A structural basis for four distinct elution profiles on concanavalin A--Sepharose affinity chromatography of glycopeptides.

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc.Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.     

Synthesis of 1-N-glycyl beta-oligosaccharide derivatives. Reactivity of Lens culinaris lectin with a fluorescent labeled streptavidin pseudoglycoprotein and immobilized neoglycolipid.

The lectin from Lens culinaris (lentil) has a binding specificity for glycopeptides bearing 6-O-linked fucose on the reducing terminus on complex-type N-linked oligosaccharides. Lentil lectin therefore provides an excellent example of a carbohydrate binding protein in which high-affinity interactions are dependent on the integrity of the oligosaccharide core structure. We report here the synthesis of the 1-N-glycyl beta-derivative of Gal beta 4GlcNAc beta 2Man alpha 6(Gal beta 4GlcNAc beta 2Man alpha 3)Man beta 4GlcNAc beta 4(Fuc alpha 6)-GlcNAc (Gal-2F) and its subsequent biotinylation and palmitoylation. The biotin derivative when bound to a streptavidin-fluorescein isothiocyanate (FITC) conjugate was able to bind to both concanavalin A (ConA) and lentil lectin affinity columns. In contrast, synthesis of the biotin derivative of the glycamine derivative of Gal-2F and subsequent binding to streptavidin-FITC afforded reactivity to a ConA affinity column but not to a lentil lectin affinity column. Lentil lectin also bound to plastic microtiter plates containing the adsorbed palmitoyl-1-N-glycyl beta-derivative. No binding occurred when the homologous glycamine neoglycolipid was used. These results suggest the 1-N-glycyl beta-derivative of oligosaccharides may have general utility as an intermediate in the synthesis of novel glycoconjugate probes.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.     

Studies on the structure of the oligosaccharide chains of alpha 1-acid glycoprotein associated with rough membranes from rat liver

The high mannose form of rat alpha 1-acid glycoprotein was isolated from rough membranes of rat liver using methods described previously. The high mannose glycopeptides were prepared by Pronase digestion, and oligosaccharides were isolated following digestion with endohexosaminidase-H. The structure of the carbohydrate chains of the high mannose glycopeptide and the oligosaccharides was examined by 300 MHz nuclear magnetic resonance spectroscopy. The glycopeptide contained a mixture of about equal amounts of AsnGlcNAc2Man9 and AsnGlcNAc2Man8. Analysis of the oligosaccharide fraction showed that it consisted of about equal amounts of GlcNAc Man9 and GlcNAc Man8; the GlcNAc Man8 fraction contained 85% of the "A" isomer (which was missing the terminal mannose from the middle antenna). The results suggested that mannose processing of alpha 1-acid glycoprotein in rough membranes of rat liver in vivo occurred only as far as the Man8 structure and that the "A" isomer was the main isomer formed.     

Proteolytic processing of pro-alpha and pro-beta precursors from human beta-hexosaminidase. Generation of the mature alpha and beta a beta b subunits

There are two major isozymes of human lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52), hexosaminidase A, alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). The alpha subunit contains a single polypeptide chain, while the beta subunit is composed of two nonidentical chains (beta a and beta b) derived from a common pro-beta precursor. The mature subunits, like those of most lysosomal enzymes, are produced through the proteolytic processing of propolypeptides once they enter the lysosome. In order to define the structure of the alpha and beta subunits generated in the lysosome, the alpha, beta a, and beta b polypeptides of hexosaminidase A and B were separated by a combination of molecular sieve and ion exchange high performance liquid chromatography, and amino-terminal sequences were determined. These were localized to the deduced amino acid sequences of previously isolated cDNAs coding for the prepro-alpha and beta polypeptides. From this analysis, the sites of hydrolysis generating the mature alpha, beta a, and beta b chains from hexosaminidase A and B could be determined. First, the signal peptide, required for processing of the pre-propolypeptides through the rough endoplasmic reticulum was predicted from the first in-frame Met residue on the cDNA. Second, amino acid sequencing defined the amino termini of the mature polypeptide chains and identified the pro-sequences removed from both the pro-alpha and pro-beta polypeptides. Third, an internal cleavage resulted in the removal of a tetrapeptide, Arg-Gln-Asn-Lys, and tripeptide, Arg-Gln-Asn, from the pro-beta chain of hexosaminidase A and B, respectively , to generate the beta b and beta a chains. This result localized the beta b and beta a chains to the amino-terminal and carboxyl-terminal halves of the pro-beta sequence, respectively. Finally, we previously reported minimal or no carboxyl-terminal processing of the pro-beta chain in the lysosome. On the other hand, we suggest that there is trimming at the carboxyl terminus of the pro-alpha chain based on comparison of molecular weights of deglycosylated alpha with the isolated beta b and beta a chains comprising the mature beta subunit with those predicted from the cDNA. Thus, in the lysosome the pro forms of hexosaminidase A and B undergo extensive proteolytic processing which, while specific in nature, has the appearance of removing easily accessible, nonessential domains, rather than contributing to biosynthetic maturation of function.     

Processing of N-linked oligosaccharides in soybean cultured cells

Evidence, based on both in vivo and in vitro studies with suspension-cultured soybean cells, is presented to demonstrate the processing of the oligosaccharide chain of plant N-linked glycoproteins. Following a 1-h incubation of soybean cells with [2-3H]mannose, the predominant glycopeptide obtained by pronase digestion of the membrane fraction was a Man7- or Man8GlcNAc2-Asn (GlcNAc, N-acetylglucosamine). However, the major oligosaccharide isolated from the lipid-linked oligosaccharides of these cells was a Glc2- or Glc3Man9GlcNAc2. Soybean cells were incubated with [2-3H]mannose and the incorporation of mannose into Pronase-released glycopeptides was followed during a 2-h chase. During the first 10 min of labeling, the radioactivity was mostly in a large-sized glycopeptide that appeared to be a Glc1Man9GlcNAc2-peptide. During the next 60 to 90 min of chase, this radioactivity was shifted to smaller and smaller-sized glycopeptides indicating that removal of sugars (i.e., processing) had occurred. Both glucosidase and mannosidase activity was detected in membrane preparations of soybean cells. Nine different glycopeptides were isolated from Pronase digests of soybean cell membrane fractions. These glycopeptides were purified by repeated gel filtration on columns of Bio-Gel P-4. Partial characterization of these glycopeptides by endoglucosaminidase H and alpha-mannosidase digestion, and by analysis of the products, suggested the following glycopeptides: Glc1Man9GlcNAc2-Asn, Man8GlcNAc2-Asn, Man7GlcNAc2-Asn, Man6GlcNAc2-Asn, and Man5GlcNAc2-Asn.     

Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution 1H-NMR spectroscopy

Mouse myeloma immunoglobulin IgM heavy chains were cleaved with cyanogen bromide into nine peptide fragments, four of which contain asparagine-linked glycosylation. Three glycopeptides contain a single site, including Asn 171, 402, and 563 in the intact heavy chain. Another glycopeptide contains two sites at Asn 332 and 364. The carbohydrate containing fragments were treated with Pronase and fractionated by elution through Bio-Gel P-6. The major glycopeptides from each site were analyzed by 500 MHz 1H-NMR and the carbohydrate compositions determined by gas-liquid chromatography. The oligosaccharide located at Asn 171 is a biantennary complex and is highly sialylated. The amount of sialic acid varies, and some oligosaccharides contain alpha 1,3-galactose linked to the terminal beta 1,4-galactose. The oligosaccharides at Asn 332, Asn 364, an Asn 402 are all triantennary and are nearly completely sialylated on two branches and partially sialylated on the triantennary branch linked beta 1,4 to the core mannose. The latter is sialylated about 40% of the time for all three glycosylation sites. The major oligosaccharide located at Asn 563 is of the high mannose type. The 1H-NMR determination of structures at Asn 563 suggests that the high mannose oligosaccharide contains only three mannose residues.     

Cytosolic glycosidases: do they exist?

The substrate specificity of the alpha-D-mannosidases of rat liver lysosome and cytosol was examined using oligosaccharides of the oligomannosidic type. The hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Both catabolic pathways occur in ordered ways, but are quite different. In fact, the lysosomal pathway is a two-step process: the first step involves a Zn(2+)-independent alpha-1,2-mannosidase activity, whereas the second involves a Zn(2+)-dependent alpha-1,3- and alpha-1,6-mannosidase activity. The final product is the disaccharide Man(beta 1-4)GlcNAc. In contrast, the cytosolic pathway leads, in one step, to a unique hexasaccharide (Man5GlcNAc) which has the same structure as the polyprenolic intermediate synthesized on the cytosolic face of the rough endoplasmic reticulum during the biosynthesis of N-glycosylprotein glycans: Man(alpha 1-2)-Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)] Man(beta 1-4)GlcNAc(beta 1-4)-GlcNAc(alpha)P-P-Dol. In addition, the enzymatic parameters of lysosome, endoplasmic reticulum and cytosol alpha-D-mannosidases are quite different. These results lead to the conclusion that the cytosol contains specific alpha-D-mannosidases which do not originate from lysosomes nor from endoplasmic reticulum. The discovery of cytosolic endo-N-acetyl-beta-D-glucosaminidase active on 'immature complex glycans' (glycopeptides of the oligomannosidic type and of the desialylated N-acetyllactosaminic type) as well as on the glycosyl-dolichol pyrophosphate intermediates allows us to hypothesize that these enzymes belong to a control system of N-glycosylprotein biosynthesis, their role being to destroy unfinished glycans. The fate of the formed oligosaccharide structures is discussed: are they destroyed by cytosolic or lysosomal exoglycosidases, or do they carry an 'oligosaccharin-like activity'?     

Requirement of the core structure of a complex-type glycopeptide for the binding to immobilized lentil- and pea-lectins

Structural requirements for the binding of oligosaccharides and glycopeptides to immobilized lentil- and pea-lectins were investigated by use of radioactively-labeled glycopeptides and oligosaccharides. The results indicate that an intact 2- acetamido-2-deoxy-β-d-glucopyranosyl residue at the reducing end of a complex-type oligosaccharide is essential for high-affinity binding to lentil lectin-Sepharose but not to concanavalin A-Sepharose and that an asparagine residue is required for the binding of a complex-type glycopeptide to pea lectin-Sepharose. In addition, interaction of a complex-type oligosaccharide with lentil lectin-Sepharose was enhanced by exposure of nonreducing, terminal 2-acetamido-2-deoxy-β-d-glucopyranosyl groups, whereas interaction with pea lectin-Sepharose was enhanced only after exposure of nonreducing, terminal α-d-mannopyranosyl groups.     

A library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins reveals that conglutinin binds to certain complex-type as well as high mannose-type oligosaccharide chains

This report describes the preparation of a library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins, characterization of the probes by liquid secondary ion mass spectrometry, and investigation of their reactions with 125I-labeled bovine serum conglutinin by chromatogram binding assays. The results, together with additional binding studies using neoglycolipids derived from purified complex type bi-, tri-, and tetraantennary oligosaccharides from urine, or their glycosidase-treated products, have shown that the combining specificity of conglutinin includes structures not only on high mannose-type oligosaccharides but also on hybrid- and complex-type chains. With high mannose-type oligosaccharides there is increased reactivity from the Man5 to the Man8 structures, indicating a preference for the terminal Man alpha 1-2 sequence. With complex- and hybrid-type oligosaccharides, the requirements for binding are the presence of nonreducing terminal N-acetylglucosamine or mannose residues, but the presence of a bisecting N-acetylglucosamine residue may inhibit binding. From these results it is deduced that the reactivity of conglutinin with the complement glycopeptide iC3b rather than the intact glycoprotein C3 is due to the oligosaccharide accessibility rendered by proteolysis in the complement cascade.     

Glycoprotein biosynthesis in quiescent and stimulated thymocytes and a T-cell lymphoma.

Quiescent thymocytes, mitogen-stimulated thymocytes and acute-leukaemic lymphoblasts provide a model for the study of protein glycosylation in quiescent cells, mitotically active non-malignant and malignant cells respectively. The biosynthesis of both complex and high-mannose-type oligosaccharides was monitored by metabolic labelling with [6-3]fucose and [2-3H]mannose. Bio-Gel P6 elution profiles of [6-3H]fucose-labelled glycopeptides showed that quiescent thymocytes and stimulated thymocytes synthesized qualitatively and quantitatively similar glycopeptides; however, higher-molecular-weight glycopeptides were synthesized by the acute-leukaemic lymphoblasts. The amount of [2(-3)H]mannose incorporated into glycopeptide by quiescent thymocytes was less than 10% of that incorporated by stimulated thymocytes. The Bio-Gel P6 elution profile of [2(-3)H]mannose-labelled glycopeptides from acute leukaemic lymphoblasts was qualitatively similar to that of stimulated thymocytes, with about 40% of the radioactivity incorporated into one glycopeptide peak. This glycopeptide was characterized by Bio-Gel P6 and concanavalin A affinity chromatography, radioactive-sugar analysis, sensitivity to alpha-mannosidase and endoglycosidase H and resistance to beta-glucosaminidase as containing a high-mannose oligosaccharide, possible of Man7-8GlcNAc2 structure. Pulse/chase experiments indicated that this high-mannose oligosaccharide was an end product and not a biosynthetic intermediate. It is concluded that higher-molecular-weight fucose-labelled glycopeptides are characteristic of the malignant cell type, and the synthesis of high-mannose oligosaccharide, Man7-8GlcNAc2, in stimulated thymocytes and acute-leukaemic lymphoblasts is associated with mitotically active cells.     

Control of glycoprotein synthesis. UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III, an enzyme in hen oviduct which adds GlcNAc in beta 1-4 linkage to the beta-linked mannose of the trimannosyl core of N-glycosyl oligosaccharides

Hen oviduct membranes are shown to catalyze the following enzyme reaction: GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP-GlcNAc leads to GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc-Asn + UDP. The enzyme catalyzing this reaction has been named UDP-GlcNAc:glycopeptide beta 4-N-acetylglucosaminyltransferase III (GlcNAc-transferase III) to distinguish it from two other GlcNAc-transferases (I and II) present in hen oviduct and previously described in several mammalian tissues. GlcNAc-transferases I and II, respectively, attach GlcNAc in beta 1-2 linkage to the Man alpha 1-3 and Man alpha 1-6 arms of Asn-linked oligosaccharide cores. A specific assay for GlcNAc-transferase III was devised by using concanavalin A/Sepharose columns to separate the product of transferase III from other interfering radioactive glycopeptides formed in the reaction. The specific activity of GlcNAc-transferase III in hen oviduct membranes is about 5 nmol/mg of protein/h. Substrate specificity studies have shown that GlcNAc-transferase III requires both terminal beta 1-2-linked GlcNAc residues in its substrate for maximal activity. Removal of the GlcNAc residue on the Man alpha 1-6 arm reduces activity by at least 85% and removal of both GlcNAc residues reduces activity by at least 93%. Two large scale preparations of product were subjected to high resolution proton NMR spectroscopy to establish the incorporation by the enzyme of a GlcNAc in beta 1-4 linkage to the beta-linked Man. This GlcNAc residue is called a "bisecting" GlcNAc and appears to play important control functions in the synthesis of complex N-glycosyl oligosaccharides. Several enzymes in the biosynthetic scheme are unable to act on glycopeptide substrates containing a bisecting GlcNAc residue.     

Structural basis for the recognition of complex-type biantennary oligosaccharides by Pterocarpus angolensis lectin

The crystal structure of Pterocarpus angolensis lectin is determined in its ligand-free state, in complex with the fucosylated biantennary complex type decasaccharide NA2F, and in complex with a series of smaller oligosaccharide constituents of NA2F. These results together with thermodynamic binding data indicate that the complete oligosaccharide binding site of the lectin consists of five subsites allowing the specific recognition of the pentasaccharide GlcNAc beta(1-2)Man alpha(1-3)[GlcNAc beta(1-2)Man alpha(1-6)]Man. The mannose on the 1-6 arm occupies the monosaccharide binding site while the GlcNAc residue on this arm occupies a subsite that is almost identical to that of concanavalin A (con A). The core mannose and the GlcNAc beta(1-2)Man moiety on the 1-3 arm on the other hand occupy a series of subsites distinct from those of con A.     

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).     

The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.     

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.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.     

Elicitors and suppressors of the defense response in tomato cells. Purification and characterization of glycopeptide elicitors and glycan suppressors generated by enzymatic cleavage of yeast invertase.

Cleavage of yeast invertase by alpha-chymotrypsin produced a number of small glycopeptides that were highly active as elicitors of ethylene biosynthesis and phenylalanine ammonia-lyase in suspension-cultured tomato cells. Five of these elicitors were purified and their amino acid sequence determined. They all had sequences corresponding to known sequences of yeast invertase, and all contained an asparagine known to carry a N-linked small high mannose glycan. The most active glycopeptide elicitor induced ethylene biosynthesis and phenylalanine ammonia-lyase half-maximally at a concentration of 5-10 nM. Structure-activity relationships of the peptide part were analyzed by further cleavage of a defined glycopeptide elicitor with various proteolytic enzymes. Removal of the C-terminal phenylalanine enhanced the elicitor activity, whereas removal of N-terminal arginine impaired it. A glycopeptide with the peptide part trimmed to the dipeptide arginine-asparagine was still fully active as elicitor. Glycopeptides with identical amino acid sequences were further separated into fractions differing in the oligosaccharide side chain. A given peptide had high elicitor activity when carrying a glycan with 10-12 mannosyl residues (Man10-12GlcNAc2), a 3-fold lower activity when carrying Man9GlcNAc2 and a 100-fold lower activity when carrying Man8GlcNAc2. The oligosaccharides, released by endo-beta-N-acetylglucosaminidase H from the pure glycopeptide elicitors, acted as suppressors of elicitor-induced ethylene biosynthesis and phenylalanine ammonia-lyase activity. A series of such oligosaccharides in the size range of Man8-13GlcNAc was purified. The structure and composition of the purified oligosaccharides corresponded to the known small high mannose glycans of yeast invertase as verified by 1H NMR spectroscopy at 600 MHz. The highest suppressor activities were obtained with the oligosaccharides containing 10-12 mannosyl residues (Man10-12GlcNAc). The oligosaccharide Man8 GlcNAc was ineffective as a suppressor. Thus, the structural requirements for the free oligosaccharides to act as efficient suppressors were the same as for the oligosaccharide side chains of the glycopeptides for high elicitor activity. We propose that the glycan suppressors bind to the same recognition site as the glycopeptide elicitors without inducing a response.     

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.