Recognition and receptor-mediated uptake of phosphorylated high mannose- type oligosaccharides by cultured human fibroblasts

The intracellular transport of newly synthesized lysosomal hydrolases to lysosomes requires the presence of one or more phosphorylated high mannose-type oligosaccharides per enzyme. A receptor that mediates mannose-6-PO4-specific uptake of lysosomal enzymes is expressed on the surface of fibroblasts and presumably accounts for the intracellular transport of newly synthesized enzymes to the lysosome. In this study, we examined the internalization of lysosomal enzyme-derived phosphorylated oligosaccharides by cultured human fibroblasts. Oligosaccharides of known specific activity bearing a single phosphate in monoester linkage were internalized with Kuptake of 3.2 X 10(-7) M, whereas oligosaccharides bearing two phosphates in monoester linkage were internalized with a Kuptake of 3.9 X 10(-8) M. Thus, phosphorylated high mannose-type oligosaccharides appear to be the minimal structure required for recognition and uptake by the fibroblast receptor. The finding that the Kuptake for monophosphorylated oligosaccharides is 100-fold less than the reported Ki for mannose-6-phosphate indicates that the fibroblast phosphomannosyl receptor contains a binding site that recognizes features of the oligosaccharide in addition to mannose-6-phosphate.     

Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta- glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.     

Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors

We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.     

Human beta-glucuronidase pinocytosis and binding to the immobilized phosphomannosyl receptor. Effects of treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase

beta-D-Glucuronidase (EC 3.2.1.31) was purified to homogeneity from human spleen, and enzyme fractions from CM-Sephadex were examined for uptake by fibroblasts and retention by a column of immobilized phosphomannosyl receptor. Uptake and binding were enhanced by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase, greatly reduced by prior treatment with alkaline phosphatase, and restored by subsequent treatment with alpha-N-acetylglucosaminyl phosphodiesterase. Immobilized phosphomannosyl receptor was used to separate high and low uptake enzyme forms. About 25% of the total beta-glucuronidase was retained by the receptor column and eluted with mannose 6-phosphate. The rate of uptake of retained enzyme was 2.5-3.0-fold greater than that of the enzyme applied to the receptor column. The fraction retained by the column was reduced to 5-10% by prior treatment of the enzyme with alkaline phosphatase. This phosphatase-resistant, receptor-retained fraction was taken up at only 24% the rate of non-phosphatase-treated, receptor-retained enzyme. However, its uptake was increased 7-fold by treatment with alpha-N-acetylglucosaminyl phosphodiesterase. The enhanced rate of pinocytosis conferred by treatment of the enzyme with alpha-N-acetylglucosaminyl phosphodiesterase was destroyed by a subsequent treatment with alkaline phosphatase. These studies demonstrate that although most of the "high uptake" enzyme in beta-glucuronidase from human spleen binds to receptors through phosphomonoesters of mannose, a significant fraction can interact with immobilized phosphomannosyl receptor and be taken up by fibroblasts through interactions involving mannose 6-phosphate in diester linkage with N-acetyl-D-glucosamine.     

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.     

Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.     

Structural basis for recognition of phosphorylated high mannose oligosaccharides by the cation-dependent mannose 6-phosphate receptor.

Mannose 6-phosphate receptors (MPRs) play an important role in the targeting of newly synthesized soluble acid hydrolases to the lysosome in higher eukaryotic cells. These acid hydrolases carry mannose 6-phosphate recognition markers on their N-linked oligosaccharides that are recognized by two distinct MPRs: the cation-dependent mannose 6-phosphate receptor and the insulin-like growth factor II/cation-independent mannose 6-phosphate receptor. Although much has been learned about the MPRs, it is unclear how these receptors interact with the highly diverse population of lysosomal enzymes. It is known that the terminal mannose 6-phosphate is essential for receptor binding. However, the results from several studies using synthetic oligosaccharides indicate that the binding site encompasses at least two sugars of the oligosaccharide. We now report the structure of the soluble extracytoplasmic domain of a glycosylation-deficient form of the bovine cation-dependent mannose 6-phosphate receptor complexed to pentamannosyl phosphate. This construct consists of the amino-terminal 154 amino acids (excluding the signal sequence) with glutamine substituted for asparagine at positions 31, 57, 68, and 87. The binding site of the receptor encompasses the phosphate group plus three of the five mannose rings of pentamannosyl phosphate. Receptor specificity for mannose arises from protein contacts with the 2-hydroxyl on the terminal mannose ring adjacent to the phosphate group. Glycosidic linkage preference originates from the minimization of unfavorable interactions between the ligand and receptor.     

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.     

Identification of membrane proteins mediating the interaction of human platelets

Mild acid hydrolysis of phosphomannan secreted by the yeast hansenula holstii (NRRL Y- 2448) produces two phosphomannyl fragments which differ strikingly in their potency as inhibitors of pinocytosis of human β-glucuronidase by human fibroblasts. The larger molecular weight polyphosphomonoester fragment is 100,000-fold more potent an inhibitor of enzyme uptake than the smaller penta-mannosyl-monophosphate fragment. Binding to attached fibroblasts at 3 degrees C was much greater with the polyphosphomonoester fragment than with the pentamannosyl-monophosphate. The larger molecular weight fragment was also subject to adsorptive pinocytosis and was taken up by fibroblasts at a rate 30- fold greater than the rate of uptake of pentamannosyl-monophosphate. Evidence that the polyphosphomonoester fragment is taken up by the phosphomannosyl-recognition system that mediates uptake of lysosomal enzymes includes: (a) its pinocytosis is inhibited by the same compounds that competitively inhibit enzyme pinocytosis (mannose-6-phosphate and phosphomannan from saccharomyces cerevisiae mutant mnn-1); (b) alkaline phosphatase treatment greatly reduces its susceptibility to pinocytosis; (c) its pinocytosis is competitively inhibited by high-uptake human β-glucuronidase; and (d) this inhibition by high-uptake enzyme is dramatically reduced by prior treatment of the enzyme with alkaline phosphatase or endoglycosidase-H. Endoglycosidase-H treatment human β-glucuronidase dramatically reduced its susceptibility to pinocytosis by fibroblasts. The phosphomannosyl components of high- uptake enzyme released by endoglycosidase-H treatment were much less effective inhibitors of polyphosphomonoester pinocytosis than when present on the phosphomannyl-enzyme. These results suggest that high-uptake acid hydrolases may be polyvalent ligands analogous to the polyphosphomonoester mannan fragment whose pinocytosis depends on interaction of more than one phospho-mannosyl recognition marker with pinocytosis receptors on fibroblasts.     

The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

Interaction of Dictyostelium discoideum lysosomal enzymes with the mammalian phosphomannosyl receptor. The importance of oligosaccharides which contain phosphodiesters

Mammalian cell lysosomal enzymes or phosphorylated oligosaccharides derived from them are endocytosed by a phosphomannosyl receptor (PMR) found on the surface of fibroblasts. Various studies suggest that 2 residues of Man-6-P in phosphomonoester linkage but not diester linkage (PDE) are essential for a high rate of uptake. The lysosomal enzymes of the slime mold Dictyostelium discoideum are also recognized by the PMR on these cells; however, none of the oligosaccharides from these enzymes contain 2 phosphomonoesters. Instead, most contain multiple sulfate esters and 2 residues of Man-6-P in an unusual PDE linkage. In this study I have tried to account for the unexpected highly efficient uptake of the slime mold enzymes. The results show that nearly all of the alpha-mannosidase molecules contain the oligosaccharides required for uptake, and that each tetrameric, holoenzyme molecule has sufficient carbohydrate for an average of 10 Man8GlcNAc2 oligosaccharides. None of the oligosaccharides or glycopeptides from the lysosomal enzymes bind to an immobilized PMR, but those with 2 PDE show slight interaction. Competition of 125I-beta-glucosidase uptake by various carbohydrate-containing fractions indicates that the best inhibitors are those with 2 PDE, either with or without sulfate esters. Furthermore, the uptake of a lysosomal enzyme isolated from a mutant strain (modA), which produces oligosaccharides with only 1 but not 2 PDE, is about 10-fold less than the uptake of wild-type enzyme which has predominantly 2 PDE. Complete denaturation of 125I-labeled wild-type beta-glucosidase in sodium dodecyl sulfate/dithiothreitol also reduces its uptake by about 10-fold. Taken together, these results suggest that the interactions of multiple, weakly binding oligosaccharides, especially those with 2 PDE, are important for the high rate of uptake of the slime mold enzymes. The conformation of the protein may be important in orienting the oligosaccharides in a favorable position for binding to the PMR.     

Structure of the high mannose oligosaccharides of a human IgM myeloma protein. I. The major oligosaccharides of the two high mannose glycopeptides.

The structures of the predominant high mannose oligosaccharides present in a human IgM myeloma protein (Patient Wa) have been determined. The IgM glycopeptides, produced by pronase digestion, were fractionated on DEAE-cellulonalysis shows that glycopeptide I contains Asn, Pro, Ala, Thr, and His and glycopeptide II contains Asn, Val, and Ser, which are the same amino acids found in the sequences around Asn 402 and Asn 563 respectively, to which high mannose oligosaccharides are attached in IgM (Patient Ou) (Putnman, F.W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (1973) Science 182, 287-290). The high mannose glycopeptides in IgM (Wa) exhibit heterogeneity in the oligosaccharide portion. Structural analysis of the major oligosaccharides indicates that the simplest structure is: (see article of journal). The larger oligosaccharides present have additional mannose residues linked alpha 1 yields 2 to terminal mannose residues in the above structure. Glycopeptide I contains primarily Man5 and Man6 species, while glycopeptide II contains Man6 and Man8 species. The two Man6 oligosaccharides have different branching patterns.     

The mod A mutant of Dictyostelium discoideum is missing the alpha 1,3-glucosidase involved in asparagine-linked oligosaccharide processing

The recessive mutation, mod A, in the Dictyostelium discoideum strain M31 results in an alteration in the post-translational modification of lysosomal enzymes. We now report studies which indicate that mod A is deficient in glucosidase II, an enzyme which is involved in the processing of asparagine-linked oligosaccharides. [2-3H]Mannose-labeled glycopeptides were prepared from three purified mod A lysosomal enzymes and compared to the equivalent glycopeptides from parental enzymes. The mod A glycopeptides were deficient in high mannose oligosaccharides containing two phosphomannosyl residues and accumulated oligosaccharides with one phosphomannosyl residue. The phosphate was present in the form of an acid-stable phosphodiester in both instances. There was also an increase in the amount of nonphosphorylated high mannose oligosaccharides mod A and these were larger than the corresponding material from the parental enzymes. In addition, the nonphosphorylated oligosaccharides were only partially degraded by alpha-mannosidase, indicating the presence of a blocking moiety. In vitro enzyme assays demonstrated that the mod A cells cannot remove the inner 1 leads to 3-linked glucose from a glucosylated high mannose oligosaccharide. The cells are also deficient in membrane-bound neutral p-nitrophenyl-alpha-D-glucosidase activity. This activity has been attributed to glucosidase II in other systems. Removal of the outer 1 leads to 2-linked glucose from Glc3Man9Glc-NAc2 is normal, demonstrating the presence of glucosidase I activity. We conclude from these data that M31 cells are deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the glucosylated oligosaccharides of newly glycosylated proteins. This defect can explain the mod A phenotype and is proposed to be the primary genetic defect in these cells.     

Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

Over 99% of thyroxine (T4), the major form of thyroid hormone in plasma, is bound to the plasma glycoprotein thyroxine-binding globulin (TBG). The carbohydrate composition of TBG (14.6% by weight) consists of mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid in the molar ratios of 11:9:16:10 per mol of glycoprotein. No fucose or N-acetylgalactosamine were detected. Amino acid analyses were performed. Glycopeptides, prepared by exhaustive pronase treatment of the glycoprotein, were separated by gel filtration and ion exchange chromatography. All glycopeptides contained the four sugars present in the native glycoprotein. One-fourth of the glycopeptide fraction was resolved into a discrete component, glycopeptide I. The remaining glycopeptides were a mixture termed glycopeptides II and III. Glycopeptides II and III were resolved into two discrete carbohydrate units, termed oligosaccharides A and B, by alkaline-borohydride treatment and DEAE-cellulose chromatography. We propose that TBG contains four oligosaccharide chains as calculated from the molecular weights of the glycopeptides and from compositional data assuming 1 asparagine residue/glycopeptide. The carbohydrate structures of the glycopeptides and relative affinities of TBG, glycopeptides and oligosaccharides for hepatocyte plasma membrane binding are presented in the accompanying paper (Zinn, A.B., Marshall, J.S., and Carlson, D.M. (1978) J. Biol. Chem. 253, 6768-6773.     

Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human beta-hexosaminidase

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.     

Cell- and ligand-specific dephosphorylation of acid hydrolases: evidence that the mannose 6-phosphatase is controlled by compartmentalization

Mouse L cells that possess the cation-independent mannose 6-phosphate (Man 6-P)/insulin-like growth factor (IGF) II receptor change the extent to which they dephosphorylate endocytosed acid hydrolases in response to serum (Einstein, R., and C. A. Gabel. 1989. J. Cell Biol. 109:1037-1046). To investigate the mechanism by which dephosphorylation competence is regulated, the dephosphorylation of individual acid hydrolases was studied in Man 6-P/IGF II receptor-positive and -deficient cell lines. 125I-labeled Man 6-P-containing acid hydrolases were proteolytically processed but remained phosphorylated when endocytosed by receptor-positive L cells maintained in the absence of serum; after the addition of serum, however, the cell-associated hydrolases were dephosphorylated. Individual hydrolases were dephosphorylated at distinct rates and to different extents. In contrast, the same hydrolases were dephosphorylated equally and completely after entry into Man 6-P/IGF II receptor-positive Chinese hamster ovary (CHO) cells. The dephosphorylation competence of Man 6-P/IGF II receptor-deficient mouse J774 cells was more limited. beta-Glucuronidase produced by these cells underwent a limited dephosphorylation in transit to lysosomes such that diphosphorylated oligosaccharides were converted to monophosphorylated species. The overall quantity of phosphorylated oligosaccharides associated with the enzyme, however, did not decrease within the lysosomal compartment. Likewise, beta-glucuronidase was not dephosphorylated when introduced into J774 cells via Fc receptor-mediated endocytosis. The CHO and J774 cell lysosomes, therefore, display opposite extremes with respect to their capacity to dephosphorylate acid hydrolases; within CHO cell lysosomes acid hydrolases are rapidly and efficiently dephosphorylated, but within J774 cell lysosomes the same acid hydrolases remain phosphorylated. This difference in processing indicates that lysosomes themselves exist in a dephosphorylation-competent and -incompetent state. Man 6-P-bearing acid hydrolases endocytosed by the L+ cells in the absence of serum were not distributed uniformly throughout the lysosomal compartment. The change in the dephosphorylation competence of L cells in response to serum suggests, therefore, that these cells contain multiple populations of lysosomes that differ with respect to their content of a mannose 6-phosphatase, and that serum factors affect the distribution of hydrolases between the different compartments.     

Changes in surface glycopeptides after malignant transformation of rat liver cells and during the regression of hepatoma cells

Normal liver cells, Zajdela's hepatoma cells, and regressing hepatoma cells were metabolically labeled with either radioactive glucosamine or mannose. Glycopeptides obtained by exhaustive pronase digestion of these cells were compared after fractionation by gel filtration on Bio-Gel P-6. Chemical analysis, affinity chromatography on immobilized lectins, alkaline treatment, and susceptibility toward endo-beta-N-acetylglucosaminidase and tunicamycin revealed dramatic changes in the glycopeptide patterns of transformed cells during the recovery of normal phenotype. The most prominent feature was the presence on the surface of hepatoma cells of a large glycopeptide, which was absent from normal liver cells and disappeared almost completely during the regression of hepatoma cells. This large glycopeptide had a Mr of 70,000, contained essentially O-glycosidically linked glycan chains, and did not result from a hypersialylation. N-glycosidically linked glycopeptides, high-mannose, and complex-type oligosaccharides were present in distinct proportions according to the differentiation state. Transformation of liver cells led to a reduction of high-mannose type oligosaccharides and an increase in the degree of branching of complex-type oligosaccharides. In addition, "bisected" glycopeptides were present only on hepatoma cells. The pattern of N-linked glycopeptides of normal liver cells was recovered during the regression of hepatoma cells. The origin of glycopeptide differences between normal and transformed cells and the evidence of a relation between carbohydrate changes, in particular the appearance of a large glycopeptide, and tumorigenicity are discussed.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Dexamethasone increases expression of mannose receptors and decreases extracellular lysosomal enzyme accumulation in macrophages

Macrophages express a mannose-specific pinocytosis receptor that binds and internalizes lysosomal hydrolases. Treatment of rat bone marrow-derived macrophages with dexamethasone resulted in a concentration- and time-dependent increase in mannose-receptor activity. The dexamethasone effect was maximal at 24 h. Half-maximal effects were observed at a dexamethasone concentration of 2.5 X 10(-9) M. With 125I-beta-glucuronidase as ligand, a 2.5-fold increase in uptake rate was observed in dexamethasone-treated cells, with no change in Kuptake (2.5 X 10(-7) M beta-glucuronidase). Cell surface binding (4 degrees C) was elevated 2.6-fold following dexamethasone treatment. The increase in ligand binding appeared to be due to an increase in number of sites with no change in affinity. Cycloheximide suppressed the dexamethasone-mediated rise in receptor number, while cycloheximide alone had little effect on receptor activity over 16 h. These results suggest that dexamethasone stimulates synthesis of mannose receptors in macrophages. Extracellular accumulation of hexosaminidase was sharply reduced by dexamethasone treatment, and corresponded with the rise in mannose-receptor activity. Extracellular levels of hexosaminidase from untreated macrophages were modestly increased by the presence of mannan, while the extracellular activity from dexamethasone-treated cells was increased significantly by mannan. Extracellular hexosaminidase, released from zymosan-treated macrophages, was dramatically reduced by dexamethasone pretreatment. Enzyme released from zymosan-stimulated macrophages was efficiently endocytosed by dexamethasone-treated cells in co-culture experiments, and this endocytosis was blocked by the addition of mannan. These results suggest that the mannose receptor of macrophages may play a role in regulating extracellular levels of lysosomal enzymes via a secretion-recapture mechanism.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins.

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with alpha-methylmannoside, constitute about 25--30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chloride columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with alpha-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.