Inhibition by lysosomal enzymes and mannose-6-phosphate of the phosphorylation of the lysosomal enzyme binding receptor protein from monkey brain

The lysosomal enzymes beta-glucuronidase and alpha-L-fucosidase and mannose-6-phosphate inhibited the phosphorylation of the lysosomal enzyme binding receptor protein prepared from monkey brain. Inhibition of both serine and tyrosine phosphorylation was observed. A non-lysosomal glycoprotein enzyme butyrylcholinesterase, mannose or glucose did not inhibit phosphorylation. Tyrosine phosphorylation of histone by the receptor protein was also inhibited by the lysosomal enzymes and mannose-6-phosphate.     

Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463)

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.     

Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly

Two related transmembrane proteins in mammalian cells bind the mannose 6-phosphate recognition marker present on all soluble lysosomal hydrolases. Recent studies of cells that express only one or neither of these proteins have shed light not only on their function in directing lysosomal enzymes into the endocytic pathway but also on their critical role in transport vesicle formation in the trans Golgi network. One of these proteins also binds insulin-like growth factor II (IGF-II) and may be an important part of the IGF-dependent system that regulates development.     

Polarized secretion of lysosomal enzymes: co-distribution of cation- independent mannose-6-phosphate receptors and lysosomal enzymes along the osteoclast exocytic pathway

The osteoclast is a polarized cell which secretes large amounts of newly synthesized lysosomal enzymes into an apical extracellular lacuna where bone resorption takes place. Using immunocytochemical techniques, we have localized the cation-independent mannose-6-phosphate (Man6P) receptor and lysosomal enzymes in this cell type in order to determine the expression and distribution of this receptor and its ligands. The results demonstrate that the osteoclast expresses large amounts of immunoreactive cation-independent Man6P receptors, despite the fact that most of the lysosomal enzymes it synthesizes are secreted. The lysosomal enzymes and the receptors are co-distributed along the exocytic pathway, i.e., the endoplasmic reticulum, including the perinuclear envelope, the Golgi stacks as well as numerous small transport vesicles that appear to fuse with the ruffled border membrane. Within the Golgi complex, the receptors and lysosomal enzymes were found distributed in two predominant patterns; (a) in all the cisternae, from cis to trans, or (b) predominantly in cis- and trans-Golgi cisternae, with the middle Golgi cisternae being unstained or depleted in antigen. This pattern suggests that enzymes and receptors traverse the Golgi from cis to trans and preferentially accumulate in cis- and in trans-cisternae. This study therefore suggests that, in the osteoclast, Man6P receptors are involved in the vectorial transport and targeting of newly synthesized lysosomal enzymes, presumably via a constitutive pathway, to the apical membrane where they are secreted into the bone-resorbing compartment. This mechanism could insure polarized secretion of lysosomal enzymes into the bone-resorbing lacuna.     

Interactions of the receptor for insulin-like growth factor II with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329, 301-307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84, 2233-2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr-250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.     

Mannose 6-phosphate receptor in porcine thyroid follicle cells. Localization and possible implications for the intracellular transport of thyroglobulin

Thyroglobulin has been shown to be phosphorylated and to carry the mannose 6-phosphate (M6P) signal in terminal position. In order to investigate whether the cation-independent mannose 6-phosphate receptor (CI-MPR) can possibly play a role in the transport of thyroglobulin the localization of the receptor was analyzed in thyroid follicle cells. The immunocytochemical observations showed that the CI-MPR is primarily located in elements of the endocytic pathway such as coated pits and endosomes. This localization of the CI-MPR in thyrocytes differs from the receptor sites in other cell types by the rare occurrence of the CI-MPR in cisternae of the Golgi complex. The observations are interpreted as an indication that the relatively small amount of receptor in the Golgi complex might be occupied primarily by lysosomal hydrolases. The CI-MPR in thyrocytes might, therefore, be unable to bind and to convey thyroglobulin efficiently. The receptor is, however, a binding site for thyroglobulin at the apical plasma membrane and may, therefore, be involved in the binding of thyroglobulin and its transfer from the follicle lumen to lysosomes.     

Suppression of the 'uncovering' of mannose-6-phosphate residues in lysosomal enzymes in the presence of NH4Cl

The uncovering ratio of phosphate groups in lysosomal enzymes is defined as the percentage of phosphomonoester groups in the oligosaccharide side chains based on the sum of phosphomonoester and phosphodiester groups. Using a new procedure for the specific and complete hydrolysis of uncovered phosphomonoester groups in denatured immunoprecipitates of human cathepsin D, we show that the uncovering ratio varies between different forms of the enzyme and may be used as an indicator of the maturation of its carbohydrate side chains. The uncovering ratio in the total (cellular and secreted) cathepsin D from U937 promonocytes is greater than 95%. It is only slightly decreased in cells incubated in the presence of 1 alpha,25-dihydroxycholecalciferol, in which the rate of synthesis of cathepsin D is several times higher than in the control cells. In U937 cells and also in fibroblasts, the uncovering is nearly complete in intermediate and mature forms of the intracellular cathepsin D but less extensive in the intracellular and secreted precursor. In both cell types, incubation with 10 mM NH4Cl results in a decrease in the uncovering ratio of total cathepsin D. However, the activity of the uncovering enzyme, N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, as determined with UDP-N-acetylglucosamine is not affected with up to 60 mM NH4Cl. Our results suggest that NH4Cl, in addition to its known effects on the acidic-pH-dependent functions of lysosomal compartments and of mannose-6-phosphate receptors, impairs the processing or transport of lysosomal enzyme precursors at, or proximally to, the site of the uncovering of their mannose-6-phosphate residues.     

Assembly of the mannose-6-phosphate receptor into reconstituted clathrin coats.

In ionic conditions in which clathrin coats are stable, the mannose-6-phosphate receptor associates with the 100-kd/50-kd coat complexes purified from bullock brain coated vesicles. These aggregates exist as striking spherical structures of 300-1000 A diameter. When clathrin is included in the assembly mixture, cages are formed which apparently encapsulate these aggregates, giving, in the absence of lipid, structures resembling full coated vesicles.     

Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.     

Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation

The biosynthesis of the mannose 6-phosphate recognition marker has been studied in transport-impaired mouse lymphoma cells to determine the subcellular location of the processing enzymes and to characterize the biosynthetic intermediates. Cells were labeled with [2-3H]mannose and chased at a low temperature (15 or 20 degrees C) or at 37 degrees C in the presence of m-chlorocarbonylcyanide phenylhydrazone to disrupt transport of the pulse-labeled molecules within the secretory apparatus. Both treatments inhibited the migration of the pulse-labeled glycoproteins to the Golgi apparatus as measured by the production of complex-type asparagine-linked oligosaccharides. Despite this inhibition in protein transport, acid hydrolases were phosphorylated. Structural analysis of the phosphorylated oligosaccharides indicated that the transport-impaired cells produced a single species of phosphorylated high mannose oligosaccharide; essentially all of the molecules contain a single phosphodiester group that is restricted to the alpha 1,6 branch of the oligosaccharide. The results suggest that synthesis of mannose 6-phosphate-bearing high mannose oligosaccharides occurs in an ordered, compartmentalized posttranslational process. The initial phosphorylation of newly synthesized acid hydrolases occurs at a pre-Golgi site and results in the production of high mannose-type units that contain a single phosphodiester group. In a subsequent compartment, probably within the Golgi apparatus, the monophosphorylated units may be converted to diphosphorylated forms. Finally, at a site distal to the phosphorylation reactions the diesters are hydrolyzed to reveal the mannose 6-phosphate recognition marker.     

Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.

We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.     

Role of mannose-6-phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission.

Herpes simplex virus (HSV) glycoprotein D (gD) is essential for virus entry into cells, is modified with mannose-6-phosphate (M-6-P), and binds to both the 275-kDa M-6-P receptor (MPR) and the 46-kDa MPR (C. R. Brunetti, R. L. Burke, S. Kornfeld, W. Gregory, K. S. Dingwell, F. Masiarz, and D. C. Johnson, J. Biol. Chem. 269:17067-17074, 1994). Since MPRs are found on the surfaces of mammalian cells, we tested the hypothesis that MPRs could serve as receptors for HSV during virus entry into cells. A soluble form of the 275-kDa MPR, derived from fetal bovine serum, inhibited HSV plaques on monkey Vero cells, as did polyclonal rabbit anti-MPR antibodies. In addition, the number and size of HSV plaques were reduced when cells were treated with bovine serum albumin conjugated with pentamannose-phosphate (PM-PO4-BSA), a bulky ligand which can serve as a high-affinity ligand for MPRs. These data imply that HSV can use MPRs to enter cells; however, other molecules must also serve as receptors for HSV because a reasonable fraction of virus could enter cells treated with even the highest concentrations of these inhibitors. Consistent with the possibility that there are other receptors, HSV produced the same number of plaques on MPR-deficient mouse fibroblasts as were produced on normal mouse fibroblasts, but there was no inhibition with PM-PO4-BSA with either of these embryonic mouse cells. Together, these results demonstrate that HSV does not rely solely on MPRs to enter cells, although MPRs apparently play some role in virus entry into some cell types and, perhaps, act as one of a number of cell surface molecules that can facilitate entry. We also found that HSV produced small plaques on human fibroblasts derived from patients with pseudo-Hurler's polydystrophy, cells in which glycoproteins are not modified with M-6-P residues and yet production of infectious HSV particles was not altered in the pseudo-Hurler cells. In addition, HSV plaque size was reduced by PM-PO4-BSA; therefore, it appears that M-6-P residues and MPRs are required for efficient transmission of HSV between cells, a process which differs in some respects from entry of exogenous virus particles.     

Synthesis and receptor binding affinity of carboxylate analogues of the mannose 6-phosphate recognition marker

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is involved in multiple physiological pathways including targeting of lysosomal enzymes, degradation of IGF2, and cicatrization through TGFbeta activation. To target potential therapeutics to this membrane receptor, four carboxylate analogues of mannose 6-phosphate (M6P) were synthesized. Three of them, two isosteric carboxylate analogues and a malonate derivative, showed a binding affinity for the M6P/IGF2R equivalent to or higher than that of M6P. Contrary to M6P, all these analogues were particularly stable in human serum. Moreover, these derivatives did not present any cytotoxic activity against two human cell lines. These analogues represent a new potential for the lysosomal targeting of enzyme replacement therapy in lysosomal diseases or to prevent the membrane-associated activities of the M6P/IGF2R.     

Estradiol down-regulates the mannose-6-phosphate/insulin-like growth factor-II receptor gene and induces cathepsin-D in breast cancer cells: a receptor saturation mechanism to increase the secretion of lysosomal proenzymes.

We have studied the regulation by estradiol of the mannose-6-phosphate (Man-6-P)/insulin-like growth factor-II (IGF-II) receptor concentration in different breast cancer cell lines. The mRNA level was assayed by Northern blot using the H5.1 cDNA probe. The protein level was assayed by Western ligand blot, by binding saturation with [125I]procathepsin-D on total membrane preparations, and by immunoprecipitation of 35S-labeled proteins. In three estrogen receptor-positive cell lines (MCF7, T47D, and ZR75-1), estradiol specifically decreased the steady state level of the Man-6-P/IGF-II receptor protein and mRNA. Moreover, in different cell lines and in primary culture of normal mammary cells, the secretion of procathepsin-D was inversely correlated with the level of Man-6-P/IGF-II receptor protein and mRNA. We conclude that estradiol down-regulates the Man-6-P/IGF-II receptor in breast cancer cells. Since two of its ligands, procathepsin-D and IGF-II, are induced by estrogen, we propose that the Man-6-P/IGF-II receptor becomes saturated after estrogen treatment. This model might explain the previously described estrogen-induced secretion of procathepsin-D and other lysosomal proenzymes routed by the same transport system.     

Transplanted LDL and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor.

The internalization signals of several constitutively recycling receptors have recently been identified as regions of four or six amino acids that include an aromatic residue, usually tyrosine. Here, we show that transplanted signals from the low density lipoprotein receptor (LDLR) and cation-independent mannose-6-phosphate receptor (Man-6-PR) promote rapid internalization of the transferrin receptor (TR), directly establishing that recognition signals are interchangeable, self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. We also show that the chemical and spatial patterns of critical residues in both four- and six-residue internalization motifs are consistent with a tight turn structure. A six-residue LDLR signal is needed for activity in TR, suggesting that an amino-terminal aromatic side chain is obligatory. In contrast, the carboxy-terminal aromatic side chain in the TR signal can be replaced by a large hydrophobic residue. Thus, internalization signals apparently require an aromatic amino-terminal residue and either an aromatic or large hydrophobic carboxy-terminal residue rather than a conserved tyrosine per se. Consistent with this conclusion, predicted internalization signals from the poly-Ig receptor, YSAF, and asialoglycoprotein receptor (ASGPR) subunit H1, YQDL, also promote internalization of TR.     

A role for the insulin-like growth factor II/mannose-6-phosphate receptor in the insulin-induced inhibition of protein catabolism

Insulin and insulin-like growth factor (IGF)-I inhibit intracellular protein degradation in a variety of different cell types. In the present studies, the IGF-I-induced inhibition of protein metabolism in Chinese hamster ovary (CHO) cells was found to be blocked by polyclonal antibodies to the IGF-II/mannose-6-phosphate phosphate (Man-6-P) receptor, but not by control immunoglobulin. In contrast, these antibodies had no effect on the ability of IGF-I to stimulate glucose uptake in the same cells. The antibodies to the IGF-II/Man-6-P receptor also inhibited the effect of IGF-I and insulin on protein catabolism in human foreskin fibroblasts and human hepatoma cells, respectively. Moreover, CHO cells overexpressing a cDNA coding for the IGF-II/Man-6-P receptor were found to exhibit an increased effect of insulin on protein catabolism. In contrast, the insulin stimulation of glucose uptake is the same in these transfected cells as in the parental CHO cells. These results implicate the IGF-II/Man-6-P receptor in the insulin- and IGF-I-induced inhibition of protein catabolism.     

Rate of protein transport in thyroid follicle cells of normal, thyroxine-treated and TSH-injected rats

The transport of labeled protein in thyroid follicles was studied with quantitative electron microscopic autoradiography in normal and T4-treated rats (2d) injected with 3H-leucine 1 to 6 h before perfusion fixation. During this time interval the total amount of labeled protein in either group was unchanged, although T4-treatment caused a reduction by about 30% of the amount of 3H-leucine incorporated into protein. The autoradiographic data were corrected for the effect of scatter of radioactivity. The relative amounts of labeled, exportable protein in the compartments Er-Golgi and exocytotic vesicles were then estimated. The half-lives of labeled, exportable protein in these compartments were calculated with non-linear regression analysis. In normal rats the half-life of labeled, exportable protein in ER-Golgi was 28 min and in the exocytotic vesicles 18 min. Inhibition of TSH-secretion by injection of thyroxine decreased the rate of protein transport through the follicle cell and increased the half-lives to 63 min (ER-Golgi) and 62 min (exocytotic vesicles). TSH given to thyroxine-treated rats 20 min or 1.5 h before fixation reduced the half-lives of labeled, exportable protein in ER-Golgi to 25 to 33 min and in exocytotic vesicles to 9 min. The findings indicate that TSH regulates the rate of intracellular protein transport in rat thyroid follicle cells at the exocytotic step as well as at an earlier step in the pathway of intracellular protein transport. The mechanism and exact location of the latter TSH regulated step is at present unknown.     

The role of mannose-6-phosphate receptor and autophagy in influencing the outcome of combination therapy

Combining two different treatment modalities for targeting malignancies is gaining importance, with preclinical/clinical results indicating higher success rates in eradicating tumors or having longer remission periods. A better understanding of the synergy between the treatments helps in optimizing the dose and time of administration. We found that chemotherapy enhanced the levels of insulin-like growth factor 2 receptor/cation-independent mannose-6-phosphate receptor (IGF2R) on the surface of tumor cells, which leads to better tumor targeting by cytotoxic T cells (CTLs). Early evidence indicates that upregulation of IGF2R involves the autophagy pathway.