Characterization of glycopeptides labelled from D-[2-3H]mannose and L-[6-3H]fucose in intestinal epithelial cell membranes during differentiation.

The labelled glycopeptides obtained by Pronase digestion of rat intestinal epithelial cell membranes were examined by gel filtration after injection of D-[2-3H]mannose and L-[6-3H]fucose. Three labelled fraction were eluted in the following order from Bio-Gel P-6, Fraction I, which was excluded from the gel, was labelled mostly with [3H]fucose and slightly with [3H]mannose. Fraction II contained "complex" asparagine-linked oligosaccharides since it was labelled with [3H]mannose and [3H]fucose, was stable to mild alkali treatment, and resistant to endo-beta-N-acetyl-glucosaminidase H. Fraction III contained "high-mannose" asparagine-linked oligosaccharides, which were labelled with [3H]mannose, but not with [3H]fucose; these were sensitive to endo-beta-N-acetylglucosaminidase H, and were adsorbed on concanavalin A-Sepharose and subsequently eluted with methyl alpha-D-mannopyranoside. The time course of incorporation of [3H]mannose into these glycopeptides in microsomal fractions showed that high-mannose oligosaccharides were precursors of complex oligosaccharides. The rate of this processing was faster in rapidly dividing crypt cells than in differentiated villus cells. The ratio of radioactively labelled complex oligosaccharides to high-mannose oligosaccharides, 3h after [3H]mannose injection, was greater in crypt than in villus-cell lateral membranes. Luminal membranes of both crypt and villus cells were greatly enriched in labelled complex oligosaccharides compared with the labelling in lateral-basal membranes. These studies show that intestinal epithelial cells are polarized with respect to the structure of the asparagine-linked oligosaccharides on their membrane glycoproteins. During differentiation of these cells quantitative differences in labelled membrane glycopeptides, But no major qualitative change, were observed.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Identification of an epidermal cell-adhesion glycoprotein.

Glycoproteins which mediate intercellular adhesion were studied by comparing the effects of trypsin and the neutral proteinase, Dispase, on human keratinocytes metabolically labelled with D-[1-14C]glucosamine or L-[1-3H]fucose. Whereas digestion of keratinocytes with trypsin/EDTA resulted in loss of both cell-substratum and intercellular adhesion, only cell-substratum adhesion was disrupted by incubation with Dispase. Analysis of the radiolabelled glycoproteins by polyacrylamide-gel electrophoresis revealed that a glycoprotein of Mr 126 000 was cleaved by trypsin/EDTA, but not by Dispase. Surface labelling of keratinocytes with galactose oxidase/NaB3H4 confirmed that this glycoprotein was exposed on the cell surface. Addition of lmM-Ca2+ prevented dispersion of keratinocytes by trypsin and concomitantly protected the glycoprotein of Mr 126 000 from digestion. These results indicate that this glycoprotein has an important role in mediating intercellular adhesion of keratinocytes.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.     

The carbohydrate components of arterial basement-membrane-like material. Studies on rabbit aortic myomedial cells in culture.

The carbohydrate composition of arterial basement-membrane-like material was investigated. Basement-membrane-like material was isolated from cultures of aortic myomedial cells by a sonication/differential-centrifugation technique. Purified basement-membrane-like material contained a total of 5% sugars, comprising glucose, galactose, mannose, fucose, sialic acid, glucosamine and galactosamine in the approximate molar proportions 3.2:3.5:3.4:3.2:1:5.5:3.1. In addition, small amounts of xylose were found. Analyses for uronic acid showed that glycosaminoglycans comprised about 1% of isolated basement-membrane-like material. The carbohydrate composition indicated the presence of complex-type oligosaccharides in addition to hydroxylysine-linked disaccharides. [3H]Glucosamine-labelled glycopeptides obtained by proteinase digestion and gel filtration were resistant to endo-beta-N-acetylglucosaminidase D, but more than 10% were susceptible to alpha-mannosidase, demonstrating the presence of high-mannose-type oligosaccharides. The distribution of carbohydrates among peptides of basement-membrane-like material on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was investigated after labelling with [3H]mannose, [3H]fucose, [3H]galactose and [3H]glucosamine. Among peptides that appeared to carry carbohydrates were a proteoglycan(s) and seven glycoproteins in the molecular-weight range 120 000-700 000.     

Synthesis of cellular and extracellular glycoproteins by cultured human keratinocytes and their response to retinoids

The glycoproteins synthesized by human keratinocytes cultured on 3T3 feeder layers were studied by metabolic labelling. Keratinocytes freed of feeder cells synthesized a complex pattern of cellular and extracellular glycoproteins that was distinct from that of 3T3 cells, dermal fibroblasts and epidermal melanocytes. The effect of low concentrations of all-trans-retinoic acid and arotinoid ethyl ester on glycoprotein synthesis was examined in keratinocyte cultures depleted of vitamin A. Treatment with either retinoid resulted in a 2-3-fold increase in the amount of D-[3H]glucosamine-labelled material in the culture medium. Gel electrophoresis revealed increased incorporation of D-[3H]glucosamine into extracellular glycoproteins of Mr 245,000, 170,000, 140,000, 130,000, 120,000 and 105,000 as well as into glycosaminoglycans in retinoid-treated cultures. The labelling of extracellular glycoproteins with L-[3H]leucine and L-[35S]methionine was also increased by retinoids suggesting increased synthesis of these components rather than an effect on their glycosylation. The Mr 245 000 glycoprotein was identified as keratinocyte-derived fibronectin by immunoblotting, immunoprecipitation and specific binding to gelatin. The results show that retinoids increase the synthesis of glycoprotein as well as glycosaminoglycan components of the extracellular matrix in human keratinocyte cultures. It is suggested that retinoids select for a population of cells that synthesize relatively large amounts of glycosaminoglycan, fibronectin and other as yet unidentified extracellular glycoproteins.     

Generation of 3HOH from D-[6-3H]glucose by erythrocytes: role of pyruvate alanine interconversion

Human and rat erythrocytes were found to generate 3HOH from D-[6(N)-3H]glucose. The rate of 3HOH production represented 7-10% of the glycolytic flux. The generation of 3HOH appeared attributable, in part at least, to the detritiation of [3-3H]pyruvate during the interconversion of the 2-keto acid and L-alanine in the reaction catalyzed by glutamate-pyruvate transaminase. Indeed, purified pig heart glutamate-pyruvate transaminase, as well as homogenates prepared from rat erythrocytes or pancreatic islets, catalyzed the generation of 3HOH from L-[3-3H]alanine. When the production of tritiated pyruvate from L-[3-3H]alanine was coupled to the conversion of the 2-keto acid to L-lactate, the production of 3HOH accounted for one-third of the reaction velocity, the latter failing to display isotopic discrimination. In these experiments, the production of 3HOH was abolished by amino-oxyacetate. Likewise, in intact rat erythrocytes, aminooxyacetate inhibited the generation of 3HOH and tritiated L-alanine from D-[6-3H]glucose (or D-[1-3H]glucose), as well as the generation of 3HOH from L-[3-3H]alanine. In pancreatic islets, however, aminooxyacetate failed to affect significantly the generation of 3HOH from D-[6-3H]glucose. These findings indicate that the generation of 3HOH from D-[6-3H]glucose is mainly attributable to an intermolecular tritium transfer in transaminase reaction, at least in cells devoid of mitochondria.     

Presence of glycoproteins in the cell nucleus as shown by radioautographic studies after administration of [3H]fucose and [3H]galactose

Dorsal root ganglia were removed from adult bullfrogs and incubated with [3H]fucose for intervals from 15 min to 1 h, followed by fixation. Some ganglia were post-incubated in the absence of [3H]fucose for up to 17 h. In additional in vivo experiments, young frogs were injected with [3H]fucose, and killed 30 min or 1 h later, and then ganglia were removed and fixed. Electron microscope radioautographs of the ganglia revealed an intense radioautographic reaction over the nuclei of Schwann and satellite cells as early as 5 min after initial exposure to [3H]fucose. At time intervals up to 2 h after initial exposure to [3H]fucose, the silver grains were evenly distributed over both the periphery and internal regions of the nucleus, while at 18 h they were localized to the cell periphery. In occasional cells, the perinuclear space was expanded in some areas and was the site of reaction. In young rats, injected with [3H]galactose and killed 15 min to 5 h later, electron microscope radioautographs revealed heavy reaction over the nuclei of duodenal villous and crypt columnar cells, in which the grains were evenly distributed over both the peripheral and internal regions. In mitotic cells, grains appeared to be associated with the condensed chromatin of forming chromosomes. These results provide strong evidence that glycoproteins exist in the nuclei of the above cell types and that they are actively renewed. The rapid appearance of nuclear reaction after initial exposure to [3H]fucose or [3H]galactose indicates that either these sugars are added to glycoproteins within the nucleus itself or that they migrate rapidly to this site after having been glycosylated elsewhere.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes.

To define the role of cytoplasmic microtubules in the biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells, we studied the effect of colchicine on the incorporation of L-[1,5,6-3H]fucose into Golgi, lateral basal and microvillus membranes. Colchicine was administered intraperitoneally before or after injection of radioactive fucose. The incorporation of radioactivity into Golgi membranes was little affected by colchicine, which did not prevent the redistribution of most of the labelled glycoproteins from the Golgi complex into other parts of the villus cell. The incorporation of labelled glycoproteins into the microvillus membrane was greatly inhibited by colchicine given 2 h or 10 min before the radioactive fucose: all labelled glycoproteins present in this membrane were equally affected. In contrast, the administration of colchicine considerably increased the incorporation of radioactivity into the lateral basal part of the plasmalemma, and prevented the disappearance of most of the labelled glycoproteins from this membrane at late times after fucose injection. These results suggest that cytoplasmic microtubular structures are important for the polarization of the intestinal villus cell and the biogenesis of the microvillus membrane, although playing little or no role in the movement of membrane components from the Golgi complex to the lateral basal part of the plasmalemma.     

Patterns of cell differentiation revealed by L-[3H]fucose incorporation in Dictyostelium

A study of the incorporation of l-[6-³H]fucose and d-[6-³H]glucosamine hydrochloride was conducted during the development of the cellular slime mold Dictyostelium discoideum 1-H. Autoradiographs revealed that pulse-labeled vegetative amoebae incorporated [³H]fucose intracytoplasmically within 15 min. The majority of the cells had randomly scattered silver grains but the remainder were distinguished by a dense localized labeling which suggested that oligo or polysaccharide synthesis was occurring. The localized pattern of labeling attributed to active synthesis declines at aggregation and early conus formation. As the pseudoplasmodium makes the developmental transition from the conus to the culmination stages the localized pattern of [³H]fucose labeling was restricted to the prespore cells while the prestalk cells were devoid of label. Prespore vacuoles were not present at the onset of this transition and consequently [³H]fucose incorporation occurred in the cells prior to their differentiation into prespore cells. In contrast to cells composing earlier stages, mature spores exhibited [³H]fucose-containing substances at the cell surface. At appropriate stages certain cells actively synthesize slime and stalk sheath which were labeled with either [³H]fucose or [³H]glucosamine.Prestalk isolates were obtained by transecting migrating slugs. [³H]Fucose was incorporated within 10 min among the basal cells of the isolate in the localized pattern typically found in prespore cells. The incorporation of [³H]fucose occurred prior to prespore differentiation as certain preparations were devoid of prespore vacuoles. Prespore isolates differentiate prestalk cells which have lost the capacity to incorporate [³H]fucose. This investigation suggests that cell contacts and interactions may affect the incorporation of [³H]fucose.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 I. Electrophoretic studies and partial fractionation of the phenol-soluble proteins

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 II. Location in inner and outer membranes

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

Biosynthesis of a glycosylated keratin by human keratinocytes

Human keratinocytes, cultured in the presence of D-[1-14C]glucosamine, incorporated radioactivity into a cytoskeleton-associated glycoprotein with Mr 53,000. This glycoprotein co-purified with prekeratin when keratinocyte cytoskeletons were extracted with 0.1 M citric acid/0.1 M sodium citrate and subjected to isoelectric precipitation at pH 4.0. Analysis of the prekeratin polypeptides by two-dimensional gel electrophoresis revealed that the radioactivity was restricted to a single polypeptide with an isoelectric point in the pH range 4.5-5.5. Acid hydrolysis of prekeratin followed by paper chromatography of the hydrolysate showed that the radioactivity was incorporated as glucosamine and not by metabolic conversion to amino acids. Control experiments showed that the radioactivity associated with the glycoprotein of Mr 53,000 was not the result of adsorbed glycolipids or non-enzymatic labelling. In contrast to the incorporation of D-[1-14C]glucosamine and D-[6-3H]glucosamine, no appreciable amounts of L-[6-3H]fucose, D-[2-3H]mannose or 32PO4 were incorporated into this glycoprotein. The immunological relationship of the glycoprotein of Mr 53,000 to the keratins was demonstrated by its reactivity with both polyclonal and monoclonal antisera to keratin.     

Partial characterization of the carbohydrate units of rat intestinal sucrase--isomaltase.

Sucrase--isomaltase was purified from rat intestinal microvillus membranes after injection of D-[2-3H]mannose and L-[6-3H]fucose, using a column of monoclonal antibody-protein A-Sepharose. After Pronase digestion and gel filtration of the glycopeptides labelled from both precursors, a major part of the radioactivity was recovered in asparagine-linked complex oligosaccharides, and a smaller amount in partially alkali-labile high-molecular-weight glycopeptides. Only a small amount of [3H]mannose was found in endo-beta-N-acetylglucosaminidase H-sensitive high-mannose oligosaccharides.     

Glycoprotein biosynthesis by organ cultures of hamster trachea

Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [¹⁴C]glucosamine and [³H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as “intracellular”, gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and “intracellular” material; it labelled extensively with both glucosamine and fucose and had a molecular weight of approx. 5000 (as judged by its elution from Sephadex G-75). This fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.     

Lectin-binding proteins in central-nervous-system myelin. Detection of glycoproteins of purified myelin on polyacrylamide gels by [3h]concanavalin A binding.

Concanavalin A strongly agglutinates purified fragments of immature and mature rat brain myelin, but only weakly agglutinates mature bovine and human myelin fragments. A sensitive method involving [3H]concanavalin binding to sodium dodecyl sulphate/polyacrylamide gels was used to detect the concanavalin A-binding proteins in purified myelin. When applied to mature rat brain myelin proteins that had been labelled in vivo with [14C]fucose, the distribution of the [3H]concanavalin A on the gel was very similar to that of [14C]fucose with the major peak corresponding to the major myelin-associated glycoprotein. The technique revealed that the immature form of the myelin-associated glycoprotein with a slightly larger apparent molecular weight also bound concanavalin A, and that in purified immature rat myelin the quantitative importance of some of the other glycoproteins in binding concanavalin A was increased relative to the myelin-associated glycoprotein. The separated proteins of bovine and human myelin bound more [3H]-concanavalin A than those of rat myelin. In these species, the myelin-associated glycoprotein was a major concanavalin A-binding protein, although two higher-molecular-weight glycoproteins also bound significant quantities of [3H]concanavalin A. The results indicate that there are receptors for concanavalin A on the surface of rat, bovine and human myelin membranes and suggest that the myelin-associated glycoprotein is one of the principal receptors.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

Blood group-active carbohydrate chains on the receptor for epidermal growth factor of A431 cells

The antigens expressed on the carbohydrate chains of the receptor for epidermal growth factor of A431 cells were studied by immunoblotting with monoclonal antibodies. Blood group A and the Type 1 based blood group ALeb and Lea antigens were detected as well as antigens associated with unsubstituted, monofucosylated and difucosylated Type 2 blood group chains. The Lea and the difucosylated Type 2 antigen activities were abolished by treating the blotted receptor with endo-beta-galactosidase, indicating that they are expressed on backbone structures of poly-lacto/neolacto type. (The term 'poly-lacto/neolacto' is used here to describe oligosaccharide backbone structures consisting of repeating Type 1, Gal beta 1-3GlcNAc (lacto) or Type 2, Gal beta 1-4GlcNAc (neolacto) sequences.) The glycosidic linkage of oligosaccharides to protein was investigated using Pronase digests of the receptor biosynthetically labelled with [3H]glucosamine or [3H]fucose. The oligosaccharides were alkali-resistant, consistent with N- rather than O-glycosidically linked chains. A proportion of [3H]fucose-labelled glycopeptides was susceptible to endo-beta-galactosidase, confirming the immunoblotting experiment using antibodies against the Lea and the difucosylated Type 2 antigenic determinants. Oligosaccharides were released from the [3H]fucose- and [3H]-glucosamine-labelled glycopeptides by hydrazinolysis. Chromatography of the oligosaccharides on Bio-Gel P6 and Concanavalin A columns indicated a spectrum of oligosaccharides which include those of high mannose type labelled with [3H]glucosamine, and a mixture of oligosaccharides labelled with [3H]fucose and [3H]glucosamine of bi- and multiantennary complex types of which a subpopulation is susceptible to digestion with endo-beta-galactosidase.     

A marked increase of fucosylation of glycoproteins in IMR-90 human diploid fibroblasts during senescence in vitro

Possible changes of glycoproteins in IMR-90 human embryonic lung fibroblasts during senescence in vitro were studied by the metabolic labeling technique using radioactive precursors for carbohydrate moieties of glycoproteins. IMR-90 fibroblasts at three different population doubling level (PDL) were incubated with [3H]fucose and [3H]glucosamine for various periods of time. The radioactively labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The results indicated a marked increase, by more than eight-fold on per mg protein basis, of labeling by [3H]fucose in old IMR-90 fibroblasts (PDL = 45) as compared to young (PDL = 22) and middle-age (PDL = 30) IMR-90 fibroblasts. In contrast, no significant difference in [3H]glucosamine labeling was observed in young and old IMR-90 cells.