In vitro mutagenesis of potential N-glycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting.

The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways.     

Identification of amino acids that modulate mannose phosphorylation of mouse DNase I, a secretory glycoprotein.

We have reported that bovine DNase I, a secretory glycoprotein, acquires mannose 6-phosphate residues on 12.6% of its Asn-linked oligosaccharides when expressed in COS-1 cells and that the extent of phosphorylation increases to 79.2% when lysines are placed at positions 27 and 74 of the mature protein (Nishikawa, A., Gregory, W. , Frenz, J., Cacia, J., and Kornfeld, S. (1997) J. Biol. Chem. 272, 19408-19412). We now demonstrate that murine DNase I, which contains Lys27 and Lys74, is phosphorylated only 20.9% when expressed in the same COS-1 cell system. This difference is mostly due to the absence of three residues present in bovine DNase I (Tyr54, Lys124, and Ser190) along with the presence of a valine at position 23 that is absent in the bovine species. We show that Val23 inhibits phosphorylation at the Asn18 glycosylation site, whereas Tyr54, Lys124, and Ser190 enhance phosphorylation at the Asn106 glycosylation site. Tyr54 and Ser190 are widely separated from each other and from Asn106 on the surface of DNase I, indicating that residues present over a broad area influence the interaction with UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, which is responsible for the formation of mannose 6-phosphate residues on lysosomal enzymes.     

Phosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deficient fibroblasts

Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos.     

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.     

Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii.

The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase.     

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.     

Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.     

The role of phosphomannose isomerase in Leishmania mexicana glycoconjugate synthesis and virulence

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate, which is the first step in the biosynthesis of activated mannose donors required for the biosynthesis of various glycoconjugates. Leishmania species synthesize copious amounts of mannose-containing glycolipids and glycoproteins, which are involved in virulence of these parasitic protozoa. To investigate the role of PMI for parasite glycoconjugate synthesis, we have cloned the PMI gene (lmexpmi) from Leishmania mexicana, generated gene deletion mutants (Delta lmexpmi), and analyzed their phenotype. Delta lmexpmi mutants lack completely the high PMI activity found in wild type parasites, but are, in contrast to fungi, able to grow in media deficient for free mannose. The mutants are unable to synthesize phosphoglycan repeats [-6-Gal beta 1-4Man alpha 1-PO(4)-] and mannose-containing glycoinositolphospholipids, and the surface expression of the glycosylphosphatidylinositol-anchored dominant surface glycoprotein leishmanolysin is strongly decreased, unless the parasite growth medium is supplemented with mannose. The Delta lmexpmi mutant is attenuated in infections of macrophages in vitro and of mice, suggesting that PMI may be a target for anti-Leishmania drug development. L. mexicana Delta lmexpmi provides the first conditional mannose-controlled system for parasite glycoconjugate assembly with potential applications for the investigation of their biosynthesis, intracellular sorting, and function.     

A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency

Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P levels and certain aspects of the craniofacial phenotype and abrogated pmm2-dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including an M6P accumulation mechanism for underglycosylation.     

Protein glycosylation pathways in filamentous fungi

Glycosylation of proteins is important for protein stability, secretion, and localization. In this study, we have investigated the glycan synthesis pathways of 12 filamentous fungi including those of medical/agricultural/industrial importance for which genomes have been recently sequenced. We have adopted a systems biology approach to combine the results from comparative genomics techniques with high confidence information on the enzymes and fungal glycan structures, reported in the literature. From this, we have developed a composite representation of the glycan synthesis pathways in filamentous fungi (both N- and O-linked). The N-glycosylation pathway in the cytoplasm and endoplasmic reticulum was found to be highly conserved evolutionarily across all the filamentous fungi considered in the study. In the final stages of N-glycan synthesis in the Golgi, filamentous fungi follow the high mannose pathway as in Saccharomyces cerevisiae, but the level of glycan mannosylation is reduced. Highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures have also been identified in the filamentous fungi. O-Linked glycosylation in filamentous fungi was seen to be highly conserved with many mannosyltransferases that are similar to those in S. cerevisiae. However, highly variable and diverse O-linked glycans also exist. We have developed a web resource for presenting the compiled data with user-friendly query options, which can be accessed at www.fungalglycans.org. This resource can assist attempts to remodel glycosylation of recombinant proteins expressed in filamentous fungal hosts.     

Mr 46,000 mannose 6-phosphate specific receptor: its role in targeting of lysosomal enzymes.

Antibodies that block the ligand binding site of the cation-dependent mannose 6-phosphate specific receptor (Mr 46,000 MPR) were used to probe the function of the receptor in transport of lysosomal enzymes. Addition of the antibodies to the medium of Morris hepatoma 7777 cells, which express only the Mr 46,000 MPR, resulted in a decreased intracellular retention and increased secretion of newly synthesized lysosomal enzymes. In fibroblasts and HepG2 cells that express the cation-independent mannose 6-phosphate specific receptor (Mr 215,000 MPR) in addition to the Mr 46,000 MPR, antibodies against the Mr 46,000 MPR inhibited the intracellular retention of newly synthesized lysosomal enzymes only when added to the medium together with antibodies against the Mr 215,000 MPR. Morris hepatoma (M.H.) 7777 did not endocytose lysosomal enzymes, while U937 monocytes, which express both types of MPR, internalized lysosomal enzymes. The uptake was inhibited by antibodies against the Mr 215,000 MPR, but not by antibodies against the Mr 46,000 MPR. These observations suggest that Mr 46,000 MPR mediates transport of endogenous but not endocytosis of exogenous lysosomal enzymes. Internalization of receptor antibodies indicated that the failure to mediate endocytosis of lysosomal enzymes is due to an inability of surface Mr 46,000 MPR to bind ligands rather than its exclusion from the plasma membrane or from internalization.     

Analysis of the glycosylation and phosphorylation of the lysosomal enzyme, beta-hexosaminidase B, by site-directed mutagenesis

Lysosomal enzymes require a mannose 6-phosphate recognition marker, constructed on asparagine-linked oligosaccharide chains, for targeting to lysosomes. We have identified the glycosylation sites of human beta-hexosaminidase B and have determined the influence of individual oligosaccharides on the phosphorylation, lysosomal targeting, and catalytic activity of the enzyme. The five potential glycosylation sites of the hexosaminidase beta-chain were modified individually by site-directed mutagenesis, and the constructs were expressed in COS 1 cells. By this analysis, we determined that four of the five potential sites were glycosylated. Two of the four oligosaccharides were preferentially phosphorylated. The absence of these two preferentially phosphorylated oligosaccharides resulted in greatly reduced amounts of the lysosomal form of the enzyme with increased secretion into the medium. The catalytic activity of beta-hexosaminidase B was not significantly altered by the absence of individual oligosaccharides suggesting the folding and assembly of the enzyme was not disrupted.     

Identification of the minimal lysosomal enzyme recognition domain in cathepsin D

Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265-293 of the beta loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the beta loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.     

Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG)

Congenital Disorders of Glycosylation (CDG) are human deficiencies in glycoprotein biosynthesis. Previous studies showed that 1 mM mannose corrects defective protein N-glycosylation in cultured fibroblasts from some CDG patients. We hypothesized that these CDG cells have limited GDP-mannose (GDP-Man) and that exogenous mannose increases the GDP-Man levels. Using a well established method to measure GDP-Man, we found that normal fibroblasts had an average of 23.5 pmol GDP-Man/10(6) cells, whereas phosphomannomutase (PMM)-deficient fibroblasts had only 2.3-2.7 pmol/10(6) cells. Adding 1 mM mannose to the culture medium increased the GDP-Man level in PMM-deficient cells to approximately 15.5 pmol/10(6) cells, but had no significant effect on GDP-Man levels in normal fibroblasts. Similarly, mannose supplementation increased GDP-Man from 4.6 pmol/10(6) cells to 24.6 pmol/10(6) cells in phosphomannose isomerase (PMI)-deficient fibroblasts. Based on the specific activity of the GDP-[(3)H]Man pool present in [2-(3)H]mannose labeled cells, mannose supplementation also partially corrected the impaired synthesis of mannosylphosphoryldolichol (Man-P-Dol) and Glc(0)(-)(3)Man(9)GlcNAc(2)-P-P-Dol. These results confirm directly that deficiencies in PMM and PMI result in lowered cellular GDP-Man levels that are corrected by the addition of mannose. In contrast to these results, GDP-Man levels in fibroblasts from a CDG-Ie patient, who is deficient in Man-P-Dol synthase, were normal and unaffected by mannose supplementation even though mannose addition was found to correct abnormal lipid intermediate synthesis in another study (Kim et al. [2000] J. Clin. Invest., 105, 191-198). The mechanism by which mannose supplementation corrects abnormal protein N-glycosylation in Man-P-Dol synthase deficient cells is unknown, but this observation suggests that the regulation of Man-P-Dol synthesis and utilization may be more complex than is currently understood.     

Lysosomal acid phosphatase is not involved in the dephosphorylation of mannose 6-phosphate containing lysosomal proteins.

The mannose 6-phosphate (Man6P) residues that are necessary for the targeting of newly synthesized lysosomal proteins are dephosphorylated after delivery of lysosomal proteins to lysosomes. To examine the role of lysosomal acid phosphatase (LAP) for the dephosphorylation of Man6P residues in lysosomal proteins, the phosphorylation of endogenous lysosomal proteins and of internalized arylsulfatase A was analyzed in mouse L-cells that overexpress human LAP. Non-transfected L-cells dephosphorylate endogenous lysosomal proteins slowly (half time approximately 13 h) as well as internalized arylsulfatase A. A more than 100-fold overexpression of LAP in these cells did not affect the dephosphorylation rate. Control experiments showed that the internalized arylsulfatase A and overexpressed LAP partially colocalize and that under in vitro conditions purified LAP does not dephosphorylate arylsulfatase A. Taken together, these results indicate that LAP is not the mannose 6-phosphatase that dephosphorylates lysosomal proteins after their delivery to lysosomes.     

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 macrophages

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.     

A Temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.     

Biochemical Characterization of a Lysosomal α-Mannosidase from the Starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

Acidic α-mannosidase is an important enzyme and is reported from many different plants and animals. Lysosomal α-mannosidase helps in the catabolism of glycoproteins in the lysosomes thereby playing a major role in cellular homeostasis. In the present study lysosomal α-mannosidase from the gonads of echinoderm Asterias rubens was isolated and purified. The crude protein sample from ammonium sulfate precipitate contained two isoforms of mannosidase as tested by the MAN2B1 antibody, which were separated by anion exchange chromatography. Enzyme with 75 kDa molecular weight was purified and biochemically characterized. Optimum pH of the enzyme was found to be in the range of 4.5–5 and optimum temperature was 37 °C. The activity of the enzyme was inhibited completely by swainsonine but not by 1-deoxymannojirimycin. Ligand blot assays showed that the enzyme can interact with both the lysosomal enzyme sorting receptors indicating the presence of mannose 6-phosphate in the glycan surface of the enzyme. This is the first report of lysosomal α-mannosidase in an active monomeric form. Its interaction with the receptors suggest that the lysosomal enzyme targeting in echinoderms might follow a mannose 6-phosphate mediated pathway similar to that in the vertebrates.     

Studies of the biosynthesis of the mannose 6-phosphate receptor in receptor-positive and -deficient cell lines

Phosphomannosyl residues present on lysosomal enzymes are specifically recognized by the mannose 6-phosphate receptor protein. This interaction results in the selective targeting of lysosomal enzymes to lysosomes. While this pathway is operative in many cell types, we have found four cultured cell lines that are deficient in the ability to bind lysosomal enzymes containing phosphomannosyl residues to their intracellular or surface membranes (Gabel, C., D. Goldberg, and S. Kornfeld, 1983, Proc. Natl. Acad. Sci. USA, 80:775-779). These cells appear to segregate lysosomal enzymes by an alternate intracellular pathway. To determine the basis for the lack of mannose 6-phosphate receptor activity in these cell lines, we studied the biosynthesis of the receptor in receptor-positive (BW5147) and receptor-deficient (P388D1 and MOPC 315) cells. The cells were labeled with [2-3H]mannose or [35S]methionine and the receptor was immunoprecipitated with an antireceptor antiserum. BW5147 cells synthesize a receptor protein whose size increases after translation/glycosylation. MOPC 315 cells produce an apparently normal receptor and degrade it rapidly. P388D1 cells fail to synthesize any detectable receptor. The receptor from BW5147 and MOPC 315 cells is a glycoprotein with both high mannose and complex asparagine-linked oligosaccharides. The complex-type units become fully sialylated and remain so during long periods of chase.