Characterization of Fucosyl Oligosaccharides Associated with Synaptic Membrane and Synaptic Junctional Glycoproteins

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.     

Development of Synaptic Glycoproteins: Effect of Postnatal Age on the Synthesis and Concentration of Synaptic Membrane and Synaptic Junctional Fucosyl and Sialyl Glycoproteins

Abstract: Synaptic plasma membranes (SPM) and synaptic junctions (SJ) were isolated from the cortices of rats varying in age between 5 and 28 days. Gel electrophoresis of SPM and SJ indicated a marked increase in the concentration of the "PSD protein" (M. W. 52,000) with development. The biosynthesis of glycoproteins was measured following the intracranial injection of [³H]fucose or [³H] N '-acetylmannosamine. The incorporation of [³H]fucose into synaptic fractions decreased two- to threefold between 10 and 28 days whereas little change in the incorporation of [³H] N '-acetylmannosamine occurred over the same period. Gel electrophoretic analyses of labeled synaptic membranes indicated major increases in the relative incorporation of radiolabeled precursors into glycoproteins with apparent molecular weights of 74,000, 65,000, 50,000, and 40,000 with increasing age. Identification of fucosyl and sialyl glycoproteins following reaction with ¹²⁵I-fucose-binding protein or labeling of sialic acid with NaIO₄NaB[³H₄] demonstrated similar increases in the concentrations of these glycoproteins. Synaptic junctions contained three major glycoproteins with apparent molecular weights of 180,000, 130,000 and 110,000. The reaction of these glycoproteins with ¹²⁵I-fucose-binding protein increased one- to twofold between 10 and 28 days but little variation in their relative distribution or synthesis occurred over this period. The reaction of synaptic junctional glycoproteins GP 180 and GP 110 with ¹²⁵I-wheat germ ag-glutinin increased between 10 and 28 days. The results indicate that the molecular composition of the synapse continues to evolve after the initial synaptic contact has been formed.     

Identification of Synapse Specific Components: Synaptic Glycoproteins, Proteins, and Transmitter Binding Sites

Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X-100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for L-glutamate, L-aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ-like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for L-glutamate (L-Glu) and L-aspartate (L-Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ-like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.     

Identification of Lectin-Purified Neural Glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA Receptor Subunits: Conservation of Glycosylation at the Synapse

Abstract: The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for ∼50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N -glycosidase F and endo -β- N -acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.     

Developmental Changes in Calmodulin-Kinase II Activity at Brain Synaptic Junctions: Alterations in Holoenzyme Composition

Synaptic junctions (SJs) from rat forebrain were isolated at increasing postnatal ages and examined for endogenous protein kinase activities. Our studies focused on the postnatal maturation of the multifunctional protein kinase designated Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). This kinase is comprised of a major 50-kilodalton (kDa) and a minor 60-kDa subunit. Experiments examined the developmental properties of CaM-kinase II associated with synaptic plasma membranes (SPMs) and synaptic junctions (SJs), as well as the holoenzyme purified from cytosolic extracts. Large developmental increases in CaM-kinase II activity of SJ fractions were observed between postnatal days 6 and 20; developmental changes were examined for a number of properties including (a) autophosphorylation, (b) endogenous substrate phosphorylation, (c) exogenous substrate phosphorylation, and (d) immunoreactivity. Results demonstrated that forebrain CaM-kinase II undergoes a striking age-dependent change in subunit composition. In early postnatal forebrain the 60-kDa subunit constitutes the major catalytic and immunoreactive subunit of the holoenzyme. The major peak of CaM-kinase II activity in SJ fractions occurred at approximately postnatal day 20, a time near the end of the most active period of in vivo synapse formation. Following this developmental age, CaM-kinase II continued to accumulate at SJs; however, its activity was not as highly activated by Ca2+ plus calmodulin.     

Characterization of Axonally Transported Glycoproteins in Regenerating Garfish Olfactory Nerve

Abstract: This study examined changes in composition and concanavalin A (Con A) binding of axonally transported glycoproteins and their pronase-generated glycopeptides in regenerating garfish olfactory nerve. A previous study had demonstrated a regeneration-related increase in the proportion of [³H]glucosamine label in lower-molecular-weight Con A-binding glycopeptides derived from transported glycoproteins. Further analysis of carbohydrate composition shows that these molecules resemble mannose-rich oligosaccharides in composition and are increased in absolute amount in regenerating nerve. Subcellular analysis shows that the Con A-binding glycopeptides are enriched in membrane subfractions, particularly in a high-density fraction that morphologically resembles isolated cell surface coat. Regeneration-related changes in intact axonally transported glycoproteins were also detected. Sodium dodecyl sulfate gel electrophoresis of transport-labeled glycoproteins disclosed growth-correlated increases in radioactivity associated with 180–200K, 105–115K, and 80–90K components, while a 150–160K molecular weight class of glycoproteins was diminished in relative labeling. Intact glycoproteins displaying an affinity for Con A were also augmented in regenerating nerve, the increases occurring primarily in molecules in the 50–140K range.     

Affinity chromatographic isolation of cell surface glycoproteins from human foetal brains and their interaction with lectins.

The cell surface glycoproteins of foetal human neurons and glial cells were isolated by affinity chromatography on Con A-Sepharose 4B. Dissociation of Con A from the matrix took place independent of buffer composition and the absence of lipids and/or detergents during chromatography. It was apparently related to the nature of glyco proteins. Pretreatment of Con A-Sepharose 4B with urea or guanidine minimized this problem. The elution of glycoproteins from the affinity matrix at 4 degrees C, instead of the usual 25 degrees C, reduced both Con A and glycolipid contamination in the eluate. Dot-enzyme-linked-lectin assay was carried out with horse radish peroxidase conjugated lectins and serotonin. It was observed that total glycoproteins contained high mannose, hybrid and a limited quantity of biantennary complex type oligosaccharide chains. O-linked oligosaccharides were also present. Desialylation and sodium chloride inhibited binding to serotonin and wheat germ agglutinin indicating the presence of sialic acid residues. Fucose was attached to the innermost core GlcNAc residue, as revealed by affinity towards pea lectin.     

Calmodulin-Dependent Protein Phosphorylation in Synaptic Junctions

Synaptic junctions (SJs) from rat forebrain were examined for Ca2+/calmodulin (CaM)-dependent kinase activity and compared to synaptic plasma membrane (SPM) and postsynaptic density (PSD) fractions. The kinase activity in synaptic fractions was examined for its capacity to phosphorylate endogenous proteins or exogenous synapsin I, in the presence or absence of Ca2+ plus CaM. When assayed for endogenous protein phosphorylation, SJs contained approximately 25-fold greater amounts of Ca2+/CAM-dependent kinase activity than SPMs, and fivefold more activity than PSDs. When kinase activities were measured by phosphorylation of exogenous synapsin I, SJs contained fourfold more activity than SPMs, and 10-fold more than PSDs. The phosphorylation of SJ proteins of 60- and 50-kilodalton (major PSD protein) polypeptides were greatly stimulated by Ca2+/CaM; levels of phosphorylation for these proteins were 23- and 17-fold greater than basal levels, respectively. Six additional proteins whose phosphorylation was stimulated 6-15-fold by Ca2+/CAM were identified in SJs. These proteins include synapsin I, and proteins of 240, 207, 170, 140, and 54 kilodaltons. The 54-kilodalton protein is a highly phosphorylated form of the major PSD protein and the 170-kilodalton component is a cell-surface glycoprotein of the postsynaptic membrane that binds concanavalin A. The CaM-dependent kinase in SJ fractions phosphorylated endogenous phosphoproteins at serine and/or threonine residues. Ca2+-dependent phosphorylation in SJ fractions was strictly dependent on exogenous CaM, even though SJs contained substantial amounts of endogenous CaM (15 micrograms CaM/mg SJ protein). Exogenous CaM, after being functionally incorporated into SJs, was rapidly removed by sequential washings. These observations suggest that the SJ-associated CaM involved in regulating Ca2+-dependent protein phosphorylation may be in dynamic equilibrium with the cytoplasm. These findings indicate that a brain CaM-dependent kinase(s) and substrate proteins are concentrated at SJs and that CaM-dependent protein phosphorylation may play an important role in mechanisms that underlie synaptic communication.     

Molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with rat brain synaptic functions.

The composition and biosynthesis of fucosyl glycoproteins present in rat brain synaptic membranes and synaptic junctions were investigated. Reaction with 125I-labelled fucose-binding protein (Lotus tetragonolobus) following sodium dodecyl sulphate gel electrophoresis identified 6--8 fucosyl glycoproteins in synaptic membranes but only three major high molecular classes (Mr = 180 000, 130 000 and 110 000) in synaptic junctions. Affinity chromatography on concanavalin A-Sepharose resolved each of the synaptic junctional fucosyl glycoproteins into concanavalin A-positive and negative components indicating the presence of at least six high molecular weight fucosyl glycoproteins in synaptic junctions. Following the administration of [3H]fucose synaptic membranes, synaptic junctions and post-synaptic densities incorporated isotope, the order of relative specific activities being synaptic membranes greater than synaptic junctions greater than post-synaptic densities. Fractionation of [3H]fucose-labelled synaptic junctions on concanavalin A-Sepharose revealed a time-dependent increase in the percentage of isotope associated with the concanavalin A-positive glycoproteins. The results demonstrate both molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with synaptic junctions.     

Evidence that a Cerebellum-Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium-Dependent Manner

Abstract: Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K glycoprotein, designated GP-A. Previous results have shown that GP-A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. In the present studies, GP-A was not detected in myelin, mitochondria, purified nuclei, or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP-A is partially soluble in the non-ionic detergent Triton X-100 and is completely soluble when cerebellum SPMs are treated with the ionic detergent N-lauryl sarcosinate. The solubilization of GP-A from cerebellum membranes was shown to be a function of bound calcium ions, e.g., pretreating SPMs with 100 μM-1mM Ca²⁺ decreased the solubility of GP-A in Triton by approximately threefold. GP-A is a major concanavalin A (Con A)-binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/ 230K fodrin doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and fore-brain synaptic fractions by two-dimensional peptide mapping indicated that they were identical. The Con A-binding property of GP-A was exploited to purify it by affinity chromatography with agarose-Con A. Peptide mapping comparisons between affinity-purified GP-A and GP-A in SPM and SJ fractions indicated that GP-A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP-A and 235K fodrin polypeptide indicated that these two synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJs reacted with anti-fodrin antisera on Western blots; however, GP-A failed to cross-react. These observations, together with results from previous studies, indicate that GP-A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. Moreover, GP-A is enriched in SJs relative to SPM fractions, is related to fodrin, and is most likely a cell-surface glycoprotein at asymmetric synapses in cerebellum. GP-A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of calcium in synaptic transmission, together with the decreased solubility of GP-A in Triton that results from micromolar concentrations of calcium, suggest that GP-A may play a role in stabilizing cerebellar synaptic junctions.     

Identification of intrinsic sialidase and sialoglycoprotein substrates in rat brain synaptic junctions

Sialidase activity associated with rat brain synaptic junctions (SJ) and synaptic membranes (SM) was determined. Both fractions released sialic acid from exogenous glycopeptides and gangliosides. SJ accounted for 5-10% of the total sialidase activity recovered from SM following extraction with Triton X-100, and the specific activity of SJ sialidase was 60% of that of the parent SM fraction. Intrinsic SJ sialidase hydrolysed 12-15% of the sialic acid associated with endogenous SJ glycoproteins. Sialic acid residues associated with SJ glycoproteins were labelled with sodium borotritide and SJ proteins fractionated by affinity chromatography on concanavalin A-agarose. SJ glycoproteins that reacted with concanavalin A (con A+ glycoproteins) accounted for 25% of the total SJ [3H]sialic acid. Intrinsic SJ sialidase hydrolysed 20% of the [3H]sialic acid associated with these glycoproteins. Each molecular weight class of con A+ glycoprotein previously shown to be a specific component of the postsynaptic apparatus contained sialic acid and was acted on by intrinsic SJ sialidase.     

Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.

The recent finding that the E1 glycoproteins of murine coronaviruses contain only O-linked oligosaccharides suggested that this unusual modification might be a distinguishing feature of coronaviruses and might play an essential role in the life cycle of this family of viruses. To examine these possibilities, we analyzed the oligosaccharide moieties of the membrane proteins of the avian coronavirus infectious bronchitis virus. In addition, we determined the effect of inhibiting the glycosylation of these proteins on viral maturation and infectivity. Infectious bronchitis virus virions contain nine proteins. Four of these proteins, GP36, GP31, GP28, and P23, are closely related structurally and appear to be homologous to the E1 proteins of murine coronaviruses. We found that the oligosaccharides of GP31 and GP28 could be removed with endoglycosidase H and that neither of these glycoproteins was detectable in tunicamycin-treated cells. These two results indicated that GP31 and GP28 contain N-linked oligosaccharides. Therefore, O-linked oligosaccharides are not a universal feature of the small coronavirus membrane glycoproteins. Tunicamycin inhibited glycosylation of all of the viral glycoproteins but did not inhibit production of virions by infectious bronchitis virus-infected cells. The virions released by these cells contained only the three non-glycosylated viral proteins P51, P23, and P14. These particles were not infectious. Therefore, it appears that glycosylated infectious bronchitis virus polypeptides are not required for particle formation. However, the viral glycoproteins are apparently indispensible for viral infectivity.     

Oligosaccharide chains of herpes simplex virus type 2 glycoprotein gG.2

gG.2 glycoprotein was purified by H966 monoclonal antibodies linked to Sepharose from herpes simplex virus type 2-infected HEp-2 cells labeled with [3H] glucosamine. The glycoprotein was subjected to Pronase digestion and the glycopeptides were fractionated by Con A-Sepharose in a major fraction (88.5% of total radioactivity) unbound to the lectin gel and in a minor species which bound to the lectin as a N-linked diantennary oligosaccharide. Mild and strong acid hydrolysis of Con A-unbound and Con A-bound fractions revealed that (i) both species were highly sialylated; (ii) the Con A-unbound fraction contained mainly labeled N-acetylgalactosamine, as is the case for O-linked oligosaccharides; and (iii) the Con A-bound fraction carried the vast majority of the labeled N-acetylglucosamine present in gG.2. Three size classes of oligosaccharides were separated from mild alkaline borohydride-treated Con A-unbound glycopeptides, which accounted for about 80% of the radioactivity present in the fraction. Galactosaminitol was recovered as the major labeled product in the strong acid hydrolyzates of the oligosaccharides generated by reductive beta-elimination, indicating that they were O-glycosidically linked to the peptide backbone. Thin-layer and DEAE-Sephacel chromatography of the three O-linked oligosaccharide species indicated that disialylated tetrasaccharides and monosialylated trisaccharides were the major components, whereas neutral disaccharide was a minor component. Digestion with neuraminidase and beta-galactosidase of the O-linked oligosaccharides supported the idea that the common disaccharide core was mainly of the structure beta-galactosyl-N-acetylgalactosamine. The large occurrence of O-linked oligosaccharides differentiates this type 2-specific herpes simplex virus glycoprotein from the type-common herpesvirus glycoproteins gB, gC, and gD.     

In Vivo Phosphorylation of the Postsynaptic Density Glycoprotein gp180

Rats received intraventricular injections of [32P]PO4 and were killed after 30 min for the preparation of postsynaptic densities (PSDs). Gel electrophoretic analysis identified a number of PSD proteins that incorporated 32P under these conditions. Major proteins that were labelled with 32P had Mr of 185,000, 165,000, 140,000, 92,000, and 51,000. Of these p185, p165, and p140 were also labelled when PSDs were incubated with [gamma-32P]ATP in vitro. In contrast p92 and p51 were relatively poorly labelled under in vitro conditions. Analysis of glycoproteins isolated by chromatography on concanavalin A (Con A)-agarose demonstrated that greater than 70-80% of the 32P present in the glycoproteins eluted from Con A-agarose with alpha-methyl-D-mannopyranoside (Con A+ glycoproteins) was associated with the PSD specific glycoprotein gp180 following both in vivo and in vitro labelling. Phosphopeptide maps and phosphoamino acid analysis of gp180 indicated that similar sites were labelled in vitro and in vivo. Analysis of the subcellular distribution of glycoproteins that incorporated 32P during in vivo labelling demonstrated that gp180 was highly concentrated in PSDs, in accord with the previously suggested exclusive association of this glycoprotein with postsynaptic structures.     

Differential and cell-type specific microheterogeneity of high mannose-type Asn-linked oligosaccharides of human transferrin receptor.

The structural analysis of high mannose-type Asn-linked (N-linked) oligosaccharides of the human transferrin receptor (hTR) from D-[2-3H]mannose metabolic-radiolabeled human cells--A431, K562, BeWo, and HL60--was investigated. The radiolabeled hTR glycopeptides were prepared and fractionated by a lectin chromatography of Concanavalin A-Sepharose. The composition analysis of hTR glycopeptides revealed that Con A-I contains both mannose and fucose, whereas Con A-III has mannose exclusively. The Con A-III glycopeptides were treated with Endo H. The released oligosaccharides were charge-fractionated by QAE-Sephadex. The neutral oligosaccharides were further size-fractionated by an amine absorption high performance liquid chromatography (HPLC). Our results demonstrate that the high mannose-type oligosaccharides of hTR ranged in size from Man5-R to Man9-R with cell-type specific patterns. A relative amount of each component was found to be differentially heterogeneous among the four different human cell lines.     

Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long-Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD-GP180

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M_r 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M_r 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N₂ instead of O₂ and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca²⁺ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca²⁺ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca²⁺-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.     

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.     

Alterations in N-linked oligosaccharides of glycoproteins during rat liver regeneration

[3H]Mannose-labeled glycopeptides in the slices after partial hepatectomy were characterized by column chromatography using Sephadex G-50, DE-52 and Con A-Sepharose, and further by digestion with alpha-mannosidase and endo-beta-N-acetylglucosaminidase H. They contained both 'complex type' and 'high-mannose type' oligosaccharides. A higher proportion of 'complex type' oligosaccharides was contained in regenerating liver 24 h after partial hepatectomy than in control. This tendency was increased gradually with time and was most pronounced at 144 h. In our previous studies, the activities of microsomal N-acetylglucosaminyltransferase towards endogenous and exogenous acceptors at 144 h after partial hepatectomy were shown to exceed most prominently that in control. No differences in the oligosaccharides were observed at 240 h when the deficit of liver had been restored. The oligosaccharides of glycopeptides in the incubation media were mostly 'complex type' and the differences between regenerating liver and control were observed only at 144 h. These results suggest that oligosaccharide processing of glycoproteins is regulated at the transfer step of peripheral N-acetylglucosamine to core oligosaccharides 144 h after partial hepatectomy, and that these alterations in oligosaccharides of glycoproteins may be related to hypertrophy and hyperplasia of hepatic cells in liver regeneration.     

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: II. The primary structures of the low molecular weight carbohydrate chains of the glycoproteins.

The primary structures of the O-glycosidically linked oligosaccharides isolated from glycoproteins GP I and GP II of Fusarium sp. M7-1 were established. The oligosaccharides released by alkaline borohydride treatment from the glycoproteins were purified by Bio-Gel P-4 and HPLC. This approach resulted in one monosaccharide and seven oligosaccharides. Their primary structures were resolved mainly by NMR spectrometry in combination with methylation mass spectrometry and fast atom bombardment mass spectrometry. The following structures have been determined. [formula: see text].