Mannose 6-phosphate Receptor Pathway Research Articles

The effect of bladder cancer cell 5637 invasiveness by heparanase through the cation-independ mannose 6-phosphate receptor pathway

Objective To study the relationship between heparanase and cation-independ mannose 6-phosphate receptor (CD222) expression in bladder cancer 5637 cell line.And find out the effect of cell invasiveness by their combination.Methods Immunofluorescence assay were used to detect the position fixing and interaction of heparanase and CD222 in bladder cancer 5637 cell line.The CD222 mRNA-targeted small interfering RNA (siRNA) were designed and constructed.5637 cells were cultured and transfected with siRNA-CD222.The expression of CD222 mRNA and protein were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.After the recombinant human heparanase were given.Western blotting were performed to detect the expression of CD222 and p-Akt in 5637 cell line before and after tansfection.The invasiveness of above cells were determined by matrigel invasion assays.Results Heparanase and CD222 staining on the cell cytoplasm.Heparanase binds CD222 and cells treated with recombinant human heparanase enhance the binding.Inhibition of CD222 expression in 5637 by siRNA could decrease invasion capacity and the level of Akt phosphorylation which was induced by recombinant human heparanase.Conclusion Heparanase binding to CD222 were localized to the cell cytoplasm and heparanase facilitates bladder cancer cell invasiveness by binding to CD222 and increasing Akt phosphorylation. Key words: Bladder cancer; Hparanase; Cation-independ mannose 6-phosphate receptor; Small interfering RNA; Invasiveness

The interactomics of sortilin: an ancient lysosomal receptor evolving new functions.

The delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptor (MPR). The MPR has been demonstrated to attain the early endosomes via a process that requires the interaction of its cytosolic domain with the GGA and AP-1 adaptor proteins. Additionally, the MPR can be recycled back to the trans-Golgi network (TGN) through its interaction with the retromer complex. Interestingly, in I-cell disease (ICD), in which the MPR pathway is non-functional, many soluble lysosomal proteins continue to traffic to the lysosomes. This observation led to the discovery that sortilin is responsible for the MPR-independent targeting of the sphingolipid activator proteins (SAPs) and acid sphingomyelinase (ASM). More recently, our laboratory has tested the hypothesis that sortilin is also capable of sorting a variety of cathepsins that exhibit varying degrees of MPR-independent transport. We have demonstrated that the transport of cathepsin D is partially dependent upon sortilin, that cathepsin H requires sortilin, and that cathepsins K and L attain the lysosomes in a sortilin-independent fashion. As a type-1 receptor, sortilin also has numerous cytosolic binding partners. It has been observed that like the MPR, the anterograde trafficking of sortilin and its cargo require both GGAs and AP-1. Similarly, the retrograde recycling pathway of sortilin also involves an interaction with retromer through a YXXphi site in the cytosolic tail of sortilin. In conclusion, the cytosolic domains of sortilin and MPR possess a high degree of functional homology and both receptors share a conserved trafficking mechanism.

Sortilin mediates the lysosomal targeting of cathepsins D and H

Delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptor (M6PR). However, in I-cell disease (ICD), in which the M6PR pathway is non-functional, some soluble lysosomal proteins continue to traffic to the lysosomes. In this paper, we tested the hypothesis that cathepsins D and H, two soluble proteases that exhibit M6PR-independent trafficking, are targeted to the lysosomes by sortilin. Using a dominant-negative sortilin construct and small interfering RNA (siRNA) we demonstrated that while cathepsin D transport is partially dependent upon sortilin, cathepsin H requires exclusively sortilin for its transport to the lysosomes. Our results suggest that sortilin functions as an alternative sorting receptor to the M6PR for these soluble hydrolases.

The 46-kDa mannose 6-phosphate receptor does not depend on endosomal acidification for delivery of hydrolases to lysosomes

In mammalian cells, the mannose 6-phosphate receptor pathway accounts for the transport of most soluble acid hydrolases to lysosomes. It is believed that dissociation of mannose 6-phosphate receptors and their ligands is entirely driven by the acidic environment in endosomal compartments. Indeed, pH-perturbing substances such as ammonium chloride and monensin have been shown to inhibit lysosomal enzyme targeting in cells that express both known mannose 6-phosphate receptors. We now demonstrate that ammonium chloride and monensin exert modest effects on the intracellular retention of lysosomal hydrolases in murine cells that synthesize only the 46-kDa mannose 6-phosphate receptor. Neither ammonium chloride nor monensin induces changes to the subcellular localization of lysosomal hydrolases and the 46-kDa mannose 6-phosphate receptor in these cells. This suggests that endosomal dissociation of the receptor and its ligands still occurs in the presence of these agents. We conclude that the murine 46-kDa mannose 6-phosphate receptor has the capacity to deliver its cargo proteins to lysosomes even in the absence of endosomal acidification.

Thematic review series: Lipid Posttranslational Modifications. Lysosomal metabolism of lipid-modified proteins

Much is now understood concerning the synthesis of prenylated and palmitoylated proteins, but what is known of their metabolic fate? This review details metabolic pathways for the lysosomal degradation of S-fatty acylated and prenylated proteins. Central to these pathways are two lysosomal enzymes, palmitoyl-protein thioesterase (PPT1) and prenylcysteine lyase (PCL). PPT1 is a soluble lipase that cleaves fatty acids from cysteine residues in proteins during lysosomal protein degradation. Notably, deficiency in the enzyme causes a neurodegenerative lysosomal storage disorder, infantile neuronal ceroid lipofuscinosis. PCL is a membrane-associated flavin-containing lysosomal monooxygenase that metabolizes prenylcysteine to prenyl aldehyde through a completely novel mechanism. The eventual metabolic fates of other lipidated proteins (such as glycosylphosphatidylinositol-anchored and N-myristoylated proteins) are poorly understood, suggesting directions for future research.

Intercellular transfer of the virally derived precursor form of acid alpha-glucosidase corrects the enzyme deficiency in inherited cardioskeletal myopathy Pompe disease.

Pompe disease is a lethal cardioskeletal myopathy in infants and results from genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Genetic replacement of the cDNA for human GAA (hGAA) is one potential therapeutic approach. Three months after a single intramuscular injection of 10(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level. Direct transduction of a target muscle, however, may not correct all deficient cells. Therefore, the amount of enzyme that can be transferred to deficient cells from virally transduced cells was studied. Fibroblasts from an affected patient were transduced with AdhGAA, washed, and plated on transwell culture dishes to serve as donors of recombinant enzyme. Deficient fibroblasts were plated as acceptor cells, and were separated from the donor monolayer by a 22-microm pore size filter. Enzymatic and Western analyses demonstrate secretion of the 110-kDa precursor form of hGAA from the donor cells into the culture medium. This recombinant, 110-kDa species reaches the acceptor cells, where it can be taken up by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks to lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme. Patient fibroblasts receiving recombinant hGAA by this transfer mechanism reach levels of enzyme activity that are comparable to normal human fibroblasts. Skeletal muscle cell cultures from an affected patient were also transduced with Ad-hGAA. Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunocytochemistry, and enzyme activity is transferred to deficient skeletal muscle cells grown in coculture. Transfer of the precursor protein between muscle cells again occurs via mannose 6-phosphate receptors, as evidenced by competitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA-knockout mice demonstrate that hepatic transduction with adenovirus encoding either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism. Taken together, these data show that the mannose 6-phosphate receptor pathway provides a useful strategy for cell-to-cell distribution of virally derived recombinant GAA.

Role of sphingolipids in the transport of prosaposin to the lysosomes

Prosaposin is the precursor of four lysosomal saposins that promote the degradation of glycosphingolipids (GSLs) by acidic hydrolases. GSLs contain a hydrophobic ceramide moiety, which acts as a membrane anchor, and a hydrophilic oligosaccharide chain that faces the lumen of the Golgi apparatus and extracellular spaces. By using fumonisin B1, PDMP and D609, we tested the hypothesis that sphingolipids mediate the transport of prosaposin to the lysosomes. Fumonisin B1 interferes with the synthesis of ceramide, PDMP blocks the formation of glucosylceramide and D609 blocks the formation of sphingomyelin. Fumonisin B1 produced a 59–85% decrease in the density of gold particles in the lysosomes of CHO and NRK cells immunolabeled with anti-prosaposin antibody, and a 55% reduction in the lysosomes of CHO cells stably transfected with an expression vector containing a human prosaposin cDNA. To examine whether the mannose 6-phosphate receptor pathway was affected by this treatment, NRK and CHO cells treated or not with fumonisin B1 were labeled with anti-cathepsin A antibody. The results showed no significant differences in labeling of the lysosomes, suggesting that the effect of fumonisin B1 was specific. When fumonisin B1 and D609 were added to the media of transfected CHO cells, a decrease in immunofluorescence with anti-prosaposin antibody was observed by confocal microscopy. PDMP did not cause any reduction in immunoreactivity, indicating that sphingolmyelin appears to be involved in this process.▪In conclusion, our data support the hypothesis that sphingolipids, possibly sphingomyelin, are involved in the transport of prosaposin to the lysosomes.—Lefrancois, S., L. Michaud, M. Potier, S. Igdoura, and C. R. Morales. Role of sphingolipids in the transport of prosaposin to the lysosomes. J. Lipid Res. 1999. 40: 1593–1603.

Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer.

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.

Phylogenetic conservation of cysteine proteinases. Cloning and expression of a cDNA coding for human cathepsin S.

A 1.8-kilobase full-length cDNA of human cathepsin S, a lysosomal cysteine proteinase, has been isolated. The single long open reading frame encodes a polypeptide of 331 amino acids consisting of a 15-amino acid NH2-terminal signal peptide, a propeptide of 99 amino acids, and a mature polypeptide of 217 amino acids. The deduced amino acid sequence contains only one potential N-glycosylation site located in the propeptide. The NH2-terminal amino acid sequence of the mature polypeptide was confirmed by sequencing cathepsin S purified from human spleen. The cDNA detects a 1.9-kilobase transcript in poly(A)+ RNA from human fibroblasts. Expression of human cathepsin S in transfected baby hamster kidney cells resulted in up to more than 300-fold cathepsin S activity as compared to untransfected controls. In the expressing baby hamster kidney cells, human cathepsin S is transported to the lysosomes via the mannose 6-phosphate receptor pathway as shown by density gradient centrifugation, immunofluorescence, and detection of the 37-kDa cathepsin S precursor in the medium in the presence of NH4Cl. The deduced amino acid sequence of human cathepsin S exhibits a substantial degree of similarity with other human cysteine proteinases and papain indicating that they have a common ancestral gene and are members of a gene family.