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.     

Purification and characterization of rat liver glycosylasparaginase.

1. Rat liver glycosylasparaginase [N4-(beta-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26] was purified to homogeneity by using salt fractionation, CM-cellulose and DEAE-cellulose chromatography, gel filtration on Ultrogel AcA-54, concanavalin A-Sepharose affinity chromatography, heat treatment at 70 degrees C and preparative SDS/polyacrylamide-gel electrophoresis. The purified enzyme had a specific activity of 3.8 mumol of N-acetylglucosamine/min per mg with N4-(beta-N-acetylglucosaminyl)-L-asparagine as substrate. 2. The native enzyme had a molecular mass of 49 kDa and was composed of two non-identical subunits joined by strong non-covalent forces and having molecular masses of 24 and 20 kDa as determined by SDS/polyacrylamide-gel electrophoresis. 3. The 20 kDa subunit contained one high-mannose-type oligosaccharide chain, and the 24 kDa subunit had one high-mannose-type and one complex-type oligosaccharide chain. 4. N-Terminal sequence analysis of each subunit revealed a frayed N-terminus of the 24 kDa subunit and an apparent N-glycosylation of Asn-15 in the same subunit. 5. The enzyme exhibited a broad pH maximum above 7. Two major isoelectric forms were found at pH 6.4 and 6.6. 6. Glycosylasparaginase was stable at 75 degrees C and in 5% (w/v) SDS at pH 7.0.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

The structure of carbohydrate unit B of porcine thyroglobulin.

The oligosaccharide fraction was obtained from porcine thyroglobulin by hydrazinolysis. Four fractions of unit B-type oligosaccharides were purified by successive chromatographies on columns of DEAE-cellulose and concanavalin A-Sepharose, and their structures were investigated by the combination of endo- and exo-glycosidase digestions, methylation analysis and Smith degradation. From the results of these studies, the structures of the unit B oligosaccharides were proposed to be as follows: see formula in text. Thus the glycoprotein was found to have triantennary and biantennary complex-type oligosaccharides as acidic sugar chains. Concerning the triantennary oligosaccharides, the following structural features were shown: (1) the sialic acid residues were not localized on certain specific branches but distributed on all three branches; (2) however, alpha (2 leads to 3)-linked sialic acid residues were exclusively located on the terminal of the branch arising from C-4 of the branching alpha-mannose residue, whereas alpha (2 leads to 6)-linked sialic acid residues occupied terminals of the other branches; (3) the outer branching alpha-mannose residue was attached to C-3 or C-6 of an inner branching beta-linked mannose residue, and both types were observed to exist.     

Heterogeneity of bovine corneal proteokeratan sulfate

Proteokeratan sulfate was extracted and purified from bovine corneal stroma and then characterized by chemical and biochemical analyses. It was fractionated into several fractions by affinity chromatography on a concanavalin A-Sepharose column or by hydrophobic chromatography on a phenyl-Sepharose column. These fractions differed widely from one another in carbohydrate content, though no significant differences of their amino acid compositions were observed. One fraction (ca. 25%, on a dry weight basis) tightly bound to a concanavalin A-Sepharose column, compared with another fraction (ca. 65%) weakly bound to the same column, was poor in galactose and N-acetylglucosamine, but contained mannose in a high proportion. Fractions (ca. 30%) tightly bound to a phenyl-Sepharose column, in contrast to the one (ca. 66%) weakly bound, had low carbohydrate contents, like the fraction tightly bound to a concanavalin A-Sepharose column. Additionally, the fractions tightly bound to these affinity columns exhibited strong inhibitory actions on erythrocyte-concanavalin A agglutination. To obtain further details of the carbohydrate moiety of the proteokeratan sulfate, an attempt was made to separate and characterize peptidokeratan sulfate and Asn-linked oligosaccharide derived from some proteokeratan sulfate fractions. The present work revealed that the proteokeratan sulfate contains keratan sulfate and high mannose-type oligosaccharide in an approximate chain number ratio of 3.5:1.0, the keratan sulfate content varies widely and the oligosaccharide content increases with decrease of the keratan sulfate content, and the protein core is homogeneous at least with respect to the amino acid composition.     

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.     

Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride.

Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.     

Preparative purification of dipeptidyl peptidase IV.

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

Increase in beta-N-Acetylglucosaminidase Activity during Germination of Cotton Seeds

A marked increase in beta-acetylglucosaminidase (2-acetamido-2-deoxy-beta-d-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) activity was observed in the germinating cotyledon of cotton seeds. The enzyme was isolated from cotton seedlings and purified to study its physiological function in the germination of cotton seeds. The purification procedure involves ammonium sulfate fractionation, ion-exchange chromatography, gel filtrations, and concanavalin A-Sepharose 4B chromatography, and the purified beta-N-acetylglucosaminidase was shown to be homogeneous by disc electrophoresis. The molecular weight was estimated to be about 125,000 by gel filtration. The enzyme hydrolyzed both p-nitrophenyl-N-acetyl-beta-d-glucosamine and p-nitrophenyl-N-acetyl-beta-d-galactosamine. When p-nitrophenyl-N-acetyl-beta-d-glucosamine was used as substrate, K(m) and V(max) were 0.625 nanomolar and 228 moles per minute per milligram, respectively, and optimum activity was at pH 5.6. The enzyme liberated beta-linked N-acetyl-glucosamine from chitin, ovalbumin, and pronase-digested wheat germ lectin.     

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.     

Preparative Purification of Dipeptidyl Peptidase IV

Abstract

Dipeptidyl-Peptidase IV was purified from pig kidney by ammonium sulfate fractionation, gel filtration, QAE-cellulose chromatography and affinity columns with Gly-Pro- and Concanavalin A-Sepharose. The specific activity of the purified enzyme is 41.8 units/mg. Polyacrylamide gel electrophoresis and silver staining show a single band. The enzyme preparation is free of aminopeptidase and dipeptidase activity, proved fluorimetrically and by gas chromatography/mass spectrometry. The most important procedure for removal of contaminating enzyme activities is a stepwise NaCl-gradient on a QAE-ZetaPrep ion exchange disk.     

The effect of peptide deletions on the glycosylation of murine immunoglobulin M heavy chains

The glycosylation and processing of the asparagine-linked oligosaccharides at individual glycosylation sites on the mu-chain of murine immunoglobulin M were investigated using variant cell lines that synthesize and secrete IgM heavy chains with known peptide deletions. Normal murine IgM has five N-linked oligosaccharides in the constant region of each heavy or mu-chain. Each mu-chain has four complex-type oligosaccharides as well as a single high mannose-type oligosaccharide near the carboxyl terminus of the molecule. The peptide deletion of the C mu 1 constant region domain in the heavy chains synthesized by one variant cell line did not prevent subsequent glycosylation at more distal glycosylation sites. In fact, the presence of this deletion resulted in more complete glycosylation at the C-terminal glycosylation site. Evaluation of glycopeptides containing individual glycosylation sites by Concanavalin A-Sepharose indicated that this deletion had no significant effect on the processing of structures from high mannose-type to complex-type oligosaccharide chains. In contrast, a deletion of the C-terminal peptide region of the heavy chain of IgM synthesized by a second variant cell line resulted in intracellular processing to more highly branched oligosaccharide structures at several of the glycosylation sites not involved in the deletion.     

Guidelines for the preparative fractionation of human serum proteins on gradient-eluted columns of concanavalin A-sepharose: elution positions of fourteen well-characterized proteins and evidence for concanavalin A-reactive albumin-IgA and -IgG complexes

Systematic studies on the fractionation of serum proteins on gradient-eluted columns of concanavalin A-Sepharose have been carried out to determine if the oligosaccharide residues were sufficiently different to permit a reasonable separation and to determine where in the chromatogram these proteins would be eluted. Human whole serum and ammonium sulfate fractions derived therefrom were used in conjunction with 2.1 x 75 cm columns of concanavalin A-Sepharose and a 4 x 400 ml gradient (Varigard) with 0.5 M methyl alpha-D-glucopyranoside as limit buffer. The elution positions and chromatographic limits of 14 well-characterized human serum proteins have been determined by double diffusion of aliquots of the effluent fractions (10X concentrated) in agarose gel against specific antibody and the general chromatographic distribution of the proteins by immunoelectrophoresis. Overall, the results demonstrate that the composition of the oligosaccharide side chain, like differences in molecular size, solubility, and charge density, is a useful parameter in the chromatographic separation of protein from serum. Although it is well-known that albumin is a nonglycoprotein, 1.0% of the protein was tightly bound by concanavalin A-Sepharose. Subsequent experiments showed that albumin binding was due to complex formation with IgA and IgG both of which possess the necessary complement of concanavalin A-reactive residues for strong binding. Sodium dodecylsulfate polyacrylamide gel electrophoresis of 2-mercaptoethanol-reduced albumin-IgA and -IgG complexes produced bands corresponding to the molecular weights of albumin and the heavy and light chains of IgA and IgG whereas unreduced samples were not dissociated. When these complexes were reacted with concanavalin A-Sepharose and treated with 2-mercaptoethanol, free albumin was eluted. The remaining adsorbed glycoprotein(s), IgA and IgG, could be eluted with methyl alpha-D-glucopyranoside. These results strongly suggest that these proteins and albumin are linked via a disulfide bond(s).     

Characterization of the two oligosaccharides present in the preferential hormonogenic domain of human thyroglobulin

The N-terminal fragment of human thyroglobulin (residues 1 to 171) contains the preferential hormonogenic site of the molecule and 2 potential sites of N-glycosylation (Asn57 and Asn91). This fragment was isolated from a human thyroglobulin purified from a single goiter. The tryptic peptides bearing the glycosylation sites were separated by Bio-Gel P-30 and HPLC columns. The oligosaccharides borne at each site were analyzed, after tritium labeling, by concanavalin A-Sepharose and HPLC. At both sites the structures observed are heterogenous, with a majority of biantennary complex type structures.     

Proteomic analysis of N-glycosylation in mosquito dopachrome conversion enzyme

A novel dopachrome conversion enzyme (DCE) is present in insects and involved in their melanization pathway. DCE shares no sequence homology with any noninsect species from bacteria to humans. Several DCE sequences have been available, but enzyme structure and catalytic mechanism are unclear. This study concerns DCE PTMs, especially glycosylation. A mosquito DCE was purified and its monosaccharide composition, N-glycosylation site, and oligosaccharide structures were determined. Results showed that N-acetyl D-glucosamine and D-mannose are the major monosaccharides and L-fucose, D-xylose, and D-arabinose are the minor ones in mosquito DCE. Glycosylation site and oligosaccharide structures were elucidated from MS and MS/MS spectra of trypsin-digested DCE glycopeptides. A single N-glycosylation site (Asn285 -Glu-Thr) was identified in DCE and was proven to be fully glycosylated. Man3GlcNAc2, Man3(Fuc)1-2GlcNAc2, and their truncated structures were the dominant oligosaccharides. In addition, high mannose-type structures (Man4-7(Fuc)GlcNAc2) were also identified. Removal of DCE N-oligosaccharides with peptide N-glycosidase (PNGase F) decreased its activity and thermal stability. However, partial DCE deglycosylation with alpha-mannosidase or alpha-fucosidase somewhat stimulated its activity and improved its thermal stability. During mass spectrometric analysis of DCE glycopeptides, their CID patterns were highly intriguing, in that some glycopeptides underwent both C-terminal rearrangement and formation of dimeric structures during CID. Results of this study provide an interesting example in terms of potential complexity of the glycopeptide CID fragmentation pattern.     

Phosphatidylinositol-specific phospholipase C of murine lymphocytes

Phosphatidylinositol-specific phospholipase C (PI-phospholipase C) was found primarily in the cytosolic fraction of murine splenic lymphocytes. However, small but significant amounts of the activity of the enzyme were detected in the microsome and plasma membrane fractions. Both the cytosolic and membrane-bound phospholipases C specifically hydrolyzed inositol phospholipids, phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. PI-Phospholipase C activity was detected in the cytosolic and microsome fractions from both T-cell-enriched and B-cell-enriched spleen cells. The membrane-bound enzyme was distinguishable from the cytosolic enzyme in the following properties. The cytosolic PI-phospholipase C showed optimal activity at pH 6.0 while the membrane-bound enzyme had two pH optima between pH 5.0 and 7.0. The activity of the cytosolic enzyme was first detected at 1 microM Ca2+, and maximum activity was observed at 100 microM Ca2+, while the membrane-bound PI-phospholipase C required higher Ca2+ concentrations, of millimolar order. The membrane-bound enzyme could hardly be extracted with 1 M NaCl but was extracted with 0.4% cholate.A portion of the membrane-bound PI-phospholipase C activity in the cholate extract was absorbed by concanavalin A-Sepharose and specifically eluted with an alpha-methylmannoside solution. The cytosolic enzyme, which was water soluble, did not bind to concanavalin A-Sepharose. Trypsinization of lymphocytes before subcellular fractionation caused a significant decrease in the PI-phospholipase C activity in the microsome fraction but almost no loss at all of the cytosolic enzyme activity.     

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-β-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two l-glutamine residues. A substance P derivative with an N-acetyl-d-glucosamine residue attached to the fifth or sixth l-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-d-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the l-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the l-glutamine residue of the peptide was not liberated by peptide-N⁴-(N-acetyl-β-d-glucosaminyl) asparagine amidase F.     

Regulation of Myelination: Schwann Cell Transition from a Myelin-Maintaining State to a Quiescent State After Permanent Nerve Transection

Permanent nerve transection of the adult rat sciatic nerve forces Schwann cells in the distal nerve segment from a myelin-maintaining to a quiescent state. This transition was followed by serial morphometric evaluation of the percentage fascicular area having myelin (myelin percent of area) in transverse sections of the distal nerve segment and revealed a rapid decline from a normal value of 36.6% to 3.2% by 14 days for the sciatic nerve to less than 1.0% throughout the remaining time course (up to 105 days). No evidence of axonal reentry into the distal nerve segment or new myelin formation was observed at times under 70 days. In some of the distal nerve segments at 70, 90, and 105 days, new myelinated fibers were observed that usually consisted of only a few myelinated fibers at the periphery and in the worst case amounted to 1.6% (myelin percent of area). Radioactive precursor incorporation of [3H]mannose into endoneurial slices at 4 and 7 days after transection revealed two species of the major myelin glycoprotein, P0, with Mr of 28,500 and 27,700. By 14 days after nerve transection, only the 27,700 Mr species remained. Incorporation of [3H]mannose into the 27,700 Mr species increased progressively to 35 days after transection and then began to decline at 70 and 105 days. Alterations in the oligosaccharide structure of this down-regulated myelin glycoprotein accounted for the progressive increase in mannose incorporation. Lectin affinity chromatography of pronase-digested P0 glycopeptides on concanavalin A-Sepharose revealed that the 28,500 Mr species of P0 had the complex-type oligosaccharide as the predominant oligosaccharide structure (92%). In contrast, the high mannose-type oligosaccharide was the predominate structure for the 27,700 Mr form, which increased to 70% of the total radioactivity by 35 days after nerve transection. Since the biosynthesis of the complex-type oligosaccharide chains on glycoproteins involves high mannose-type intermediates, the mechanism of down-regulation in the biosynthesis of this major myelin glycoprotein, therefore, results in a biosynthetic switch from the complex-type oligosaccharide structure as an end product to the predominantly high mannose-type oligosaccharide structure as a biosynthetic intermediate. This biosynthetic switch occurs gradually between 7 and 14 days after nerve transection and likely reflects a decreased rate of processing through the Golgi apparatus. It remains to be determined if the high mannose-type oligosaccharide chain on P0 can undergo additional processing steps in this permanent nerve transection model.     

Purification and characterization of UDP-GlcNAc: GlcNAcbeta 1-6(GlcNAcbeta 1-2)Manalpha 1-R [GlcNAc to Man]-beta 1, 4-N-acetylglucosaminyltransferase VI from hen oviduct

A new beta1,4-N-acetylglucosaminyltransferase (GnT) responsible for the formation of branched N-linked complex-type sugar chains has been purified 64,000-fold in 16% yield from a homogenate of hen oviduct by column chromatography procedures using Q-Sepharose FF, Ni(2+)-chelating Sepharose FF, and UDP-hexanolamine-agarose. This enzyme catalyzes the transfer of GlcNAc from UDP-GlcNAc to tetraantennary oligosaccharide and produces pentaantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-6 arm. It requires a divalent cation such as Mn(2+) and has an apparent molecular weight of 72,000 under nonreducing conditions. The enzyme does not act on biantennary oligosaccharide (GnT I and II product), and beta1,6-N-acetylglucosaminylation of the Manalpha1-6 arm (GnT V product) is essential for its activity. This clearly distinguishes it from GnT IV, which is known to generate a beta1-4-linked GlcNAc residue only on the Manalpha1-3 arm. Based on these findings, we conclude that this enzyme is UDP-GlcNAc:GlcNAcbeta1-6(GlcNAcbeta1-2)Manalpha1-R [GlcNAc to Man]-beta1,4-N-acetylglucosaminyltransferase VI. This is the only known enzyme that has not been previously purified among GnTs responsible for antenna formation on the cores of N-linked complex-type sugar chains.