The mannose-6-phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae

Mannose-6-phosphate (Man-6-P) receptors for lysosomal enzymes were localized by immunocytochemistry in several secretory and adsorptive cell types using monospecific antireceptor antibodies. By immunofluorescence, the receptors were found in the Golgi region of polarized cells. When localized by immunoperoxidase at the electron microscope level, they were detected in Golgi cisternae, coated vesicles, endosomes, and lysosomes of all cell types examined (hepatocytes, exocrine pancreatic and epididymal epithelia). Within the Golgi complex, immunoreactive receptors were restricted in their distribution to one or two cisternae on the cis side of the Golgi stacks. They were not detected in trans Golgi or GERL cisternae. Based on their high concentration of Man-6-P receptors, we propose that the cis Golgi cisternae represent the site where the secretory and lysosomal pathways diverge: lysosomal enzymes bearing the Man-6-P recognition marker bind to Man-6-P receptors in this location and are delivered to endosomes and lysosomes via coated vesicles.     

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.     

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.     

Mannose 6-phosphate receptor dependent secretion of lysosomal enzymes.

BHK and mouse L cells transfected with the cDNA for the human 46 kd mannose 6-phosphate receptor (MPR 46) secrete excessive amounts of newly synthesized mannose 6-phosphate containing polypeptides. The secretion is dependent on the amount, the recycling and the affinity for ligands of MPR 46. Incubation of transfected cells with antibodies blocking the binding site of MPR 46 reduces the secretion, and cotransfection with the cDNA for the human 300 kd mannose 6-phosphate (MPR 300) restores it to normal values. These results indicate that the two mannose 6-phosphate receptors compete for binding of newly synthesized ligands. In contrast to ligands bound to MPR 300, those bound to the MPR 46 are transported to and released at a site, e.g. early endosomes or plasma membrane, from where they can exit into the medium. Since antibodies blocking the binding site of MPR 46 reduce secretion also in non-transfected BHK and mouse L cells, at least part of the basal secretion of M6P-containing polypeptides is mediated by the endogenous MPR 46.     

Polymorphism of mannose-6-phosphate isomerase in cattle

Enzymo-electrophoresis and enzymo-electrofocusing of white cells and of muscular extracts of cattle has revealed four kinds of phenotypic patterns. Evidence presented here indicates that three of these phenotypes are controlled by two codominant, autosomal alleles, MPI ^B and MPI ^C. The allele MPI ^C occurs with a frequency of about 0.10 in most of the breeds studied and of about 0.03 in the Charolais breed. The fourth, very rare, two-banded phenotype, encountered mostly in purebred and crossbred Charolais, is believed to be under the dependence of a third allele MPI ^A but the breeding tests are lacking.     

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.     

Differential sorting of lysosomal enzymes in mannose 6-phosphate receptor-deficient fibroblasts.

In higher eukaryotes, the transport of soluble lysosomal enzymes involves the recognition of their mannose 6-phosphate signal by two receptors: the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). It is not known why these two different proteins are present in most cell types. To investigate their relative function in lysosomal enzyme targeting, we created cell lines that lack either or both MPRs. This was accomplished by mating CD-MPR-deficient mice with Thp mice that carry a CI-MPR deleted allele. Fibroblasts prepared from embryos that lack the two receptors exhibit a massive missorting of multiple lysosomal enzymes and accumulate undigested material in their endocytic compartments. Fibroblasts that lack the CI-MPR, like those lacking the CD-MPR, exhibit a milder phenotype and are only partially impaired in sorting. This demonstrates that both receptors are required for efficient intracellular targeting of lysosomal enzymes. More importantly, comparison of the phosphorylated proteins secreted by the different cell types indicates that the two receptors may interact in vivo with different subgroups of hydrolases. This observation may provide a rational explanation for the existence of two distinct mannose 6-phosphate binding proteins in mammalian cells.     

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.     

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.     

Mannose-6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes

We have examined the distribution of mannose-6-phosphate (Man6P) receptors (215 kD) for lysosomal enzymes in cultured Clone 9 hepatocytes at various times after the addition or removal of lysosomotropic weak bases (chloroquine or NH4Cl). Our previous studies demonstrated that after treatment with these agents, Man6P receptors are depleted from their sorting site in the Golgi complex and accumulate in dilated vacuoles that could represent either endosomes or lysosomes (Brown, W. J., E. Constantinescu, and M. G. Farquhar, 1984, J. Cell Biol., 99:320-326). We have now investigated the nature of these vacuoles by labeling NH4Cl-treated cells simultaneously with anti-Man6P receptor IgG and lysosomal or endosomal markers. The structures in which the immunolabeled receptors are found were identified as endosomes based on the presence of endocytic tracers (lucifer yellow and cationized ferritin). The lysosomal membrane marker, lgp120, was associated with a separate population of swollen vacuoles that did not contain detectable Man6P receptors. When cells were allowed to recover from weak base treatment, the receptors reappeared in the Golgi cisternae of most cells (approximately 90%) within approximately 20 min, indicating that as the intra-endosomal pH drops and lysosomal enzymes dissociate, the entire population of receptors rapidly recycles to Golgi cisternae. When NH4Cl-treated cells were allowed to endocytose Man6P, a competitive inhibitor of lysosomal enzyme binding, the receptors also recycled to the Golgi cisternae, suggesting that lysosomal enzymes can dissociate from the receptors under these conditions (high pH + presence of competitive inhibitor). From these results it can be concluded that the intracellular itinerary of the 215-kD Man6P receptor involves its cycling via coated vesicles between the Golgi complex and endosomes, ligand dissociation is both necessary and sufficient to trigger the recycling of Man6P receptors to the Golgi complex, and endosomes rather than secondary lysosomes represent the junction where endocytosed material and primary lysosomes carrying receptor-bound lysosomal enzymes meet.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arf regulates interaction of GGA with mannose-6-phosphate receptor

The role of ADP-ribosylation factor (Arf) in Golgi associated, γ-adaptin homologous, Arf-interacting protein (GGA)-mediated membrane traffic was examined. GGA is a clathrin adaptor protein that binds Arf through its GAT domain and the mannose-6-phosphate receptor through its VHS domain. The GAT and VHS domains interacted such that Arf and mannose-6-phosphate receptor binding to GGA were mutually exclusive. In vivo , GGA bound membranes through either Arf or mannose-6-phosphate receptor. However, mannose-6-phosphate receptor excluded Arf from GGA-containing structures outside of the Golgi. These data are inconsistent with predictions based on the model for Arf's role in COPI veside coat function. We propose that Arf recruits GGA to a membrane and then, different from the current model, 'hands-off' GGA to mannose-6-phosphate receptor. GGA and mannose-6-phosphate receptor are then incorporated into a transport intermediate that excludes Arf .     

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.     

Thyroglobulin, the major and obligatory exportable protein of thyroid follicle cells, carries the lysosomal recognition marker mannose-6-phosphate.

Thyroglobulin (TG), the major exportable protein of thyroid follicle cells, is conveyed to lysosomes on a complex secretion, storage and recapture pathway by as yet unknown transport mechanisms. This report establishes that the dimeric porcine TG-molecule carries an average of six phosphate residues. Endoglycosidase digestion showed that two phosphate residues are bound to the high-mannose carbohydrate side chains (CHO), while two others are linked to the complex CHO. These four residues are also sensitive to alkaline phosphatase treatment, indicating their terminal linkage. Immunoprecipitation analyses showed that TG obtained from microsomal fractions is already phosphorylated. Most important, an enzymatic assay applied to hydrolysates of TG established that the two phosphate residues at the high mannose CHO are present as mannose-6-phosphate (M-6-P). Alkaline phosphatase treatment of biosynthetically radiophosphorylated CHO followed by hydrolysis and t.l.c. indicated that M-6-P is present at least in part in phosphomonoester linkage. Furthermore, porcine TG binds specifically to the M-6-P receptor of Chinese hamster ovary cells. It is concluded that the M-6-P residues of TG are exposed and able to operate as a ligand for the M-6-P receptor. It is unknown why the lysosomal recognition-marker M-6-P does not convey TG directly on an intracellular route to lysosomes. We propose that for the secretion of newly synthesized TG into the follicle lumen an additional export signal dominating over the M-6-P recognition-marker is required.     

Four new alleles at the mannose-6-phosphate isomerase locus in rabbit

Four new alleles at the rabbit ( Oryctolagus cuniculus ) mannose-6-phosphate isomerase (E. C. 5. 3. 1. 8, MPI) locus are proposed to account for phenotypes observed after starch gel electrophoresis and enzymatic staining of red cell lysates and tissues. Population data from various wild and domestic rabbit populations are presented.     

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.     

Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags—2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)—were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses.     

Endocytosis of mannose-6-phosphate binding sites by mouse T-lymphoma cells

The endocytosis and intracellular transport of mannose-6-phosphate conjugated to bovine serum albumin (Man-6-P:BSA) by mouse T-lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man-6-P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man-6-P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent- or 125I-labeled Man-6-P:BSA at 0 degree C revealed a uniform distribution of the Man-6-P binding sites over the cell surface. Competition experiments indicate that the Man-6-P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37 degrees C, endocytosis of Man-6-P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5-15-min incubation of cells at 37 degrees C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man-6-P:BSA in the Golgi region after 15-30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man-6-P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30-min incubation at 37 degrees C, the internalized Man-6-P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co-stained for acid phosphatase. These results demonstrate a temporal participation of clathrin-containing coated vesicles during the initial endocytosis of Man-6-P binding sites and that one step in the Man-6-P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.