Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Structure of the mannose-rich oligosaccharide chains of concanavalin A-binding glycopeptides derived from beef brain glycoproteins

Abstract: A neutral, mannose-rich, concanavalin A (Con A)-binding glycopeptide fraction was obtained by proteolytic digestion of defatted beef brain tissue. Hydrazinolysis followed by gel filtration of the reaction products provided three oligosaccharides. A portion of each oligosaccharide was treated by exhaustive digestion with α-mannosidase. Another portion was subjected to selective acetolysis of Manαl-6Man linkages, providing two fragments that were recovered by gel filtration. The structure of the intact oligosaccharides, as well as the fragments obtained by selective acetolysis and enzymatic treatment, were resolved by gas-liquid chromatographic-mass spectrometric analysis. The structures of the three oligosaccharides were: (a) Manαl-2Manαl-6(Manαl -3)Manαl-6(Manαl-2Manαl-2Manαl 3)Manβ1-4- N -acetylglucosamine (GlcNAc)β - 4N- acetylglucosaminitol (GlcOLNAc); (b) Manαl -2Manαl -6(Manαl -3)Manαl-6(Manαl-2Manαl-3)-Manβ1-4GlcNAcβl -4GlcOLNAc; and (c) Manαl -6(Manαl-3) Manαl - 6(Manαl - 3)Manβl -4GlcNAc-βl - 4GlcOLNAc. These structures account for 15–20% of the glycoprotein-carbohydrate of whole beef brain and most of the oligosaccharides that demonstrate a high affinity for Con A. In view of the large number of Con A-binding glycoproteins in brain tissue, it appears that many of these different glycoproteins must contain structurally identical oligosaccharides.     

Characterization of concanavalin A-binding neuronal and glial surface glycoproteins from human foetal brain.

Neuronal and glial surface glycoproteins have been isolated from human foetal brains by affinity chromatography on 8 M urea or 6 M guanidine-treated Con A-Sepharose 4B at 4 degrees C and three groups of glycoproteins of molecular mass 65-73 kDa, 52-63 kDa and 43-48 kDa have been identified on SDS/PAGE. These glycoproteins exhibited anomalous behaviour on SDS/PAGE, indicating the existence of a gradation of mutually interconvertible protein-SDS aggregates in dynamic equilibrium with one another. Deglycosylation and deacylation did not alter the SDS/PAGE multiple band pattern. Purified glycoproteins contained 160 +/- 90 micrograms carbohydrate/mg protein, and a sialic acid content of 25 +/- 5 nmole/mg protein. The N-terminals were blocked. The glycoproteins moved preferentially on acid/urea/PAGE. Sepharose 6B gel filtration in the absence of lipid and detergents resolved the glycoproteins into an excluded peak I and a low molecular mass peak II. Peaks I and II were non-interconvertible on Sepharose 6B gel filtration or on reversed phase HPLC in an isopropanol/water/TFA gradient system. Both peaks rendered a single fast moving band of identical mobility on acid/urea/PAGE, suggesting that peak I was possibly a micellar aggregate of the monomeric peak II. The glycoproteins were refractory to digestion by trypsin or pronase and reacted identically towards various lectins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of concanavalin A-binding neurospecific glycoproteins from various regions of the rat brain during experimental neurosis

The polypeptide composition of neurospecific glycoproteins in different areas of the rat brain under experimental neurosis is characterized using SDS-PAG-electrophoresis followed by electroblot and immunofixation on nitrocellulose membranes. The soluble and membrane-bound glycoproteins are purified by Con A-Sepharose column chromatography. Changes in the glycoprotein polypeptide composition in different areas of the rat brain under experimental neurosis are qualitative. Soluble glycopolypeptide 27 kDa and membrane glycopolypeptide 32 kDa are not revealed in the midbrain and corpus striatum. Soluble polypeptide 47 kDa is absent in the cerebral cortex and hippocampus. It is suggested that the above mentioned glycopolypeptides are important for the CNS physiological functioning.     

Separation of glycopeptides derived from brain glycoproteins by gel filtration.

Glycopeptides obtained from rat brain by proteolytic digestion with papain have been separated from glycosaminoglycans by means of gel filtration. The glycosaminoglycans appear in the void volume, whereas the glycopeptides are retarded. Glycopeptides of groups A+B (Brunngraber et al., 1973) (MW = 3800-500) C+D (MW = 2000) which were partially resolved by the method, were identified in the elution profile. Nucleic acids, also solubilized by papain, are eluted together with the glycosaminoglycans.     

Identification and characteristics of concanavalin A-binding neurospecific glycoproteins in human brain and brain tumors

Soluble and membrane-bound neurospecific Con A-binding glycoproteins from human brain and tumours were identified and characterized, using a procedure which included stepwise extraction with low and high ionic strength buffers, buffered. Triton X-100 and sodium deoxycholate followed by ConA-Sepharose column chromatography, SDS-PAAG electrophoresis and immunoblotting. Adsorbed antisera against different types of neurospecific glycoproteins were used. The bulk of neurospecific glycoproteins (11 and 13) were revealed in protein fractions extracted with low ionic strength buffers and Triton X-100. In astrocytomas and glyoblastomas, some neurospecific glycoproteins were absent. Some glycoproteins were found in tumours, but were absent in brain tissue. Soluble, 77 kD glycoprotein, 11 and 16 kD glycoproteins solubilized with high ionic strength buffers and intrinsic membrane-bound 51, 57, 61, 74 and 77 kD glycoproteins can be viewed as stable neurospecific markers in malignant brain tumours.     

Selective solubilization and purification of cell-surface concanavalin A-binding glycoproteins from Novikoff hepatocellular carcinoma cells

Novikoff hepatocellular carcinoma cells possess cell-surface glycoproteins that bind the lectin, concanavalin A. A subset of Con A-binding plasma membrane glycoproteins was solubilized by addition of n-butanol to a suspension of Novikoff cells. Glycoproteins solubilized into the n-butanol-saturated aqueous phase of the two-phase mixture were purified by sequential chromatography on DEAE-cellulose and Sepharose-conjugated concanavalin A. Glycoproteins specifically bound to the Sepharose-conjugated Con A exhibited apparent M_r = 72,000 to 125,000. The plasma membrane localization of these components was inferred by their isolation from cells surface labeled with NaIO₄/ NaB³H₄. A xenoantiserum, raised against glycoproteins specifically bound to Sepharose-conjugated concanavalin A was employed to identify reactive components in nonionic detergent extracts of Novikoff tumor cells or rat hepatocytes surface labeled using lactoperoxidase-catalyzed iodination (¹²⁵I). Major reactive peptides in extracts of Novikoff cells exhibited apparent M_r = 74,000, 82, 000, 110,000, and 135,000, while those in extracts of hepatocytes possessed apparent M_r = 98,000 and 105,000. The reactivity of the antiserum with extracts of ¹²⁵I-labeled Novikoff cells was abolished by absorption of the antiserum with hepatocytes, indicating that the qualitative differences observed may result from structural modification of one or more cell-surface glycoproteins, rather than the expression of new or inappropriate glycoproteins. This antiserum will provide a useful probe to investigate alterations in the expression or structure of glycoproteins that occur as a consequence of malignant transformation or adaptation of malignant cells to growth in the ascitic form.     

Studies on glycopeptides derived from acidic glycoproteins of guinea-pig serum

1. Fraction I, a fraction containing acidic glycoproteins, isolated from guinea-pig serum, was digested with Pronase after removal of sialic acid and a major and a minor glycopeptide fraction were isolated by chromatography with Sephadex G-25 and G-50. 2. The major fraction was examined by various methods and shown to contain several glycopeptides. Estimates of molecular weight of the glycopeptide fractions were obtained. Although some variation appeared to occur, the glycopeptides were not grossly heterogeneous with respect to size. An average prosthetic group was estimated to contain about 15 sugar residues. 3. Aspartic acid was the principal amino acid present in the fractions and in all subfractions of the major fraction investigated. Where examined, ammonia was liberated on acid hydrolysis in approximately equimolar amounts to the aspartic acid present. The carbohydrate composition of the fractions was also determined. 4. The glycopeptides showed relatively little degradation in alkaline solution. 5. These results suggest that an N-acylglycosylamine bond involving aspartic acid forms the major type of linkage between carbohydrate and polypeptide. The isolation of a compound with the composition and chromatographic properties of 2-acetamido-1-(l-beta-aspartamido)-1,2-dideoxy-beta-d-glucose supports this view, and indicates that N-acetylglucosamine is the sugar involved in at least many linkages. 6. Fraction I contains some glycoproteins that are susceptible to Pronase and one or more others that resist digestion before the removal of sialic acid. A brief examination revealed some similarities between prosthetic groups derived from both kinds of glycoprotein.     

Glycopeptides from rat brain glycoproteins

The lipid-free protein residue of rat brain tissue was treated with papain to solubilize the heteropolysaccharide chains of the tissue glycoproteins. The glycopeptides were separated into non-dialyzable and dialyzable glycopeptide preparations. Each preparation was then sorted out into groups of glycopeptides by means of electrophoresis and gel filtration. The quantitatively predominant glycopeptides were the alkali-stable glycopeptides (Group A) which accounted for 64% of the glycopeptide carbohydrate recovered from rat brain. Most of the group A glycopeptides appeared in the non-dialyzable preparation. The molecular weight of the glycopeptides of Group A ranged from approximately 5200–3700. The largest glycopeptide molecule in this mixture possessed the highest electrophoretic mobility and contained one fucose, four N-acetylneuraminic acid (NANA), six N-acetylglucosamine, four galactose, and three mannose residues per molecule. The spectrum of glycopeptides isolated in this group showed a progressive decrease in NANA rsidues, NANA and galactose residues, and NANA, galactose, and N-acetylglucosamine residues which could be correlated with a progressive decline in molecular weight and electrophoretic mobility. Some of the glycopeptides in each fraction recovered from this group of glycopeptides contained sulfate ester groups.A second group of glycopeptides (Group C glycopeptides) accounted for 25% of the total glycoprotein carbohydrate recovered from rat brain. These were recoverd from the dialyzable glycopeptide preparation, and resolved into three fractions by column electrophoresis. These glycopeptides do not contain sulfate, are composed predominately of mannose and N-acetylglucosamine, and possess a molecular weight of approximately 3000.Several minor groups of glycopeptides were detected. Alkali-labile glycopeptides (Group B) appeared in the non-dialyzable glycopeptide preparation. The dialyzable glycopeptide preparation contained glycopeptides (Group E) which contained N-acetylgalactosamine and glucose. These had a molecular weight of approximately 2000. Group D glycopeptides recovered from the dialyzable glycopeptide preparation contained variable amounts of NANA, mannose, galactose, N-acetylglucosamine, and sulfate. These possessed a molecular weight of approximately 2900.     

Preparation and properties of sialomucopolysaccharides obtained from rat brain

Sialomucopolysaccharides were released from the defatted protein residue by the proteolytic action of papain after extraction of rat whole brains with chloroform-methanol (2:1, v/v). Further purification is achieved by dialysis to remove low-molecular-weight fragments and by precipitation of nucleic acids and glucuronic acid-containing mucopolysaccharides by treatment with cetylpyridinium chloride. Gel filtration of the sialomucopolysaccharides through Sephadex G-200 removes the major portion of the impurities that absorb light in the ultraviolet region. The sialomucopolysaccharides were fractionated on DEAE-Sephadex to yield a population of sialomucopolysaccharides that show an increase in N-acetylneuraminic acid content and a decrease in fucose content as the concentration of chloride required to elute the individual components is increased. On gel filtration on Sephadex G-200, those sialomucopolysaccharide molecules rich in N-acetylneuraminic acid and poor in fucose appear to be larger molecules than those rich in fucose and poor in N-acetylneuraminic acid. A structure is proposed in which all sialomucopolysaccharide molecules are assumed to possess the same repeating unit consisting of hexosamine and hexose. The molecules differ from each other in the number of fucose and N-acetylneuraminic acid residues attached to the basic structure. Most of the hexosamine is present as glucosamine, although one fraction was obtained that appeared to contain galactosamine. Most of the hexose present is accounted for as galactose and mannose, although small amounts of glucose were found in some fractions. Methods of analysis for the N-acetylneuraminic acid and hexosamine components of the sialomucopolysaccharides were defined.     

Partial characterization of rat hepatoma cell-surface glycopeptides possessing concanavalin A receptor activity.

Papain digestion of Novikoff or AS-30D rat hepatoma cells released glycopeptides from the cell surface. That portion of the glycopeptides accessible to Sephadex G-50 was digested with pronase and the component glycopeptides partially resolved by ion-exchange chromatography. Each tumor type yielded two well resolved sialoglycopeptide fractions which possessed concanavalin A receptor activity. The amino acid and saccharide composition of these low molecular weight (3,100 ± 300 daltons) sialoglycopeptides was determined.     

Developmental changes of proteins and concanavalin A reactive glycoproteins in growth cones from rat forebrain.

Growth cones were isolated from the forebrains of 1, 5 and 9 days-old rats. The ultrastructural characterization of the obtained subcellular fractions reveals that two of them (GC1 and GC2) contain predominantly growth cones. It was found that the protein content of the membranes contained in these fractions increases 7.5 times, while in whole forebrain the increase is only 3 times, showing that during the studied developmental period there is a predominant protein enrichment of the specialized brain structures (e.g. growth cones). Electrophoretic studies show that there are characteristic changes of the Coomassie Brilliant Blue R250 staining and concanavalin A reactive protein profiles. Comparison of the protein patterns of growth cones to those of synaptosomes from mature forebrain reveal a number of bands, which appear to be characteristic for one of these structures. The possible roles of the developmentally controlled proteins in the processes of synaptogenesis is discussed.     

Preparation and properties of mitochondria derived from synaptosomes.

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

Preparation of glycopeptides and oligosaccharides from thyroxine-binding globulin.

Over 99% of thyroxine (T4), the major form of thyroid hormone in plasma, is bound to the plasma glycoprotein thyroxine-binding globulin (TBG). The carbohydrate composition of TBG (14.6% by weight) consists of mannose, galactose, N-acetylglucosamine, and N-acetylneuraminic acid in the molar ratios of 11:9:16:10 per mol of glycoprotein. No fucose or N-acetylgalactosamine were detected. Amino acid analyses were performed. Glycopeptides, prepared by exhaustive pronase treatment of the glycoprotein, were separated by gel filtration and ion exchange chromatography. All glycopeptides contained the four sugars present in the native glycoprotein. One-fourth of the glycopeptide fraction was resolved into a discrete component, glycopeptide I. The remaining glycopeptides were a mixture termed glycopeptides II and III. Glycopeptides II and III were resolved into two discrete carbohydrate units, termed oligosaccharides A and B, by alkaline-borohydride treatment and DEAE-cellulose chromatography. We propose that TBG contains four oligosaccharide chains as calculated from the molecular weights of the glycopeptides and from compositional data assuming 1 asparagine residue/glycopeptide. The carbohydrate structures of the glycopeptides and relative affinities of TBG, glycopeptides and oligosaccharides for hepatocyte plasma membrane binding are presented in the accompanying paper (Zinn, A.B., Marshall, J.S., and Carlson, D.M. (1978) J. Biol. Chem. 253, 6768-6773.