Biomedical subjects
S Kornfeld
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Cation-dependent mannose 6-phosphate receptor contains two internalization signals in its cytoplasmic domain.
The signals required for rapid internalization of the bovine cation-dependent mannose 6-phosphate receptor have been localized to two distinct regions of the 67-amino acid cytoplasmic domain. One signal includes phenylalanine 13 and phenylalanine 18, while the other involves tyrosine 45. The former signal is more potent than the latter, but both must be present for the maximal rate of receptor internalization. Each signal shares similarities with the known internalization signals of other recycling receptors.
Mannose 6-phosphate receptors and lysosomal enzyme targeting.
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The cation-dependent mannose 6-phosphate receptor. Structural requirements for mannose 6-phosphate binding and oligomerization.
The structural requirements for oligomerization and the generation of a functional mannose 6-phosphate (Man-6-P) binding site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) were analyzed. Chemical cross-linking studies on affinity-purified CD-MPR and on solubilized membranes containing the receptor indicate that the CD-MPR exists as a homodimer. To determine whether dimer formation is necessary for the generation of a Man-6-P binding site, a cDNA coding for a truncated receptor consisting of only the signal sequence and the extracytoplasmic domain was constructed and expressed in Xenopus laevis oocytes. The expressed protein was completely soluble, monomeric in structure, and capable of binding phosphomannosyl residues. Like the dimeric native receptor, the truncated receptor can release its ligand at low pH. Ligand blot analysis using bovine testes beta-galactosidase showed that the monomeric form of the CD-MPR from bovine liver and testes is capable of binding Man-6-P. These results indicate that the extracytoplasmic domain of the receptor contains all the information necessary for ligand binding as well as for acid-dependent ligand dissociation and that oligomerization is not required for the formation of a functional Man-6-P binding site. Several different mutant CD-MPRs were generated and expressed in X. laevis oocytes to determine what region of the receptor is involved in oligomerization. Chemical cross-linking analyses of these mutant proteins indicate that the transmembrane domain is important for establishing the quaternary structure of the CD-MPR.
Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis.
The cation-independent mannose 6-phosphate receptor (Cl-MPR) sorts newly synthesized lysosomal enzymes in the Golgi and endocytoses extracellular lysosomal enzymes. To determine the role of the 163 amino acid cytoplasmic domain of the Cl-MPR in these functions, receptor-deficient mouse L cells were transfected with normal bovine Cl-MPR cDNA or cDNAs mutated in the cytoplasmic domain. The normal Cl-MPR functioned in sorting and endocytosis. Mutant receptors with 40 and 89 residues deleted from the carboxyl terminus of the cytoplasmic tail functioned normally in endocytosis, but were partially impaired in sorting. Mutant receptors with larger deletions leaving only 7 and 20 residues of the cytoplasmic tail were defective in endocytosis and sorting. A mutant receptor containing alanine instead of tyrosine residues at positions 24 and 26 was defective in endocytosis, and partially impaired in sorting. Receptors deficient in endocytosis accumulated at the cell surface. These results indicate that the cytoplasmic domain of the Cl-MPR contains different signals for rapid endocytosis and efficient lysosomal enzyme sorting.
Ligand interactions of the cation-independent mannose 6-phosphate receptor. The stoichiometry of mannose 6-phosphate binding.
The interaction of the bovine cation-independent mannose 6-phosphate receptor with a variety of phosphorylated ligands has been studied using equilibrium dialysis and immobilized receptor to measure ligand binding. The dissociation constants for mannose 6-phosphate, pentamannose phosphate, bovine testes beta-galactosidase, and a high mannose oligosaccharide with two phosphomonoesters were 7 X 10(-6) M, 6 X 10(-6) M, 2 X 10(-8) M, and 2 X 10(-9) M, and the mol of ligand bound/mol of receptor monomer were 2.17, 1.85, 0.9, and 1.0, respectively. We conclude that the cation-independent mannose 6-phosphate receptor has two mannose 6-phosphate-binding sites/polypeptide chain.
Ligand interactions of the cation-dependent mannose 6-phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor.
The interactions of the bovine cation-dependent mannose 6-phosphate receptor with monovalent and divalent ligands have been studied by equilibrium dialysis. This receptor appears to be a homodimer or a tetramer. Each mole of receptor monomer bound 1.2 mol of the monovalent ligands, mannose 6-phosphate and pentamannose phosphate with Kd values of 8 X 10(-6) M and 6 X 10(-6) M, respectively and 0.5 mol of the divalent ligand, a high mannose oligosaccharide with two phosphomonoesters, with a Kd of 2 X 10(-7) M. When Mn2+ was replaced by EDTA in the dialysis buffer, the Kd for pentamannose phosphate was 2.5 X 10(-5) M. By measuring the affinity of the cation-dependent and cation-independent mannose 6-phosphate receptors for a variety of mannose 6-phosphate analogs, we conclude that the 6-phosphate and the 2-hydroxyl of mannose 6-phosphate each contribute approximately 4-5 kcal/mol of Gibb's free energy to the binding reaction. Neither receptor appears to interact substantially with the anomeric oxygen of mannose 6-phosphate. The receptors differ in that the cation-dependent receptor displays no detectable affinity for N-acetylglucosamine 1'-(alpha-D-methylmannopyranose 6-monophosphate) whereas this ligand binds to the cation-independent receptor with a poor, but readily measurable Kd of about 0.1 mM. The spacing of the mannose 6-phosphate-binding sites relative to each other may also differ for the two receptors.
The chicken liver cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for insulin-like growth factor II.
The chicken liver cation-independent mannose 6-phosphate receptor has been purified to apparent homogeneity by affinity chromatography on pentamannose phosphate-Sepharose and tested for its ability to bind iodinated human IGF-I, human IGF-II, and chicken IGF-II. In contrast to the bovine, rat, and human cation-independent mannose 6-phosphate receptors, which bind human IGF-II and IGF-I with nanomolar and micromolar affinities, respectively, the chicken receptor failed to bind either radioligand at receptor concentrations as high as 1 microM. The bovine receptor binds chicken IGF-II with high affinity while the chicken receptor binds this ligand with only low affinity, which we estimate to be in the micromolar range. These data demonstrate that the chicken cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for IGF-II. These results provide an explanation for the failure of previous investigators to identify the type II IGF receptor by IGF-II cross-linking to chicken cells and indicate that the mitogenic activity of IGF-II in chick embryo fibroblasts is most likely mediated via the type I IGF receptor.
Expression of the yeast aspartyl protease, proteinase A. Phosphorylation and binding to the mannose 6-phosphate receptor are altered by addition of cathepsin D sequences.
Proteinase A, a yeast aspartyl protease that is highly homologous to the mammalian lysosomal aspartyl protease, cathepsin D, was expressed in Xenopus oocytes and its biosynthesis and post-translational modifications were characterized. While 29-45% of the proteinase A was secreted from oocytes, approximately 37% of the cell-associated proteinase A underwent proteolytic cleavage, characteristic of delivery to a lysosomal organelle. Although proteinase A is not targeted to the yeast vacuole by a mannose 6-phosphate receptor-dependent pathway, 2-5% of the proteinase A molecules expressed in oocytes bound to a Man-6-P receptor column. However, analysis of its [2-3H]mannose-labeled oligosaccharides revealed that 14-23% of these units contain phosphomannosyl residues. A hybrid molecule (H6), in which the propiece and first 12 amino acids of proteinase A were changed to the cathepsin D sequence, was also expressed in oocytes. The binding of H6 to the Man-6-P receptor was approximately 12-fold greater than observed for proteinase A. This increased level of receptor binding could be accounted for by three factors: 1) a small increase in the total amount of phosphorylated oligosaccharides, 2) an increase in the number of oligosaccharides which acquire two phosphomonoesters, and 3) the presence of a greater percentage of oligosaccharides with one phosphomonoester which exhibit high affinity binding to the Man-6-P receptor. These results demonstrate that proteinase A is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase. However, this interaction is altered by the addition of cathepsin D sequences, resulting in the generation of a higher affinity ligand for binding to the Man-6-P receptor.
Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors.
We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti-peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD-MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR-containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.
The cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II.
The bovine cation-independent mannose 6-phosphate receptor (CI-MPR) and the human insulin-like growth factor II (IGF-II) receptor have recently been shown to be 80% identical in their amino acid sequences as deduced from cDNA clones (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). We have studied the binding of IGF-II to affinity-purified CI-MPR in order to obtain direct evidence that the same protein binds mannose 6-phosphate-containing ligands and IGF-II. In equilibrium binding studies, the CI-MPR bound 0.95 mol of IGF-II/mol of receptor with a Kd of 0.2 nM. The pH optimum of binding was 7.4. The addition of mannose 6-phosphate did not affect binding, indicating that the two ligands interact with different binding sites on the receptor. IGF-I bound to the receptor with a much lower affinity (Kd of 0.4 microM), and insulin binding could not be detected. IGF-II did not bind to the cation-dependent mannose 6-phosphate receptor. We conclude that the cation-independent mannose 6-phosphate receptor and the IGF-II receptor are the same protein.
Cloning and sequence analysis of the cation-independent mannose 6-phosphate receptor.
We have isolated and sequenced cDNA clones encoding the entire sequence of the bovine cation-independent mannose 6-phosphate receptor. The deduced 2499-amino acid precursor has a calculated molecular mass of 275 kDa. Analysis of the sequence indicates that the protein has a 44-residue amino-terminal signal sequence, a 2269-residue extracytoplasmic region, a single 23-residue transmembrane region, and a 163-residue carboxyl-terminal cytoplasmic region. The extra-cytoplasmic region consists of 15 contiguous repeating domains, one of which contains a 43-residue insertion that is similar to the type II repeat of fibronectin. The 15 domains have an average size of 147 amino acids and a distinctive pattern of 8 cysteine residues. Alignment of the 15 domains and the extracytoplasmic domain of the cation-dependent mannose 6-phosphate receptor shows that all have sequence similarities and suggests that all are homologous.
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.
Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus.
We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.
Studies of the biosynthesis of the cation-dependent mannose 6-phosphate receptor in murine cell lines.
We have studied the biosynthesis of the cation-dependent mannose 6-phosphate receptor in murine BW5147 lymphoma cells and MOPC 315 plasmacytoma cells. The cells were labeled with [35S]methionine or [2-3H]mannose and the receptor immunoprecipitated with an anti-receptor antiserum. The receptor was first detected as a glycoprotein with an apparent molecular mass of 40 kDa. This intermediate was rapidly processed to a mature form which was stable during 22 h of chase. In these cells, the mature receptor has an apparent molecular mass of 43 kDa. The 3-kDa increase occurs as a result of processing of Asn-linked high-mannose oligosaccharides to complex-type units.
46 kd mannose 6-phosphate receptor: cloning, expression, and homology to the 215 kd mannose 6-phosphate receptor.
We have isolated cDNA clones encoding the entire sequence of the bovine 46 kd cation-dependent mannose 6-phosphate (CD Man-6-P) receptor. Translation of CD Man-6-P receptor mRNA in Xenopus laevis oocytes results in a protein that binds specifically to phosphomannan-Sepharose, thus demonstrating that our cDNA clones encode a functional receptor. The deduced 279 amino acid sequence reveals a single polypeptide chain that contains a putative signal sequence and a transmembrane domain. Trypsin digestion of microsomal membranes containing the receptor and the location of the five potential N-linked glycosylation sites indicate that the receptor is a transmembrane protein with an extracytoplasmic amino terminus. This extracytoplasmic domain is homologous to the approximately 145 amino acid long repeating domains present in the 215 kd cation-independent Man-6-P receptor.
The high mannose oligosaccharides of Dictyostelium discoideum glycoproteins contain a novel intersecting N-acetylglucosamine residue.
Previous studies have shown that the majority of the asparagine-linked high mannose oligosaccharide units present on the lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-sulfate and mannose 6-phosphate residues, the latter being diesterified to methyl groups. Using a variety of techniques, including NMR spectroscopy and fast atom bombardment mass spectrometry, we have found that many of these oligosaccharides also contain an N-acetylglucosamine residue linked beta 1-4 to the mannose residue linked alpha 1-6 to the beta-linked core mannose. (Formula: see text) As far as we are aware, this is the first description of an intersecting N-acetylglucosamine residue in this position on a high mannose oligosaccharide.