Mouse "protective protein":cDNA cloning, sequence comparison, and expression

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes β-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects β-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.</p

Mouse "protective protein":cDNA cloning, sequence comparison, and expression

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes β-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects β-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.</p

The gene encoding human protective protein (PPGB) is on chromosome 20

Normal lymphocyte prometaphase chromosome spreads were hybridized in situ using single- and double-color fluorescence techniques. The results obtained with either the 1.8-kb protective protein cDNA or a 12-kb genomic fragment of the human protective protein gene as probe demonstrate that the PPGB gene is localized on the long arm of chromosome 20. This assignment was confirmed by hybridization with whole chromosome DNA libraries.</p

Human Lysosomal Protective Protein Has Cathepsin A-like Activity Distinct from Its Protective Function

The protective protein was first discovered because of its deficiency in the metabolic storage disorder galactosialidosis. It associates with lysosomal β-galactosidase and neuraminidase, toward which it exerts a protective function necessary for their stability and activity. Human and mouse protective proteins are homologous to yeast and plant serine carboxypeptidases. Here, we provide evidence that this protein has enzymatic activity similar to that of lysosomal cathepsin A: 1) overexpression of human and mouse protective proteins in COS-1 cells induces a 3-4-fold increase of cathepsin A-like activity; 2) this activity is reduced to ∼1% in three galactosialidosis patients with different clinical phenotypes; 3) monospecific antibodies raised against human protective protein precipitate virtually all cathepsin A-like activity in normal human fibroblast extracts. Mutagenesis of the serine and histidine active site residues abolishes the enzymatic activity of the respective mutant protective proteins. These mutants, however, behave as the wild-type protein with regard to intracellular routing, processing, and secretion. In contrast, modification of the very conserved Cys60 residue interferes with the correct folding of the precursor polypeptide and, hence, its intracellular transport and processing. The secreted active site mutant precursors, endocytosed by galactosialidosis fibroblasts, restore β-galactosidase and neuraminidase activities as effectively as wild-type protective protein. These findings indicate that the catalytic activity and protective function of the protective protein are distinct.</p

Human Lysosomal Protective Protein Has Cathepsin A-like Activity Distinct from Its Protective Function

The protective protein was first discovered because of its deficiency in the metabolic storage disorder galactosialidosis. It associates with lysosomal β-galactosidase and neuraminidase, toward which it exerts a protective function necessary for their stability and activity. Human and mouse protective proteins are homologous to yeast and plant serine carboxypeptidases. Here, we provide evidence that this protein has enzymatic activity similar to that of lysosomal cathepsin A: 1) overexpression of human and mouse protective proteins in COS-1 cells induces a 3-4-fold increase of cathepsin A-like activity; 2) this activity is reduced to ∼1% in three galactosialidosis patients with different clinical phenotypes; 3) monospecific antibodies raised against human protective protein precipitate virtually all cathepsin A-like activity in normal human fibroblast extracts. Mutagenesis of the serine and histidine active site residues abolishes the enzymatic activity of the respective mutant protective proteins. These mutants, however, behave as the wild-type protein with regard to intracellular routing, processing, and secretion. In contrast, modification of the very conserved Cys60 residue interferes with the correct folding of the precursor polypeptide and, hence, its intracellular transport and processing. The secreted active site mutant precursors, endocytosed by galactosialidosis fibroblasts, restore β-galactosidase and neuraminidase activities as effectively as wild-type protective protein. These findings indicate that the catalytic activity and protective function of the protective protein are distinct.</p

Human lysosomal protective protein:Glycosylation, intracellular transport, and association with β-galactosidase in the endoplasmic reticulum

In lysosomes β-galactosidase and neuraminidase acquire a stable and active conformation through their association with the protective protein. The latter is homologous to serine carboxypeptidases and has cathepsin A-like activity which is distinct from its protective function towards the two glycosidases. To define signals in the human protective protein important for its intracellular transport, and to determine the site of its association with β-galactosidase, we have generated a set of mutated protective protein cDNAs carrying targeted base substitutions. These mutants were either singly transfected into COS-1 cells or cotransfected together with wild type human β-galactosidase. We show that all point mutations cause either a complete or partial retention of the protective protein precursor in the endoplasmic reticulum. This abnormal accumulation leads to degradation of the mutant proteins probably in this compartment. Only the oligosaccharide chain on the 32-kDa subunit acquires the mannose 6-phosphate recognition marker, the one on the 20-kDa subunit seems to be merely essential for the stability of the mature protein. In cotransfection experiments, wild type β-galactosidase and protective protein appear to assemble already as precursors, soon after synthesis, in the endoplasmic reticulum. Mutated protective protein precursors that are retained in the endoplasmic reticulum or pre-Golgi complex interact with and withhold normal β-galactosidase molecules in the same compartments, thereby preventing their normal routing.</p

Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres.

Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs

CTCF induces histone variant incorporation, erases the H3K27me3 histone mark and opens chromatin

Insulators functionally separate active chromatin domains frominactive ones. The insulator factor, CTCF, has been found to bind to boundaries and to mediate insulator function. CTCF binding sites are depleted for the histone modification H3K27me3 and are enriched for the histone variant H3.3. In order to determine whether demethylation of H3K27me3 and H3.3 incorporation are a requirement for CTCF binding at domain boundaries or whether CTCF causes these changes, we made use of the LacI DNA binding domain to control CTCF binding by the Lac inducer IPTG. Here we show that, in contrast to the related factor CTCFL, the N-terminus plus zinc finger domain of CTCF is sufficient to open compact chromatin rapidly. This is preceded by incorporation of the histone variant H3.3, which thereby removes the H3K27me3 mark. This demonstrates the causal role for CTCF in generating the chromatin features found at insulators. Thereby, spreading of a histone modification from one domain through the insulator into the neighbouring domain is inhibited

Distinct Functions for Mammalian CLASP1 and-2 During Neurite and Axon Elongation

Mammalian cytoplasmic linker associated protein 1 and -2 (CLASP1 and -2) are microtubule (MT) plus-end tracking proteins that selectively stabilize MTs at the edge of cells and that promote MT nucleation and growth at the Golgi, thereby sustaining cell polarity. In vitro analysis has shown that CLASPs are MT growth promoting factors. To date, a single CLASP1 isoform (called CLASP1α) has been described, whereas three CLASP2 isoforms are known (CLASP2α, -β, and -γ). Although CLASP2β/γ are enriched in neurons, suggesting isoform-specific functions, it has been proposed that during neurite outgrowth CLASP1 and -2 act in a redundant fashion by modulating MT dynamics downstream of glycogen synthase kinase 3 (GSK3). Here, we show that in differentiating N1E-115 neuroblastoma cells CLASP1 and CLASP2 differ in their accumulation at MT plus-ends and display different sensitivity to GSK3-mediated phosphorylation, and hence regulation. More specifically, western blot (WB) analysis suggests that pharmacological inhibition of GSK3 affects CLASP2 but not CLASP1 phosphorylation and fluorescence-based microscopy data show that GSK3 inhibition leads to an increase in the number of CLASP2-decorated MT ends, as well as to increased CLASP2 staining of individual MT ends, whereas a reduction in the number of CLASP1-decorated ends is observed. Thus, in N1E-115 cells CLASP2 appears to be a prominent target of GSK3 while CLASP1 is less sensitive. Surprisingly, knockdown of either CLASP causes phosphorylation of GSK3, pointing to the existence of feedback loops between CLASPs and GSK3. In addition, CLASP2 depletion also leads to the activation of protein kinase C (PKC). We found that these differences correlate with opposite functions of CLASP1 and CLASP2 during neuronal differentiation, i.e., CLASP1 stimulates neurite extension, whereas CLASP2 inhibits it. Consistent with knockdown results in N1E-115 cells, primary Clasp2 knockout (KO) neurons exhibit early accelerated neurite and axon outgrowth, showing longer axons than control neurons. We propose a model in which neurite outgrowth is fine-tuned by differentially posttranslationally modified isoforms of CLASPs acting at distinct intracellular locations, thereby targeting MT stabilizing activities of the CLASPs and controlling feedback signaling towards upstream kinases. In summary, our findings provide new insight into the roles of neuronal CLASPs, which emerge as regulators acting in different signaling pathways and locally modulating MT behavior during neurite/axon outgrowth

The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. RESULTS: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. CONCLUSIONS: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes