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.     

A fascination with sugars

We now recognize that a large number of membrane and soluble proteins contain covalently linked oligosaccharides that exhibit a vast array of structures and participate in a wide variety of biological processes. Nowhere is this better illustrated than the mannose 6-phosphate (Man-6-P) recognition system that mediates the trafficking of newly synthesized acid hydrolases to lysosomes in higher eukaryotes. The Asn-linked high-mannose oligosaccharides of these hydrolases facilitate folding of the nascent proteins in the endoplasmic reticulum via interaction with lectin-type chaperones and after phosphorylation in the Golgi, function as ligands for binding to Man-6-P receptors, a critical step in their transport to lysosomes. Failure to synthesize the Man-6-P recognition marker results in a serious lysosomal storage disease, one of a growing number of genetic conditions, termed congenital disorders of glycosylation, that result from faulty glycan biosynthesis.     

Antibody to the phosphomannosyl receptor inhibits recycling of receptor in fibroblasts

The 215-kd phosphomannosyl receptor is involved in the transport of newly synthesized acid hydrolases to lysosomes and also mediates the pinocytosis of lysosomal enzymes by fibroblasts in culture. Recycling of receptors to the sorting sites is an integral part of both these processes. In this report, we describe the inhibition in human fibroblasts of both functions of the phosphomannosyl receptor by a rabbit antiserum to the bovine liver receptor. This inhibition cannot be completely accounted for by inhibition of ligand-receptor interaction. Rather the antibody appears to cross-link receptors and cause a removal of receptors from the sorting sites (plasma membrane and Golgi apparatus) and their accumulation in a compartment from which they do not recycle. Removal of receptors from the recycling pool by antibody is irreversible, and return of receptors requires synthesis of new protein. Degradation of "trapped receptors" is enhanced (t1/2 = 7.5 hr), but much more gradual than their removal from the functional receptor pool (t1/2 = 30 min).     

Renin, a secretory glycoprotein, acquires phosphomannosyl residues

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP- GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine- labeled renin to the Man-6-P receptor as well as direct analysis of [2- 3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.     

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.     

The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor

We have analyzed the interaction of phosphorylated oligosaccharides and lysosomal enzymes with immobilized bovine liver cation-dependent mannose-6-P receptor. Oligosaccharides with phosphomonoesters were the only species that interacted with the receptor, and molecules with two phosphomonoesters showed the best binding. Lysosomal enzymes with several oligosaccharides containing only one phosphomonoester had a higher affinity for the receptor than did the isolated oligosaccharides, indicating the possible importance of multivalent interactions between weakly binding ligands and the receptor. The binding of a mixture of phosphorylated lysosomal enzymes to the cation-dependent Man-6-P receptor was markedly influenced by pH. At pH 6.3, almost all of the lysosomal enzymes bound to the receptor; whereas at pH 7.0-7.5, approximately one-third of the material passed through the column, one-third interacted weakly, and one-third bound tightly. The distribution of individual lysosomal enzyme activities was similar to that of the total material. The species of phosphorylated oligosaccharides present on the lysosomal enzymes which interacted poorly with the receptor were similar to those found on the tightly bound material and included species of oligosaccharides with two phosphomonoester groups. Isolated oligosaccharides of this type bound to the receptor over the entire pH range tested. These findings indicate that at neutral pH the phosphorylated oligosaccharides on some lysosomal enzyme molecules are oriented in a manner which makes them inaccessible to the binding site of the cation-dependent Man-6-P receptor. Since the same enzymes bind to the cation-independent Man-6-P receptor at neutral pH, at least a portion of the phosphomannosyl residues must be exposed. We conclude that small variations in the pH of the Golgi compartment where lysosomal enzymes bind to the receptors could potentially modulate the extent of binding to the two receptors.     

Comparative kinetics of phosphomannosyl receptor-mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them

In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6-phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I-labeled glycopeptides (Ki of 1.5 X 10(-6) M) and for the uptake of secretion acid hydrolases (Ki of 2.2 X 10(-6) M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 X 10(-5) M) compares closely to that previously determined for the pinocytosis of intact "high-uptake" acid hydrolases (3-6 X 10(-5) M). "High-uptake" fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A-binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6-phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand.     

Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting

We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to a mature form of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. greater than 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.     

Lysosomal enzyme trafficking in mannose 6-phosphate receptor-positive mouse L-cells: demonstration of a steady state accumulation of phosphorylated acid hydrolases

During their transport from the endoplasmic reticulum to lysosomes, newly synthesized acid hydrolases are phosphorylated in the Golgi apparatus to generate a common recognition marker, mannose 6-phosphate (Man 6-P). The phosphorylated acid hydrolases then bind to the Man 6-P receptor and are transported by an unknown route to lysosomes. To learn more about the delivery pathway, we examined the fate of the phosphorylated oligosaccharides synthesized by a Man 6-P receptor-positive line of mouse L-cells. In contrast to the rapid degradation of the recognition marker previously observed in mouse lymphoma cells (Gabel, C. A., D. E. Goldberg, and S. Kornfield. 1982. J. Cell Biol., 95:536-542), the number of high mannose oligosaccharides phosphorylated by the L-cells after a 30-min pulse labeling with [2-3H]mannose increased continuously during a subsequent 4-h chase period to a maximum of 9.3% of the total cell-associated structures. After 19 h of chase the absolute number of phosphorylated oligosaccharides declined, but the loss was accompanied by a general loss of cellular oligosaccharides such that 7.4% of the cell-associated high mannose oligosaccharides remained phosphorylated. The longevity of the Man 6-P recognition marker in the L-cells was verified by analyzing the ability of an individual acid hydrolase, beta-glucuronidase, to serve as a ligand for the Man 6-P receptor. At least 60% of the steady state beta-glucuronidase molecules isolated from the L-cells could undergo receptor-mediated endocytosis into enzyme-deficient human fibroblasts. Dense lysosomal granules isolated by metrizamide gradient centrifugation from [3H]mannose-labeled L-cells were found to be highly enriched in their content of phosphomonoester-containing oligosaccharides. The data indicate that acid hydrolases may retain their Man 6-P recognition markers within lysosomes, and suggest the possibility that dephosphorylation occurs at a nonlysosomal location through which the newly synthesized enzymes pass en route to lysosomes.     

Identification of a rat liver alpha-N-acetylglucosaminyl phosphodiesterase capable of removing "blocking" alpha-N-acetylglucosamine residues from phosphorylated high mannose oligosaccharides of lysosomal enzymes

We recently reported that the high mannose-type oligosaccharides of the biosynthetic intermediates of beta-glucuronidase contain phosphate groups in diester linkage between mannose residues and outer alpha-linked N-acetylglucosamine residues (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633-6639). We now describe an alpha-N-acetylglucosaminyl phosphodiesterase from rat liver that is capable of removing the N-acetyl-glucosamine residues, leaving phosphomonoester groups on the high mannose oligosaccharide units. This activity is greatly enriched in smooth membrane preparations. It can be distinguished from a lysosomal alpha-N-acetylglucosaminidase by several criteria, including subcellular localization and differential inhibition by amino sugars. In addition, human fibroblasts with mutations which lead to a deficiency of the lysosomal activity have normal levels of the alpha-N-acetylglucosaminyl phosphodiesterase. This enzyme may be involved in the "unmasking" of the phosphomannosyl recognition marker on newly synthesized acid hydrolases which could then direct the targeting of these enzymes to lysosomes.     

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.     

Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester)

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.     

Production in yeast of alpha-galactosidase A,a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease

A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ([Man-P](1-2)Man(8)GlcNAc(2)) sugar chains. Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase. Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.     

The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi

The mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor is known to cycle between the Golgi, endosomes, and the plasma membrane. In the Golgi the receptor binds newly synthesized lysosomal enzymes and transports them directly to an endosomal (prelysosomal) compartment without traversing the plasma membrane. Deletion of the carboxyl-terminal Leu-Leu-His-Val residues of the 163 amino acid cytoplasmic tail of the bovine Man-6-P/IGF-II receptor partially impaired this function, resulting in the diversion of a portion of the receptor-ligand complexes to the cell surface, where they were endocytosed. The same phenotype was observed when 134 residues of the cytoplasmic tail were deleted from the carboxyl terminus. Disruption of the Tyr24-Lys-Tyr-Ser-Lys-Val29 plasma membrane internalization signal alone had little effect on Golgi sorting, but when combined with either deletion resulted in a complete loss of this function. The mutant receptors retained the ability to recycle to the Golgi and bind cathepsin D. These results indicate that the cytoplasmic tail of the Man-6-P/IGF-II receptor contains two signals that contribute to Golgi sorting, presumably by interacting with the Golgi clathrin-coated pit adaptor proteins. The Leu-Leu-containing sequence represents a novel motif for mediating interaction with Golgi adaptor proteins.     

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.     

Strategies for carbohydrate recognition by the mannose 6-phosphate receptors

The two members of the P-type lectin family, the 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR), are ubiquitously expressed throughout the animal kingdom and are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The best-characterized function of the MPRs is their ability to direct the delivery of approximately 60 different newly synthesized soluble lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) on their N-linked oligosaccharides to the lysosome. In addition to its intracellular role in lysosome biogenesis, the CI-MPR, but not the CD-MPR, participates in a number of other biological processes by interacting with various molecules at the cell surface. The list of extracellular ligands recognized by this multifunctional receptor has grown to include a diverse spectrum of Man-6-P-containing proteins as well as several non-Man-6-P-containing ligands. Recent structural studies have given us a clearer view of how these two receptors use related, but yet distinct, approaches in the recognition of phosphomannosyl residues.     

Structural insights into the mechanism of pH-dependent ligand binding and release by the cation-dependent mannose 6-phosphate receptor

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a key component of the lysosomal enzyme targeting system that binds newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and transports them to endosomal compartments. The interaction between the MPRs and its ligands is pH-dependent; the homodimeric CD-MPR binds lysosomal enzymes optimally in the pH environment of the trans Golgi network (pH approximately 6.5) and releases its cargo in acidic endosomal compartments (相似文献   

Intracellular transport of acid alpha-glucosidase in human fibroblasts: evidence for involvement of phosphomannosyl receptor-independent system

Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.     

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.     

Postendocytic maturation of acid hydrolases: evidence of prelysosomal processing

The mannose 6-phosphate (Man 6-P) receptor operates to transport both endogenous newly synthesized acid hydrolases and extracellular enzymes to the lysosomal compartment. In a previous study (Gabel, C. A., and S. A. Foster, 1986, J. Cell Biol., 103:1817-1827), we noted that beta-glucuronidase molecules internalized by mouse L-cells via the Man 6-P receptor undergo a proteolytic cleavage and a limited dephosphorylation. In this report, we present evidence that indicates that the postendocytic alterations of the acid hydrolase molecules occur at a site through which the enzymes pass en route to the lysosomal compartment. Mouse L-cells incubated at 20 degrees C with beta-glucuronidase (isolated from mouse macrophage secretions) internalize the enzyme in a process that is inhibited by Man 6-P but unaffected by cycloheximide. As such, the linear accumulation of the ligand observed at 20 degrees C appears to occur through the continued recycling of the cell surface Man 6-P receptor. The subcellular distribution of the internalized ligands was assessed after homogenization of the cells and fractionation of the extracts by density gradient centrifugation. In contrast to the accumulation of the ligand within lysosomes at 37 degrees C, the beta-glucuronidase molecules internalized by the L cells at 20 degrees C accumulate within a population of vesicles that sediment at the same density as endocytic vesicles. Biochemical analysis of the internalized ligands indicates that: (a) the subunit molecular mass of both beta-glucuronidase and beta-galactosidase decrease upon cell association relative to the input form of the enzymes, and (b) the beta-glucuronidase molecules experience a limited dephosphorylation such that high-mannose-type oligosaccharides containing two phosphomonoesters are converted to single phosphomonoester forms. The same two post-endocytic alterations occur after the internalization of beta-glucuronidase by human I-cell disease fibroblasts, despite the low acid hydrolase content of these cells. The results indicate, therefore, that acid hydrolases internalized via the Man 6-P receptor are processed within the endocytic compartment. In that endogenous newly synthesized acid hydrolases display similar alterations during their maturation, the results further suggest that the endosomal compartment is involved in the sorting of ligands transported via both the cell surface and intracellular Man 6-P receptor.