phosphorus-radioisotopes and mannose-6-phosphate

BHK cells transfected with human cathepsin D (CD) cDNA normally segregate the autologous hamster cathepsin D while secreting a large proportion of the human proenzyme. In the present work, we have utilized these transfectants to examine to what extent the mannose-6-phosphate-dependent pathway for lysosomal enzyme segregation contributes to the differential sorting of human and hamster CD. We report that, in recipient control BHK cells, the rate of mannose-6-phosphate-dependent endocytosis of human procathepsin D secreted by transfected BHK cells is lower than that of hamster procathepsin D and much lower than that of human arylsulphatase A. The missorted human enzyme bears phosphorylated oligosaccharides and most of its phosphate residues are "uncovered", like the autologous enzyme. Thus, despite both the Golgi-associated modifications of oligosaccharides, i.e. the phosphorylation of mannose and the uncovering of mannose-6-phosphate residues, which proceed on human and hamster procathepsin D with comparable efficiency, only the latter is accurately packaged into lysosomes. Ammonium chloride partially affects the lysosomal targeting of cathepsin D in control BHK cells, whereas in transfected cells, this drug strongly inhibits the maturation of human procathepsin D and slightly enhances its secretion. These data indicate that: (1) over-expression of a lysosomal protein does not saturate the Golgi-associated reactions leading to the synthesis of mannose-6-phosphate; (2) a portion of cathepsin D is targeted independently of mannose-6-phosphate receptors in the transfected BHK cells; and (3) whichever mechanism for lysosomal delivery of autologous procathepsin D is involved, this is not saturated by the high rate of expression of human cathepsin D.

Topics: Ammonium Chloride; Animals; Cathepsin D; Cell Line; Cricetinae; Endocytosis; Enzyme Precursors; Humans; Lysosomes; Mannosephosphates; Phosphorus Radioisotopes; Phosphorylation; Receptor, IGF Type 2; Recombinant Fusion Proteins; Species Specificity; Sulfur Radioisotopes; Transfection

There are at least three stages in the targeting of soluble lysosomal enzymes: transfer of N-acetylglucosaminyl 1-phosphate to high-mannose oligosaccharide side chains, removal of N-acetylglucosamine and recognition of the "uncovered" mannose 6-phosphate residues. Defects in the transfer reaction cause mucolipidoses II and III. Those in the subsequent stages of the targeting may result in similar clinical disorders. To differentiate between possible defects of the targeting in cultured cells we have developed a procedure for a combined detection of the phosphorylation, uncovering of the transferred phosphate residues and the targeting of lysosomal enzymes. For this purpose cultured cells are metabolically labelled with [32P]phosphate and a lysosomal enzyme, such as cathepsin D, is isolated from the labelled cells and the medium by immunoprecipitation. The immunoprecipitates are dissolved with sodium dodecylsulphate and incubated in the presence and absence of calf intestine alkaline phosphatase. We show that the treatment of the denatured protein results in hydrolysis of phosphomonoester groups and that the phosphodiester and the peptide bonds remain intact. The initial and the residual radioactivity associated with the lysosomal enzyme which represent the total phosphate and the phosphodiester groups, respectively, are determined by gel-electrophoresis, fluorography and densitometry. This procedure extends one of the previously established methods for the diagnosis of mucolipidoses II and III.

Topics: Calcitriol; Cathepsin D; Cell Line; Humans; Lysosomes; Mannosephosphates; Methionine; Oligosaccharides; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Radioisotope Dilution Technique; Sulfur Radioisotopes

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.

Topics: Animals; Cell Line; ErbB Receptors; Glycosylation; Hexosephosphates; Mannose; Mannosephosphates; Phosphates; Phosphorus Radioisotopes; Protein Processing, Post-Translational; Tritium

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.

Topics: Animals; Hexosephosphates; Lysosomes; Mannosephosphates; Microsomes; Organ Culture Techniques; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Swine; Thyroglobulin; Thyroid Gland