Search

We previously reported that cultured cells incubated with beta-xylosides synthesized alpha-GalNAc-capped GAG-related xylosides, GalNAc alpha GlcA beta Gal beta Gal beta Xyl beta-R and GalNAc alpha GlcA beta GalNAc beta GlcA beta Gal beta Gal beta Xyl beta-R, where R is 4-methylumbelliferyl or p-nitrophenyl (Manzi et al., 1995; Miura and Freeze, 1998). In this study, we characterized an alpha-N-acetylgalactosaminyltransferase (alpha-GalNAc-T) that probably adds the alpha-GalNAc residue to the above xylosides. Microsomes from several animal cells and mouse brain contained the enzyme activity which requires divalent cations, and has a relatively broad pH optimal range around neutral. The apparent K(m) values were in the submillimolar range for the acceptors tested, and 19 microM for UDP-GalNAc. 1H-NMR analysis of the GlcA-beta-MU acceptor product showed the GalNAc residue is transferred in alpha 1,4-linkage to the glucuronide, which is consistent with previous results reported on alpha-GalNAc-capped Xyl-MU (Manzi et al., 1995). Various artificial glucuronides were tested as acceptors to assess the influence of the aglycone. Glucuronides with a bicyclic aromatic ring, such as 4-methylumbelliferyl beta-D-glucuronide (GlcA-beta-MU) and alpha-naphthyl beta-D-glucuronide, were the best acceptors. Interestingly, a synthetic acceptor that resembles the HNK-1 carbohydrate epitope but lacking the sulfate group, GlcA beta 1,3Gal beta 1,4GlcNAc beta-O-octyl (delta SHNK-C8), was a better acceptor for alpha-GalNAc-T than the glycosaminoglycan-protein linkage region tetrasaccharyl xyloside, GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta-MU. GlcA-beta-MU and delta SHNK-C8 competed for the alpha-GalNAc-T activity, suggesting that the same activity catalyzes the transfer of the GalNAc residue to both acceptors. Taken together, the results show that the alpha-GalNAc-T described here is not restricted to GAG-type oligosaccharide acceptors, but rather is a UDP-GalNAc:glucuronide alpha 1-4-N-acetylgalactosaminyltransferase.

In the preceding two papers, we described two new classes of sulfated N-linked oligosaccharides isolated from total cellular 35SO4-labeled macromolecules of different mammalian cell lines. The first class carries various combinations of sialic acids and 6-O-sulfate esters on typical complex-type chains, while the second carries heparin and heparan-like sequences. In this study, we have characterized a sulfophosphoglycoprotein of 140 kDa from FG-Met-2 pancreatic cancer cells whose oligosaccharides share some properties of both these classes. The molecule was localized to the cell surface by electron microscopy using a monoclonal antibody (S3-53) and by cell surface 125I-labeling. Metabolic labeling of the cells with radioactive glucosamine, methionine, inorganic sulfate, or phosphate all demonstrated a single 140-kDa molecule. Pulse-chase analysis and tunicamycin treatment indicated the glycosylation of a putative primary translation product of 110 kDa via an intermediate (120 kDa) to the mature form (140 kDa). Digestion with peptide:N-glycosidase F (PNGaseF) indicated a minimum of four N-linked glycosylation sites. PNGaseF released more than 90% of the [6-3H]GlcNH2 label and 40-70% of 35SO4 label from the immunoprecipitated 140-kDa molecule. The isolated oligosaccharides were characterized as described in the preceding two papers. The majority of [6-3H]GlcNH2-labeled molecules were susceptible to neuraminidase. More than 50% of the 35SO4 label was associated with only 5-10% of the 3H-labeled chains. Some of the sulfated chains were partly sialylated molecules with four to five negative charges. Treatment with nitrous acid released about 25% of the 35SO4 label as free sulfate, together with 6% of the [6-3H]GlcNH2 label, indicating the presence of N-sulfated glucosamine residues. Some of these oligosaccharides were degraded by heparinase and heparitinase. Therefore, while they are not as highly charged as typical heparin or heparan chains, they must share structural features that permit recognition by the enzymes. Thus, this 140-kDa glycoprotein contains at least four asparagine-linked chains substituted with a heterogeneous mixture of sulfated sequences. The heterogeneity of these molecules is as extensive as that described for whole-cell sulfated N-linked oligosaccharides in the preceding two papers.

In the preceding paper (Roux, L., Holojda, S., Sundblad, G., Freeze, H. H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889) we described the metabolic labeling and isolation of sulfated N-linked oligosaccharides from mammalian cell lines. All cell lines studied contained a class of sulfated sialylated complex-type chains with 2-6 negative charges. In this paper, we show that bovine pulmonary arterial endothelial (CPAE) and human erythroleukemia (K562) cell lines also contain a class of more highly charged sulfated but less sialylated oligosaccharides. These molecules were further characterized by ion exchange chromatography and various enzymatic and chemical treatments. In both cell lines they contained greater than 6 negative charges, but those from K562 were even more highly charged than those from CPAE. Nitrous acid, heparinase, and heparitinase degradation of K562 oligosaccharides released 88, 64, and 78%, respectively, of 35S label. Combined digestion with the two enzymes resulted in 87% release. The corresponding values for CPAE were 48, 25, and 50% (60% for the two enzymes together). Chondroitinase ABC (or AC) digestion of K562 and CPAE oligosaccharides released 10 and 5%, respectively. About 30% of the 35S-labeled oligosaccharides from CPAE were sensitive to endo-beta-galactosidase, indicating that poly-N-acetyl-lactosamine structures were present on some chains. Highly charged [3H]mannose-labeled sulfated oligosaccharides from CPAE cells became neutral after treatment with heparinase/heparitinase but were resistant to Pronase, further proving that glycosaminoglycan (GAG)-like chains were directly attached to N-linked oligosaccharides. Such neutralized oligosaccharides did not bind to concanavalin A-Sepharose, but some interacted with phytohemagglutinin L4, indicating that they were bi-, tri-, or tetra-antennary complex-type chains. Thus, K562 and CPAE cells contain different types of GAG chains directly attached to asparagine-linked oligosaccharides. Such molecules were not found in many other cell lines that synthesize the more typical O-linked GAG chains. This suggests that the occurrence of these novel N-linked chains is not a random event resulting from accidental initiation of GAG chain synthesis on N-linked intermediates in the Golgi apparatus.

The structures of sulfated N-linked oligosaccharides have been reported for a few specific proteins. We recently demonstrated that such oligosaccharides occur in many different types of tissue culture cell lines (Freeze, H. H., and Varki, A. (1986) Biochem. Biophys. Res. Commun. 140, 967-973). Here we report improved methods to metabolically label cell lines with 35SO4 and to release sulfated N-linked oligosaccharides with peptide:N-glycosidase F as well as the partial structure of some of these novel oligosaccharides. The released 35SO4-labeled chains from Chinese hamster ovary (CHO) cells and bovine pulmonary artery endothelial cells (CPAE) were characterized by gel filtration, anion exchange and lectin affinity chromatography, and various enzymatic and chemical treatments. Each cell line contains a class of sulfated oligosaccharide chains bearing from two to six negative charges in varying combinations of O-sulfate esters and sialic acids. These molecules represent a significant proportion of both the total 35SO4 label and the total anionic N-linked oligosaccharides. They are also relatively enriched in a CHO mutant that is deficient in glycosaminoglycan chain synthesis. Lectin affinity chromatography of such molecules from CPAE cells indicates that the majority are sialylated multiantennary complex-type chains. The sulfate esters are exclusively of the primary type. Sequential exoglycosidase digestions, including beta-hexosaminidase A treatment at low pH, demonstrate that at least one-third of these sulfate esters are found in the following structure, (formula; see text) where R is the remainder of the underlying oligosaccharide, and SA is sialic acid. In addition to these molecules, a more highly charged group of sulfated N-linked oligosaccharides sharing structural features with glycosaminoglycans was found in CPAE cells, but not in CHO cells. These are described in the following paper (Sundblad, G., Holojda, S., Roux, L., Varki, A., and Freeze, H. H. (1988) J. Biol. Chem. 263, 8890-8896).

Core fucosylation of N-linked oligosaccharides (GlcNAcbeta1, 4(Fucalpha1,6)GlcNAcbeta1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I. E. and Newell, P. C. (1989) Development 107, 87-94), specifically recognize fucose residues in alpha1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing alpha-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core alpha1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice.

Previous studies showed that vegetative Dictyostelium discoideum cells make a lysosomal proteinase, proteinase-1, that contains multiple GlcNAc-alpha-1-P residues in phosphodiester linkage to serine. We extended these studies and, in contrast to earlier reports, found that proteinase-1 contains 7.5 mol of Fuc, 8 mol of Man, 2 mol of Xyl, and 30 mol of GlcNAc per calculated mol of protein but no Man-6-P residues. The protein binds to concanavalin A and wheat germ agglutinin lectin affinity columns, and PNGase-F digestion released most of the mannose and xylose but little of the GlcNAc. beta-Elimination under reducing conditions released only GlcNAc-alpha-1-P. There was no evidence for the release of disaccharides or of fucitol. A rabbit antiserum and monoclonal antibodies prepared against proteinase-1 recognize GlcNAc-alpha-1-P residues in immunoblots and are specifically competed by UDP-GlcNAc or GlcNAc-alpha-1-P. Use of other monoclonal antibodies showed the presence of mannose-6-sulfate on N-linked sugar chains, and alpha-fucose residues on the protein. Thus, proteinase-1 has at least two types of modifications: Glc NAc-alpha-1-P-Ser, which we call phosphoglycosylation, and N-linked oligosaccharides. This is the first purified lysosomal enzyme in Dictyostelium that does not contain Man-6-P residues. The GlcNAc-alpha-1-P-specific antibodies also recognize a group of developmentally regulated proteins, especially enriched in vegetative cells. Some of them are also lysosomal cysteine proteinases, and all bind to the GlcNAc-alpha-1-P-specific monoclonal antibody but not to the mammalian CI-Man-6-P receptor. Conversely, lysosomal enzymes that have Man-6-P do not bind to the GlcNAc-alpha-1-P-specific antibody. An exception to this is beta-N-acetylglucosaminidase, where 15% of the activity binds to this antibody. Thus, there appear to be two sets of lysosomal enzymes with distinct post-translational modifications.

The concentration of D-mannose in serum is 20-50 micron, but its physiological significance for glycoprotein synthesis is unknown. Here, we show that the uptake of D-mannose by different mammalian cell lines involves a mannose-specific transporter(s) with a K(uptake) of about 30-70 micron and a V(max) which is probably sufficient to account for the bulk of mannose needed for glycoprotein synthesis. Mannose uptake appears to be through a facilitated transport process since it is not inhibited by cyanide. Phloretin completely inhibits mannose uptake, but phloridzin inhibits only 25-30%. Both of these inhibitors can block 2-deoxyglucose uptake in fibroblasts which occurs through the typical glucose transporters. None of 9 other sugars tested inhibited mannose transport. Most importantly, 5 mM D-glucose only inhibits mannose uptake by 50% showing that it is not an efficient competitor. These results suggest that this transporter(s) may use serum mannose for glycoprotein synthesis.

Removal of the terminal sialic acid residues from many serum glycoproteins results in exposure of their penultimate galactose residues and rapid clearance from circulation by the liver. Low-density lipoprotein is a glycoprotein containing 21 galactose and 9 sialic acid residues per particle. Studies in this laboratory and others have shown that both the liver and extrahepatic tissues contribute to the degradation of low-density lipoprotein. This study was undertaken to determine whether desialylation of pig low-density lipoprotein alters its removal from circulation. Low-density lipoprotein was incubated at 37 degrees C with an agarose-bound neuraminidase, proteinase-free, from Clostridium perfringens. After 18 h at pH 5.0, 70% of the sialic acid residues were removed. The desialylated 131I-labelled and native 125I-labelled low-density lipoproteins were simultaneously injected into a pig, and their disappearance from plasma was followed for 96 h. The turnovers of the two were identical. In contrast, neuraminidase-treated fetuin was cleared about 200-fold faster than native fetuin. Studies were also performed in cultured rat hepatocytes. Rates of degradation of native and neuraminidase-treated low-density lipoprotein were similar, whereas asialo-fetuin was degraded at six to ten times the rate of native fetuin. Thus desialylation does not appear to alter low-density-lipoprotein catabolism by hepatic or extrahepatic cells.

Mannose in N-linked oligosaccharides is assumed to be derived primarily from glucose through phosphomannose isomerase (PMI). The discovery of mammalian mannose-specific transporters that function at physiological concentrations suggested that mannose might directly contribute to oligosaccharide synthesis. To determine the relative contribution of glucose and mannose, human fibroblasts were labeled with either [2-3H]mannose or [1,5,6-3H]glucose at the same specific activity, and the N-linked chains were released by PNGase F digestion. Most of the trichloroacetic acid-precipitable [3H]mannose label was released by this digestion, but only about 10% of the trichloroacetic acid-precipitable material was released from cells labeled with [1,5,6-3H]glucose. Both sugars labeled a similar array of oligosaccharides, and acid hydrolysis of these chains showed that [2-3H]mannose contributed 65-75% of the [3H]mannose in cells labeled for 1 h, despite the 100-fold higher concentration of exogenous glucose. Mannose consumption and [2-3H]mannose utilization were within the range of rates expected for mannose transport via the mannose-specific transporter. About 7-14% of the [2-3H]mannose is used for glycosylation, while the rest (86-93%) is catabolized to 3H2O via PMI. Increasing the exogenous mannose concentration beyond mannose transporter saturation results in the conversion of >99% of [2-3H]mannose into 3H2O. Long term labeling of cells with [2-3H]mannose showed that the specific activity of mannose in glycoproteins reached 77% of the specific activity of [2-3H]mannose added to the medium. These results show that when fibroblasts are provided with physiological concentrations of mannose, they use the mannose-specific transporter to supply the majority of mannose needed for glycoprotein synthesis. PMI may normally be used to catabolize excess mannose rather than to primarily supply Man-6-P for glycoprotein synthesis.

Dictyostelium discoideum makes multiple developmentally regulated lysosomal cysteine proteinases. One of these, a lysosomal enzyme called proteinase I, contains a cluster of GlcNAc-alpha-1-P-Ser residues. We call this phosphoglycosylation. To study its function, a cDNA library from vegetative cells was screened, and two novel cysteine proteinase clones were characterized (cprD and cprE). Each of them has highly conserved regions expected for cysteine proteinases, but unlike any other, each has a serine-rich domain containing three distinct motifs, poly-S, SGSQ, and SGSG. cprD and cprE cDNAs were overexpressed in Dictyostelium and the active enzymes identified. cprD codes for a protein of approximately 36 kDa (CP4), which is recognized by monoclonal antibodies against GlcNAc-1-P and fucose. cprE corresponds to a 29-kDa protein, which is recognized by antibodies against GlcNAc-1-P. mRNA for both enzymes is present in the vegetative phase and increases during growth on bacteria but decreases throughout development. When the formation of the fruiting body is complete the mRNA for both messages is detected again but in very low levels. Having cloned cDNAs for proteins that carry GlcNAc-1-P should allow us to probe the function of the carbohydrate in these putative lysosomal enzymes.

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.

Lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-phosphate and bind with high affinity to the phosphomannosyl receptor of human fibroblasts. In this study, we have partially characterized the Asn-linked oligosaccharide units present on these enzymes. [3H]Mannose-labeled alpha-D-mannosidase, beta-D-glucosidase, and beta-D-N-acetylglucosaminidase were purified from the spent growth medium of strain AX3 and glycopeptides were prepared by pronase digestion. Approximately 75% of the glycopeptides contained sulfate residues. These could be removed by solvolysis without degrading the underlying oligosaccharide. Following solvolysis (but not before), the oligosaccharides could be released by endo-beta-N-acetylglucosaminidase H, indicating the presence of high mannose-type units. Greater than 85% of the oligosaccharides contained one or two mannose 6-phosphate residues in the form of an unusual acid-stable phosphodiester. About 3% of the oligosaccharides contained phosphomonoesters and only 6% were neutral species. The major neutral oligosaccharide eluted in the position of Man9GlcNAc when analyzed by high performance liquid chromatography whereas the minor species appeared to be 1-2 residues larger. Acetolysis of the major phosphorylated fractions revealed that molecules with a single mannose 6-phosphate contained the phosphomannosyl residue on the branch linked alpha 1,6 to the beta-linked mannose whereas molecules with two phosphomannosyl residues had the residues on this branch as well as the branch linked alpha 1,3 to the beta-linked mannose. The mechanism of mannose phosphorylation in the slime mold must differ from that of mammalian cells since the phosphomannosyl residues are present as acid-resistant phosphodiesters rather than acid-labile phosphodiesters.

Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P- and Man-6-P-bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation.

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha-mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.

Dictyostelium discoideumglycoproteins contain mannose-6-SO4in highly immunogenic N-linked oligosaccharides. To more precisely define the structural requirements of the antigenic determinant, we have analyzed the oligosaccharides synthesized by two mutant strains (HL241 and HL243) that lack it.

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.

Mannose 6-phosphate is an important recognition site involved in transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes. The current study is the first demonstration of functional mannose phosphate receptors in macrophages. The receptor appears to be similar in many respects to that expressed in fibroblasts. Binding at 4 degrees C of a mannose-6-P-containing ligand, alpha-mannosidase from Dictyostelium discoideum, was specific and saturable (KD = 1.6 nM). In the presence of permeabilizing agents (saponin and digitonin), macrophage mannose-6-P receptors gave a distribution of 15-20% on the surface and 80-85% inside. Uptake studies gave a Kuptake value of 4.9 nM. Mannose-6-P, Hansenula holstii phosphomannan, and fructose 1-phosphate were effective inhibitors of alpha-mannosidase uptake. Inhibitors of mannose uptake, such as beta-glucuronidase, mannose-bovine serum albumin, fucose-bovine serum albumin, or mannan had no effect on alpha-mannosidase uptake. Likewise, an inhibitor (fucoidin) of the macrophage receptor which recognizes negatively charged proteins did not inhibit alpha-mannosidase uptake. Uptake was linear over 90 min and inhibited by chloroquine, suggesting that surface receptors recycle. These data demonstrate that macrophages contain receptors which specifically recognize mannose-6-P units and are distinct from the well characterized mannose receptors. The finding that the mannose-6-P receptors play a role at the surface, together with the fact that most of the receptors are intracellular (similar to the mannose receptor) suggests that both carbohydrate receptors play a regulatory role at the surface and intracellularly in transport of lysosomal enzymes.

alpha-D-Mannosidase and beta-D-glucosidase from Dictyostelium discoideum are efficiently endocytosed into mutant human fibroblasts through a saturable, mannose 6-phosphate (Man-6-P)-inhibitable uptake system (Freeze, H. H., Kaplan, A., and Miller, A. L. (1980) J. Biol. Chem. 255, 11081-11084). We have extended this study using both of these active, purified enzymes and 125I-labeled beta-glucosidase for uptake into normal human fibroblasts. The pH optimum of uptake is 6.0 for both enzymes and greater than 95% is inhibited by 2 mM Man-6-P (Ki = 5 X 10(-5) M). A variety of mono-and diesterified mannans or mannan derivatives also inhibited uptake of the enzymes. Both enzymes compete with each other for uptake (Ki, 2.0 X 10(-9) M) and have Kuptake of 1.0-2.2 X 10(-9) M and a Vmax of 0.35-0.48 pmol/mg of cell protein/h. The specific binding of 125I-beta-glucosidase to fibroblasts was measured at 0-4 degrees C and found to have a Kd of 1.0 X 10(-9) M with approximately 15,900 +/- 900 receptors/cell. The receptors could be internalized every 5-7 min at saturating concentrations of enzyme at 37 degrees C. The 125I-beta-glucosidase previously bound to the cells at 4 degrees C could be released by continued incubation at 4 degrees C in the presence of Man-6-P, however, after brief warming to 37 degrees C followed by reincubation at 4 degrees C, Man-6-P could no longer release the ligand. Chloroquine inhibited 95% of the uptake of 125I-beta-glucosidase at 50 microM. Following internalization of the enzyme, it is degraded to trichloroacetic acid-soluble fragments with a half-life of approximately 6.5 h. These data suggest that the slime mold enzymes are bound to the same receptors which function in the uptake of mammalian lysosomal enzymes and make the slime mold lysosomal enzymes useful models to study uptake involving this receptor in normal human fibroblasts.

4-Methylumbelliferyl-beta-xyloside (Xyl beta MU) primes glycosaminoglycan synthesis by first serving as an acceptor for the addition of 2 galactoses and 1 glucuronic acid residue to make the typical core structure, GlcUA beta 1, 3Gal beta 1,3Gal beta 1,4Xyl beta MU. To investigate the relative localization of these biosynthetic enzymes, intact and properly oriented rat liver Golgi preparations were incubated with Xyl beta MU and 1 microM UDP-[3H]Gal and then chased with 5 microM of unlabeled UDP-Gal, UDP-GlcUA, UDP-GlcNAc, UDP-GalNAc, and CMP-Neu5Ac. Under these conditions, no intervesicular transport occurs and acceptor labeling depends entirely upon transporter-mediated delivery of the labeled sugar nucleotides into the lumen of a vesicle and co-localization of the appropriate glycosyltransferases. The labeled products were isolated from the incubation medium and from within the Golgi and their structures analyzed by C18, anion-exchange, and amine adsorption high performance liquid chromatography in combination with glycosidase digestions. Surprisingly, the major products within the Golgi were two sialylated xylosides (Sia alpha 2,3Gal beta 1,4Xyl-beta MU and Sia alpha 2,8Sia alpha 2,3Gal beta 1,4Xyl beta MU) rather than the expected group of partially completed GAG core structures. Less than 10% of the products within the Golgi are the expected core structures containing a second Gal residue or, in addition, GlcUA. The amount of the sialylated products is only partially decreased if the chase is omitted or if the chase is done in the absence of added CMP-Sia, suggesting a pool of previously transported CMP-Sia drives synthesis of the major products. Conversely, when detergent permeabilized vesicles are provided with high concentration of the same sugar nucleotides, the ratio of sialylated products is reduced and replaced by an increase in GAG-like products. These results argue that GAG core-specific Ga1 transferase I and II are not extensively co-localized within the same Golgi compartment. By contrast, glycosaminoglycan core Gal transferase I is substantially co-localized with an alpha-2,3-sialyltransferase and an alpha-2,8-sialyltransferase. Incubating intact Golgi vesicles with exogenous diffusible acceptors offers a novel method to assess the functional co-localization of glycosyltransferases of multiple pathways within the Golgi compartments.

The accompanying article (Manzi, A., Salimath, P. V., Spiro, R. C., Keifer, P. A., and Freeze, H. H. (1995) J. Biol. Chem. 270, 9154-9163) reported the complete structure of a novel molecule made by human melanoma cells incubated with 1 mM 4-methylumbelliferyl-beta Xyl (Xyl beta MU). The product resembles a common pentasaccharide core region found in chondroitin/dermatan sulfate glycosaminoglycans, except that a terminal alpha-Gal-NAc residue is found in a location normally occupied by beta-GalNAc in these chains or alpha-GlcNAc in heparan sulfate chains. In this paper we show that several other human cancer cell lines and Chinese hamster ovary cells also make alpha-GalNAc-capped xylosides. The [6-3H]galactose-labeled Xyl beta MU product binds to immobilized alpha-GalNAc-specific lectin from Helix pomatia and the binding is competed by GalNAc, but not by Glc. Binding to the lectin is destroyed by digestion with alpha-N-acetylgalactosaminidase, but not beta-hexosaminidase. The nature of the aglycone influences the amount and relative proportion of this material made, with p-nitrophenyl-beta-xyloside being a better promoter of alpha-GalNAc-terminated product than Xyl beta MU. This novel oligosaccharide accounts for 45-65% of xyloside-based products made by both human melanoma and Chinese hamster ovary cells when they are incubated with 30 microM Xyl beta MU, but at 1 mM both the total amount and the proportion decreases to only 5-10%. In both cell lines this product is replaced by a corresponding amount of Sia alpha 2,3Gal beta 4Xyl beta MU. Preferential synthesis of the alpha-GalNAc-capped material at very low xyloside concentration argues that it is a normal biosynthetic product and not an experimental artifact. This pentasaccharide may be a previously unrecognized intermediate in glycosaminoglycan chain biosynthesis. Since this alpha-GalNAc residue occurs at a position that determines whether chondroitin or heparan chains are added to the acceptor, it may influence the timing, type, and extent of further chain elongation.

We have examined the relationship of N-linked oligosaccharide structures to the proper targeting and proteolytic processing of two lysosomal enzymes, α-mannosidase and β-glucosidase, in the slime mold Dictyostelium discoideum. Two different mutant strains, HL241 and HL243, each synthesize the same nonglucosylated, truncated, lipid-linked oligosaccharide precursor, Man6GlcNAc2. [3H]Mannose-labeled N-linked oligosaccharides were studied following their release from immunoprecipitated α-mannosidase and β-glucosidase by digestion with peptide: N-glycosidase F. The oligosaccharides from both mutants resembled each other, but they were smaller and contained fewer anionic groups than those from the wild-type. The oligosaccharides from the mutants strains were reduced in sulfate and Man-6-P content, and all Man-6-P was in the form of acid-stable phosphodiesters.

We previously described a diverse family of sulfated anionic N-linked oligosaccharides released by peptide: N-glycosidase F (PNGaseF) from calf pulmonary artery endothelial (CPAE) cells (Roux, L., Holoyda, S., Sundblad, G., Freeze, H.H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889). Since a major fraction of the intact lung consists of endothelial cells, we reasoned that bovine lung might be a rich source of similar molecules. Total N-linked oligosaccharides from bovine lung acetone powder were released by PNGaseF, labeled by [3H]NaBH4 reduction, and the anionic fractions were studied with a variety of techniques. The sugar chains with lesser negative charge (designated Class I) share several properties of conventional multiantennary complex-type chains. However, unlike the case with CPAE cells, sialic acids account only for a minority of the anionic properties and only a small proportion carry sulfate esters. A variety of different treatments indicate that most of the unexplained negative charge is due to multiple carboxylic acid groups. Resistance to beta-glucuronidase and alpha-iduronidase suggests that these may be previously undescribed modifications of mammalian oligosaccharides. The most highly charged N-linked chains (designated Class II) are more similar in general structure to the corresponding ones from CPAE cells, although relatively more abundant. Their high charge is primarily due to chondroitin sulfate, heparin/heparan sulfate, or keratan sulfate glycosaminoglycan chains. Sequential digestion studies suggest that a significant proportion of these molecules have more than one type of glycosaminoglycan chain associated with them. Compositional analysis indicates the presence of xylose residues in Class II, but not Class I molecules. However, unlike the case with conventional glycosaminoglycans, these residues are not at the reducing terminus. Most previously reported structures of complex-type N-linked oligosaccharides are derived from the glycoproteins of blood cells, plasma, or the secretions of cultured mammalian cells. This library of N-linked oligosaccharides from an intact mammalian organ (lung) contains a high proportion of novel anionic sugar chains whose structures are different from conventional complex-type sialylated chains and only partially related to those from CPAE cells. Further exploration of the N-linked chains of intact mammalian tissues seems warranted.

beta-Xylosides compete with endogenous proteoglycan core proteins and act as alternate acceptors for synthesizing protein-free glycosaminoglycan chains. Their assembly on these alternate acceptors utilizes the same glycosyltransferases that make the protein-bound chains. Most studies using alternate acceptors focus on the production of sulfated glycosaminoglycan chains that are thought to be the major products. However, we previously showed that labeling melanoma cells with [6-3H]galactose in the presence of 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) beta-xylosides led to the synthesis of mostly di- to tetrasaccharide products including incomplete core structures. We have solved the structure of one of the previously unidentified products as, GalNAc alpha(1,4)GlcA beta(1,3)Gal beta(1,3)Gal beta(1,4)Xyl beta MU, based on compositional analysis by high performance liquid chromatography, fast atom bombardment, electrospray mass spectrometry, and one-dimensional and two-dimensional 1H NMR spectroscopy. The novel aspect of this molecule is the presence of a terminal alpha-Gal-NAc residue at a position that is normally occupied by beta-GalNAc in chondroitin/dermatan sulfate or by alpha-Glc-NAc in heparin or heparan sulfate chains. An alpha-GalNAc residue at this critical location may prevent further chain extension or influence the type of chain subsequently added to the common tetrasaccharide core.

The phosphorylated oligosaccharides of Dictyostelium discoideum contain methylphosphomannosyl residues which are stable to mild-acid and base hydrolysis (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we present evidence that these methyl groups are derived from [methyl-3H]methionine, in vivo and [methyl-3H]S-adenosylmethionine in vitro. About 18% of the macromolecules secreted from vegetative cells labeled with [methyl-3H]methionine are released by digestion with preparations of endoglycosidase/peptide N-glycosidase F. The majority of the released molecules are sulfated, anionic high mannose-type oligosaccharides. Strong acid hydrolysis of the [3H]methyl-labeled molecules yields [3H]methanol with kinetics of release similar to those found for the generation of Man-6-P from chemically synthesized methylphosphomannose methylglycoside. Treatment of the [3H]methyl-labeled molecules with a phosphodiesterase from Aspergillus niger which is known to cleave this phosphodiester also releases [3H]methanol from a portion of the oligosaccharides. In vitro incorporation of [methyl-3H]S-adenosylmethionine into endogenous acceptors found in membrane preparations shows that the [3H]methyl group of the methylphosphomannose residues can be derived from this molecule.

The N-linked oligosaccharides found on the lysosomal enzymes from Dictyostelium discoideum are highly sulfated and contain methylphosphomannosyl residues (Gabel, C. A., Costello, C. E., Reinhold, V. N., Kurtz, L., and Kornfeld, S. (1984) J. Biol. Chem. 259, 13762-13769). Here we report studies done on the structure of N-linked oligosaccharides found on proteins secreted during growth, a major portion of which are lysosomal enzymes. Cells were metabolically labeled with [2-3H]Man and 35SO4 and a portion of the oligosaccharides were released by a sequential digestion with endoglycosidase H followed by endoglycosidase/peptide N-glycosidase F preparations. The oligosaccharides were separated by anion exchange high performance liquid chromatography into fractions containing from one up to six negative charges. Some of the oligosaccharides contained only sulfate esters or phosphodiesters, but most contained both. Less than 2% of the oligosaccharides contained a phosphomonoester or an acid-sensitive phosphodiester typical of the mammalian lysosomal enzymes. A combination of acid and base hydrolysis suggested that most of the sulfate esters were linked to primary hydroxyl groups. The presence of Man-6-SO4 was demonstrated by the appearance of 3,6-anhydromannose in acid hydrolysates of base-treated, reduced oligosaccharides. These residues were not detected in acid hydrolysates without prior base treatment or in oligosaccharides first treated by solvolysis to remove sulfate esters. Based on high performance liquid chromatography quantitation of percentage of 3H label found in 3,6-anhydromannose, it is likely that Man-6-SO4 accounts for the majority of the sulfated sugars in the oligosaccharides released from the secreted glycoproteins.

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2-3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic-derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.

We previously reported that cultured mammalian cells incubated with 4-methylumbelliferyl (MU) or p -nitrophenyl (pNP) beta-xyloside synthesize an alpha-GalNAc-terminated pentasaccharide resembling the glycosaminoglycan-core protein linkage region. Here we show that human melanoma M21 cells and human neuroblastoma cells incubated with Xylbeta-MU/pNP also make an alpha-GalNAc-terminated heptasaccharide containing one chondroitin disaccharide repeat. High performance liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry analysis of intact or glycosidase-digested xyloside showed the structure as: GalNAcalphaGlcAbeta1,3GalNAcbeta1,4GlcAbeta1,3Galbe ta1,3Galbeta1, 4Xylbeta-MU/pNP. The alpha-GalNAc-terminated xylosides can account for approximately 10% of the total Xylbeta-MU/pNP products ( approximately 1.5 nmol/h/mg). These results show that GalNAcalphaGlcAbeta-modification is relatively abundant, but not unique to the GAG-linkage tetrasaccharide. alpha-GalNAc addition to the GlcA residue does not appear to be an extension of general phase II detoxification of xenobiotics that involve glucuronidation, since M21 cells incubated with MU synthesize only 0.3 pmol GlcAbeta-MU/h/mg protein, and undetectable amount of GalNAcalphaGlcAbeta-MU (<40 fmol/h/mg). Further, subcellular fractionation shows that the alpha- N- acetylgalactosaminyltransferase activity colocalizes in the Golgi with other glycosyl transferases and not in the ER, where xenobiotic detoxification glucuronosyltransferases are found. Although GalNAcalphaGlcAbeta-terminal modification has not been detected on naturally occurring GAG chains, the substantial amount of alpha-GalNAc transferase activity suggests that the alpha-GalNAc transferase could utilize other GlcA-containing glycoconjugates as acceptors.