Replacement of valine at residue 60 in norrin by glutamic acid, a mutation found in humans with a severe type of Norrie disease, results in a considerable reduction (50%) in the amount of norrin in the extracellular matrix of transfected COS-7 cells. Replacement of arginine at residue 121 by glutamine, which is associated with a less severe type of Norrie disease, results in a reduction in the amount of norrin R121Q in the extracellular matrix (26%). These studies suggest that norrin is a secreted protein that forms disulfide-bonded oligomers that are associated with the extracellular matrix upon secretion from cells. Moreover, the disulfide-rich motif of norrin and prepro-von Willebrand factor promotes interchain disulfide bond formation among polypeptides in which it is found.