Identification of an N-acetylglucosaminyltransferase in Dictyostelium discoideum that transfers an "intersecting" N-acetylglucosamine residue to high mannose oligosaccharides

Glycoproteins synthesized by the cellular slime mold Dictyostelium discoideum have been shown to contain asparagine-linked high-mannose oligosaccharides which have an N-acetylglucosamine group in a novel intersecting position (attached beta 1-4 to the mannose linked alpha 1-6 to the core mannose). We have used crude membrane preparations from vegetative D. discoideum (strain M4) to characterize the enzyme activity responsible for catalyzing the transfer of GlcNAc to the intersecting position of high-mannose oligosaccharides. UDP-GlcNAc:oligosaccharide beta-N-acetylglucosaminyltransferase activity in these preparations attaches GlcNAc to the mannose residue-linked alpha 1-6 to the beta-linked core mannose of the following Man9GlcNAc oligosaccharide as shown by the arrow. (formula; see text) It will also attach GlcNAc to the same intersecting position and/or to the bisecting position (beta-linked core mannose) of the following Man5GlcNAc oligosaccharide. (formula; see text) An analysis of the pH profiles, effects of heat denaturation, and substrate inhibitions on the addition of GlcNAc to either the intersecting or bisecting position of this Man5GlcNAc oligosaccharide indicates that a single enzyme activity is responsible for transferring GlcNAc to both positions. Various oligosaccharides were assayed to determine the substrate specificity of the transferase activity. These data indicate that both the mannose-attached alpha 1-3 and the mannose-attached alpha 1-6 to the mannose receiving the GlcNAc play a critical role in substrate suitability; absence of the alpha 1-6 mannose results in at least a 90% decrease in activity, while absence of the alpha 1-3 mannose results in a completely inactive substrate. This suggests that the minimal substrate is the disaccharide Man alpha 1-3Man.     

Structural analysis of N-linked oligosaccharides from glycoproteins secreted by Dictyostelium discoideum. Identification of mannose 6-sulfate

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.     

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

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

Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F

Peptide N-glycosidase from Flavobacterium meningosepticum cleaves complex as well as neutral glycoproteins (Plummer, T.H., Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. (1984) J. Biol. Chem. 259, 10700-10704). Examples of neutral glycoprotein substrates include ribonuclease B (one high mannose oligosaccharide chain) and yeast external invertase (nine chains/invertase subunit). The rate of deglycosylation by the glycosidase was greatly enhanced if the glycoprotein substrate was denatured prior to enzyme treatment, from a low of 11-fold for external invertase to a high of 844-fold for ribonuclease B. Peptide N-glycosidase F was unable to cleave the asparaginyl-N-acetylglucosamine bond in endo-beta-N-acetylglucosaminidase H-modified external invertase or ribonuclease B, although that in similarly modified glycopeptide substrate was cleaved. Ribonuclease B was digested sequentially with various exoglycosidases to produce an oligosaccharide chain of varied length. Using the resulting forms of ribonuclease B as substrates for peptide N-glycosidase F, the minimum oligosaccharide chain for cleavage was the di-N-acetyl-chitobiosyl core unit.     

The glycoproteins of Dictyostelium discoideum. Changes during development.

The glycoproteins of D. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately half-way through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.     

Characterization of a novel calmodulin from Dictyostelium discoideum

We have purified calmodulin from the eukaryotic microorganism Dictyostelium discoideum (Clarke, M., Bazari, W. L., and Kayman, S. C. (1980) J. Bacteriol. 141, 397-400) and have compared it to calmodulin purified from bovine brain. The two proteins behaved almost identically during fractionation on ion exchange and gel filtration columns and on isoelectric focusing gels. Dictyostelium calmodulin had one-third the specific activity of brain calmodulin in the Ca2+-dependent activation of brain cyclic nucleotide phosphodiesterase; this activation was inhibited for both proteins by 25 microM trifluoperazine. Dictyostelium calmodulin also activated erythrocyte (Ca2+ + Mg2+)-ATPase and interacted with the inhibitory subunit of skeletal muscle troponin. Competition radioimmune assays showed that Dictyostelium calmodulin could compete with brain calmodulin for antibodies to brain calmodulin. These similarities indicate a close relationship between Dictyostelium and brain calmodulin and suggest that the functional capabilities of the protein have been conserved even among evolutionarily distant species. However, substantial differences in primary structure were detected by amino acid analyses and peptide mapping. Most interesting is the lack of trimethyllysine in Dictyostelium calmodulin. This unusual amino acid, which is commonly found in calmodulins, is therefore not essential for interaction between calmodulin and the calmodulin-regulated proteins tested here.     

How a plant lectin recognizes high mannose oligosaccharides

The crystal structure of Pterocarpus angolensis seed lectin is presented in complex with a series of high mannose (Man) oligosaccharides ranging from Man-5 to Man-9. Despite that several of the nine Man residues of Man-9 have the potential to bind in the monosaccharide-binding site, all oligomannoses are bound in the same unique way, employing the tetrasaccharide sequence Manalpha(1-2)Manalpha(1-6)[Manalpha(1-3)]Manalpha(1-. Isothermal titration calorimetry titration experiments using Man-5, Man-9, and the Man-9-containing glycoprotein soybean (Glycine max) agglutinin as ligands confirm the monovalence of Man-9 and show a 4-times higher affinity for Man-9 when it is presented to P. angolensis seed lectin in a glycoprotein context.     

Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins

Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum.     

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

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

Maturation of asparagine-linked oligosaccharides in Dictyostelium discoideum analyzed with modification-specific probes

Lysosomal enzymes in Dictyostelium discoideum contain high mannose oligosaccharides that contain mannose 6-phosphate and several unusual structures. The synthesis and distribution of these post-translational modifications were studied using probes for different carbohydrate groups. These probes include lectin-like antibodies directed to two distinct sulfated and one nonsulfated N-linked determinants, the lectin Con A, and the mammalian 215-kDa phosphomannosyl receptor. Only Con A binds to newly synthesized alpha-mannosidase present in the rough endoplasmic reticulum. The other modifications are acquired at different rates and are first detected on protein in light density Golgi-like membranes. Mutations which prevent protein transport to Golgi membranes block synthesis of these moieties, but inhibitors which prevent later transport steps have no effect. The majority of modified proteins are in lysosomes but significant amounts are delivered to nonlysosomal destinations. Different lysosomal proteins contain unequal amounts of each modification.     

Glycoprotein biosynthesis in plants. Demonstration of lipid-linked oligosaccharides of mannose and N-acetylglucosamine.

Previous studies from this laboratory have shown that particulate preparations from maturing cotton fibers catalyze the transfer of mannose from GDP-[14C]mannose into mannosylphosphorylpolyisoprenol (Forsee, W. T., and Elbein,A. D. (1973) J. Biol. Chem. 248, 2858-2867). In this report, we show that these particulate preparations also catalyze the inocoporation of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides and into glycoprotein. The oligosaccharide-lipids were treated with dilute acid to liberate the water-soluble oligosaccharides and these oligosaccharides could then be separated into seven or eight distinct radioactive peaks by paper chromatography in isobutyric acid/NH4OH/H2betaO (57/4/39). The smallest of the oligosaccharides appears to be a trisaccharide with the structure Man leads to GlcNAc-GlcNAc. Thus the oligosaccharides attached to the lipids apparently range in size from those having 3 glycose units to those having approximately 8 to 10 glycose units. The radioactivity in the smaller-sized oligosaccharide-lipids could be chased into the larger oligosaccharide-lipids by a second incubation in the presence of unlabeled GDP-mannose. The sugar at the reducing ends of the oligosaccharides was identified as GlcNAc while some mannose (20 to 30%) was present in alpha linkages at the nonreducing ends...     

Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].     

Biosynthesis of methylphosphomannosyl residues in the oligosaccharides of Dictyostelium discoideum glycoproteins. Evidence that the methyl group is derived from methionine

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.     

Oligosaccharide heterogeneity of glycoproteins sulfated during the vegetative growth of Dictyostelium discoideum

Macromolecules are sulfated during the vegetative growth of Dictyostelium discoideum. A characterisation of the structures of sulfated oligosaccharides associated with these macromolecules indicates that the oligosaccharides are heterogeneous. Endoglycosidase and pronase digestion were used with gel-filtration chromatography to obtain two different oligosaccharide fractions and a glycopeptide fraction; these were further characterised by ion-exchange and lectin-affinity chromatography and by acid hydrolysis. The data indicate that up to 43% of the sulfate is associated with typical N-linked oligosaccharides, that up to 5% is associated with N-linked oligosaccharides that are either very large or extremely highly charged, and that the remaining sulfate is associated with oligosaccharides non-N-linked to protein. Each fraction was also shown to be heterogeneous at most other structural levels. Electrophoretic analyses following the endoglycosidase and pronase treatments indicated that all of the macromolecules are glycoproteins and suggested further that at least two of the oligosaccharide fractions are located on different groups of glycoproteins.     

Developmental regulation of processing alpha-mannosidases and "intersecting" N-acetylglucosaminyltransferase in Dictyostelium discoideum.

We have identified three developmentally regulated oligosaccharide-processing enzyme activities in Dictyostelium discoideum. Two different alpha-mannosidase activities present at extremely low levels in vegetative cells are expressed during development. The first of these activities (MI) rises sharply from 6 to 12 h of development whereas the second activity (MII) rises sharply from 12 to 18 h of development. MI acts on Man9GlcNAc, which it can degrade to Man5GlcNAc but is inactive toward p-nitrophenyl-alpha-D-mannoside (pnpMan). MII acts on pnpMan but not Man9GlcNAc. These activities are distinct from each other and from lysosomal alpha-mannosidase activity as demonstrated by pH optima, substrate specificity, sensitivity to inhibitors and divalent cations, developmental profiles, and solubility. The characteristics of these developmentally regulated alpha-mannosidase activities are similar to those of Golgi alpha-mannosidases I and II from higher eucaryotes, and they appear to catalyze the in vivo formation of processed asparagine-linked oligosaccharides by developed cells. In addition, developed cells have very low levels of a soluble alpha-mannosidase activity, which is the predominant activity in vegetative cells. This soluble vegetative alpha-mannosidase activity has properties that are reminiscent of the endoplasmic reticulum alpha-mannosidase from rat liver. The intersecting N-acetylglucosaminyltransferase activity that we have described recently in vegetative cells of D. discoideum (Sharkey, D. J., and Kornfeld, R. (1989) J. Biol. Chem. 264, 10411-10419) has a developmental profile that is distinct from that of either of the alpha-mannosidase activities. It has maximum activity at 6 h of development and decreases sharply to its minimum level by 12 h of development. The changes that occur in the levels of these three processing enzymes with development correlate well with the different arrays of asparagine-linked oligosaccharides found in early and late stages of development (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18485-18497).     

The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum

In this report we examine the accumulation of glycoconjugates in the extracellular medium and insoluble matrices surrounding developing cells of the cellular slime mold Dictyostelium discoideum. Conditions were employed which permitted advanced development (slug stage and beyond) in suspension culture. Under these conditions, up to one-third of the total culture protein appeared as non-sedimentable, extracellular material over the course of 48 h of incubation. Most of the secreted molecules expressed carbohydrate antigens (glycoantigens) as detected by Western blotting, using a panel of six monoclonal antibodies. Since the glycoantigens are secreted, immunoelectron microscopy was used to localize the glycoantigens in the extracellular matrices surrounding normally developing cells, including the slime sheath, stalk tube, inner spore coat, outer spore coat, and intercellular fluid between spores. Each glycoantigen had a characteristic distribution, and each extracellular matrix space contained a unique combination of glycoantigens. Thus, although each of these matrices (except inter-spore fluid) contains cellulose as a primary component, they could be distinguished on the basis of their glycoantigen and, by inference, glycoprotein compositions. Furthermore, there were differences between anterior and posterior regions of both slime sheaths and stalk tubes. These observations show that secretion as detected in suspension culture occurs under normal conditions as a part of the process of depositing extracellular matrices around the cells. The distributions show that the cell aggregate positionally regulates the expression and deposition of secretory glycoproteins; the resultant patterns of expression of unique protein-linked carbohydrate structures imply a functional role in matrix organization and possibly cell activity which can now be explored.     

Purification and characterization of a novel dipeptidyl peptidase from Dictyostelium discoideum

A dipeptidyl peptidase (DPP) was purified to homogeneity using lys-ala-beta-naphthylamide, the standard substrate for DPP II. The enzyme is a monomer with a Mr of 70kDa, pl 5.2, and Km 5.0 microM. Its terminal amino acid sequence was XXLLYAIQKRLF and was not identical to that of any known protein. Although initially considered to be a DPP II, the enzyme differed in some properties from classical DPP IIs. It had a pH optimum of 7.9, was not active on X-pro-naphthylamides, the usual substrates of mammalian DPP II, but was active on arg-arg- and asp-arg-naphthylamides, substrates acted on by the DPP III class of enzymes. This enzyme therefore combines properties typical of both DPP II and III and differs from all previously described DPPs. Activity on lys-ala-beta-naphthylamide was most abundant during aggregation and its activity is consistent with processing specific peptides during development.     

Analysis of a novel diacylglycerol kinase from Dictyostelium discoideum: DGKA

Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of diacylglycerols to generate phosphatidic acid and have been investigated in prokaryotic and eukaryotic organisms. Recently, a protein that is significantly similar to human DGK-theta, DGKA, was identified in Dictyostelium discoideum. It has been shown to possess DGK activity when assayed using a medium-chain diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8). A complete understanding of DGK catalytic and regulatory mechanisms, as well as physiological roles, requires an understanding of its biochemical and kinetic properties. This report presents an analysis of these properties for DGKA. The enzyme catalyzes the phosphorylation of DiC8, and another medium-chain DAG, DiC6 (1,2-dihexanoyl-sn-glycerol), in a Michaelis-Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs was dependent on substrate surface concentration and the detergent that was used. DGKA displayed Michaelis-Menten kinetics with respect to bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octyl glucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol %. At higher surface concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. In contrast, DGKA displayed sigmoidal kinetics with respect to bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. Finally, we show the catalytic activity of DGKA was significantly enhanced by phosphatidylserine (PS) and phosphatidic acid (PA).