Synthesis, transport and processing of cathepsin C in Morris hepatoma 7777 cells and rat hepatocytes

The synthesis, transport and processing of cathepsin C was studied in Morris hepatoma 7777 cells by metabolic labelling, immunoprecipitation and characterization of labelled polypeptides by gel electrophoresis and fluorography. The largest detectable precursor of cathepsin C was a polypeptide of Mr = 92 500. Even 3 min after synthesis this precursor was accompanied by four polypeptides with Mr values ranging from 63 000 to 54 000, indicating cleavage of the precursors within the endoplasmic reticulum. The early forms of cathepsin C were associated with low-buoyant-density organelles containing the markers of endoplasmic reticulum and Golgi complex. About 30% of these early forms were secreted within 3 h after synthesis. The remaining 70% were transferred into dense lysosomes and processed between 2 and 3 h after synthesis to a mixture of the least five major and nine minor polypeptides with Mr values ranging from 73 000 to 12 000. These forms remained stable for at least 3 days. In freshly isolated hepatocytes cathepsin C was processed to forms closely related to those found in the hepatoma cells. Cathepsin C was synthesized in Morris hepatoma 7777 cells as a glycoprotein with mannose-6-phosphate residues that mediated mannose-6-phosphate-specific receptor-dependent uptake in human skin fibroblasts. In contrast to hepatocytes, synthesis of mannose-6-phosphate receptors in Morris hepatoma 7777 cells was below the limit of detection. The hepatoma cells did not express at the cell surface these or other receptors mediating endocytosis of lysosomal enzymes. Further, processing and transport of newly synthesized cathepsin C was largely resistant to NH4Cl. Apparently, cathepsin C is transferred in Morris hepatoma 7777 cells by a mechanism independent of mannose-6-phosphate-specific receptors.     

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.     

Neither type of mannose 6-phosphate receptor is sufficient for targeting of lysosomal enzymes along intracellular routes

Mouse embryonic fibroblasts that are deficient in the two mannose 6- phosphate receptors (MPRs) MPR 46 and MPR 300 missort the majority (> or = 85%) of soluble lysosomal proteins into the medium. Human MPR 46 and MPR 300 were expressed in these cells to test whether overexpression of a single type of MPR can restore transport of lysosomal proteins to lysosomes. Only a partial correction of the missorting was observed after overexpression of MPR 46. Even at MPR 46 levels that are five times higher than the wild-type level, more than one third of the newly synthesized lysosomal proteins accumulates in the secretions. Two-fold overexpression of MPR 300 completely corrects the missorting of lysosomal enzymes. However, at least one fourth of the lysosomal enzymes are transported along a secretion-recapture pathway that is sensitive to mannose 6-phosphate in medium. In control fibroblasts that express both types of MPR, the secretion-recapture pathway is of minor importance. These results imply that neither overexpression of MPR 46 nor MPR 300 is sufficient for targeting of lysosomal proteins along intracellular routes.     

Molecular cloning, expression and functional characterization of the chicken cation dependent mannose 6-phosphate receptor protein

Mammalian mannose 6-phosphate (M6P) receptors function in transport of lysosomal enzymes. To understand the structural and functional significance of the chicken cation dependent mannose 6-phosphate receptor (MPR) (Mr 46kDa), a full-length cDNA for the chicken protein was cloned and expressed in mpr((-/-)) MEF cells devoid of both the receptors. The stably transfected cells express the receptor that could be affinity purified by phosphomannan chromatography. The authenticity of the receptor was confirmed by its immuno-reactivity with mammalian MPR 46 antibodies and its ability to sort cathepsin D in transfected cells (92.3%) as compared to mock transfected cells (50.2%), establishing a functional role for the chicken receptor.     

Biosynthesis of the human asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGP-R) isolated from human liver is a single polypeptide of Mr = 46,000. Monospecific polyclonal anti-human ASGP-R antibodies as well as anti-rat ASGP-R antibodies specifically inhibit binding of 125I-asialoorosomucoid to human hepatoma Hep G2 ASGP-R. These anti-ASGP-R antibodies specifically immunoprecipitate the 46,000-Da polypeptide from hepatoma cells labeled biosynthetically with 35S-amino acid. The receptor is initially synthesized as a 40,000-Da precursor which is converted to the mature 46,000-Da species with a t1/2 of approximately 45 min. The precursor species is sensitive to endo-beta-N-acetylglucosaminidase H and becomes resistant coincident with the appearance of the mature 46,000-Da receptor. In addition, the receptor synthesized in the presence of tunicamycin is approximately 34,000 Da. The newly synthesized ASGP-R reaches the cell surface after 45-60 min, where only the mature 46,000-Da species is present. In Hep G2 cells, the ASGP-R has a mean lifetime of approximately 30 h, a value which is unaltered during maximal rates of receptor-mediated endocytosis of ASGP.     

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.     

Effects of brefeldin A on the endocytic route. Redistribution of mannose 6-phosphate/insulin-like growth factor II receptors to the cell surface.

The effect of brefeldin A (BFA) on the trafficking of the mannose 6-phosphate/insulin-like growth factor II receptor within the endocytic route was analyzed. Treatment with BFA induced a redistribution of the receptor to the cell surface and increased both the binding and internalization of ligands 2-4-fold. The effect of BFA was dose- and time-dependent and reversible. Determinations of transport rates showed that BFA increases the internalization rate and the externalization rate of the receptor. This implies that the higher surface concentration is due to higher concentrations of receptor at the intracellular sites from where they recycle to the cell surface. The effect of BFA was additive to the redistribution induced by insulin-like growth factors I and II and was observed in all human and rodent cell lines analyzed. BFA increased also the cell surface expression of the Mr 46,000 mannose 6-phosphate receptor but not of the transferrin receptor. The results indicate that BFA interferes with the transport of mannose 6-phosphate receptors and affects the endocytosis of lysosomal enzymes by increasing the number of receptors available for recycling to the cell surface.     

Characterization of the mannose 6-phosphate receptor (Mr 300 kDa) protein dependent pathway of lysosomal enzyme targeting in Biomphalaria glabrata mollusc cells

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, β-hexosaminidase and α-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of ∼20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of ∼13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, β-hexosaminidase and α-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.     

Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network

Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane- associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.     

Role of cation independent mannose 6-phosphate receptor protein in sorting and intracellular trafficking of lysosomal enzymes in chicken embryonic fibroblast (CEF) cells

Delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptors (MPRs) in mammals. However, in non-mammalian cells the role of MPR300 in sorting and trafficking of acid hydrolases to lysosomes is not fully understood till now. In this paper, we tested the role of MPR300 in sorting and trafficking of lysosomal enzymes in CEF cells using a small interfering RNA (siRNA) technology. Inactivation of MPR300 resulted in the secretion of large amounts of newly synthesized hydrolases into the medium and also inhibited the endocytosis of mannose 6-phospharylated ligands. Knockdown of MPR300 in CEF cells results in missorting of fucosidase and arylsulfatse A enzymes into the medium. The results indicated that the MPR300 in CEF cells plays a key role in sorting and trafficking of these soluble hydrolases.     

The mannose 6-phosphate receptor and the biogenesis of lysosomes

Localization of the 215 kd mannose 6-phosphate receptor (MPR) was studied in normal rat kidney cells. Low levels of receptor were detected in the trans Golgi network, Golgi stack, plasma membrane, and peripheral endosomes. The bulk of the receptor was localized to an acidic, reticular-vesicular structure adjacent to the Golgi complex. The structure also labeled with antibodies to lysosomal enzymes and a lysosomal membrane glycoprotein (lgp120). While lysosome-like, this structure is not a typical lysosome that is devoid of MPRs. The endocytic marker alpha 2 macroglobulin-gold entered the structure at 37 degrees C, but not at 20 degrees C. With prolonged chase, most of the marker was transported from the structure into lysosomes. We propose that the MPR/lgp-enriched structure is a specialized endosome (prelysosome) that serves as an intermediate compartment into which endocytic vesicles discharge their contents, and where lysosomal enzymes are released from the MPR and packaged along with newly synthesized lysosomal glycoproteins into lysosomes.     

Is movement of mannose 6-phosphate-specific receptor triggered by binding of lysosomal enzymes?

Mannose 6-phosphate-specific receptors with an apparent molecular mass of 215,000 are present in fibroblasts at the cell surface and in intracellular membranes. The cell surface receptors mediate endocytosis of exogenous lysosomal enzymes and exchange with the intracellular receptors, which function in the sorting of endogenous lysosomal enzymes. In the present study, several methods independent of receptor ligands were designed in order to examine the exchange of receptors under conditions where receptor-ligand complexes do not dissociate (weak bases and monensin) or where receptor-ligand complexes are not formed due to absence of endogenous ligands as a result of inhibition of protein synthesis. Weak bases and monensin reduce the concentration of receptors at the cell surface by 20-30% and free cell surface receptors were replaced by occupied receptors. The latter continued to be exchanged with internal ligand-occupied receptors and the rates of the exchange were similar to the control values. The exchange of receptors between the cell surface and internal membranes was also not affected when the receptor ligands were depleted from the transport compartments by treating the cells with cycloheximide for up to 10 h. We conclude from these results that movement of mannose 6-phosphate-specific receptors along the endocytosis and sorting pathways is constitutive and not triggered by binding or dissociation of ligands.     

Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network.

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.     

P-type lectins

The two members of the P-type lectin family, the cation-dependent mannose 6-phosphate receptor (CD-MPR) and the insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR), are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The P-type lectins play an essential role in the generation of functional lysosomes within the cells of higher eukaryotes by directing newly synthesized lysosomal enzymes bearing the mannose 6-phosphate (M6P) signal to lysosomes. At the cell surface, the IGF-II/MPR also binds to the nonglycosylated polypeptide hormone, IGF-II, targeting this potent mitogenic factor for degradation in lysosomes. Moreover, in recent years, the multifunctional nature of the IGF-II/MPR has become increasingly apparent, as the list of extracellular ligands recognized by this receptor has grown to include a diverse spectrum of M6P-containing proteins as well as nonglycosylated ligands, implicating a role for the IGF-II/MPR in a number of important physiological pathways. Recent investigations have provided valuable insights into the molecular basis of ligand recognition by the MPRs as well as the complex intracellular trafficking pathways traversed by these receptors. This review provides a current view on the structures, functions, and medical relevance of the P-type lectins.     

Hepatoma-associated nuclear matrix nonhistone antigens

Polyclonal antibodies generated against a group of high molecular weight nonhistone proteins from Morris hepatoma 7777 were used in immunological studies of hepatoma-associated nonhistone proteins in rat and hamster. We revealed the presence of cross-reactive antigens in rat Morris hepatomas 7777 and 8994, and in hamster Kirkman-Robbins hepatoma, but not in normal rat or hamster livers. These specific nonhistone proteins were found to be preferentially localized in the nuclear matrix of rat Morris hepatoma 7777 as well as hamster Kirkman-Robbins hepatoma.     

Antibody to mannose 6-phosphate specific receptor induces receptor deficiency in human fibroblasts.

Polyclonal antibodies to the mannose 6-phosphate specific receptor from human liver inhibited the endocytosis of lysosomal enzymes in fibroblasts by greater than 95% and enhanced 3-20-fold the secretion of precursors of lysosomal enzymes in these cells. Exposing fibroblasts for 4 h to antibody resulted in loss of greater than 90% of the membrane-bound receptors. If fibroblasts were treated with the antibody in the presence of CBZ-Phe-Ala-CHN2, an inhibitor of lysosomal cysteine proteinases, the receptor and smaller degradation products are recovered in dense lysosomes. In treated cells 18-58% of total receptor-related polypeptides were recovered in dense lysosomes. In control cells less than 4% of the receptor was found in the lysosomal fraction. We conclude from these results that normally the receptor is spared from lysosomal degradation. When tagged with antibody, however, the receptor is transported into lysosomes and degraded. The loss of intracellular receptors involved in segregation of newly synthesized lysosomal enzymes indicates an exchange between the former and the plasma membrane-bound receptors.     

The novel Drosophila lysosomal enzyme receptor protein mediates lysosomal sorting in mammalian cells and binds mammalian and Drosophila GGA adaptors

Biogenesis of lysosomes depends in mammalian cells on the specific recognition and targeting of mannose 6-phosphate-containing lysosomal enzymes by two mannose 6-phosphate receptors (MPR46, MPR300), key components of the extensively studied receptor-mediated lysosomal sorting system in complex metazoans. In contrast, the biogenesis of lysosomes is poorly investigated in the less complex metazoan Drosophila melanogaster. We identified the novel type I transmembrane protein lysosomal enzyme receptor protein (LERP) with partial homology to the mammalian MPR300 encoded by Drosophila gene CG31072. LERP contains 5 lumenal repeats that share homology to the 15 lumenal repeats found in all identified MPR300. Four of the repeats display the P-lectin type pattern of conserved cysteine residues. However, the arginine residues identified to be essential for mannose 6-phosphate binding are not conserved. The recombinant LERP protein was expressed in mammalian cells and displayed an intracellular localization pattern similar to the mammalian MPR300. The LERP cytoplasmic domain shows highly conserved interactions with Drosophila and mammalian GGA adaptors known to mediate Golgi-endosome traffic of MPRs and other transmembrane cargo. Moreover, LERP rescues missorting of soluble lysosomal enzymes in MPR-deficient cells, giving strong evidence for a function that is equivalent to the mammalian counterpart. However, unlike the mammalian MPRs, LERP did not bind to the multimeric mannose 6-phosphate ligand phosphomannan. Thus ligand recognition by LERP does not depend on mannose 6-phosphate but may depend on a common feature present in mammalian lysosomal enzymes. Our data establish a potential important role for LERP in biogenesis of Drosophila lysosomes and suggest a GGA function also in the receptor-mediated lysosomal transport system in the fruit fly.     

Hepatoma-associated non-histone proteins are phosphoproteins preferentially localized in nuclear matrix

1. High molecular weight non-histone proteins (NHP) were isolated from Morris hepatoma 7777 by Sephadex G-100, S-200 chromatography. 2. Specific polyclonal antibodies were raised against these NHP in rabbits. These antibodies recognized specific NHP components present in Morris hepatoma 7777 and 8994, but not in normal rat liver. Hepatoma-associated antigens are phosphoproteins. 3. Immunologically specific NHP of Morris hepatoma are intensively concentrated in nuclear matrix fraction.     

Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts

Biosynthesis and processing of cathepsin B in cultured human skin fibroblasts were investigated using immunological procedures. Upon metabolic labeling with [35S]methionine for 10 min, a precursor form with Mr 44,500 was identified. During an 80-min chase, about 50% of it was converted to an Mr 46,000 form. Further processing yielded mature forms with Mr 33,000 and 27,000, in a final quantitative ratio of about 3:1. Processing of cathepsin B was inhibited by leupeptin, which led to an accumulation of the Mr 33,000 polypeptide. The Mr 33,000 form appeared to be the most active form and showed a half-time of about 12 h. About 5% of newly synthesized enzyme was secreted as precursor, being detectable extracellularly already after 40 min. NH4Cl enhanced the secretion of the precursor about 20-fold. The precursor and the 33-kDa form contained phosphorylated N-linked oligosaccharides. Cleavage by peptide N-glycosidase F or biosynthesis in the presence of tunicamycin yielded a precursor with Mr 39,000. Evidence of a mannose 6-phosphate-dependent transport of cathepsin B in fibroblasts was obtained on the basis of the following results: (i) cathepsin B precursor from NH4Cl-stimulated secretions was internalized in a mannose 6-phosphate inhibitable manner, and (ii) I-cell fibroblasts secreted more than 95% of newly synthesized cathepsin B precursor. In conclusion, cathepsin B from human skin fibroblasts shows an analogous biosynthetic behavior as other lysosomal enzymes.