Complex-type asparagine-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni adult males contain terminal beta-linked N-acetylgalactosamine

Many studies have shown that the human blood fluke Schistosoma mansoni contains glycoproteins whose oligosaccharide side chains are antigenic in infected hosts. We report here that adult male schistosomes synthesize glycoproteins containing complex-type N-linked chains that have structural features not commonly found in mammalian glycoproteins. Adult male worms were incubated in media containing either [3H]mannose, [3H]glucosamine, or [3H]galactose, and the metabolically radiolabeled oligosaccharides on newly synthesized glycoproteins were analyzed. Schistosomes synthesize triantennary- and biantennary-like complex-type asparagine-linked chains that contain mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. Interestingly, none of the complex-type chains contain sialic acid, and few of the chains contain galactose. Since N-acetylgalactosamine is not a common constituent of mammalian-derived N-linked chains, we investigated the position and linkage of this residue in the schistosome-derived glycopeptides. Virtually all of the N-acetylgalactosamine was beta-linked and in a terminal position. The unusual features of the S. mansoni glycoprotein oligosaccharides support the possibility that they may be involved in the host immune response to infection.     

Intracellular movement of two mannose 6-phosphate receptors: return to the Golgi apparatus

We have used Chinese hamster ovary (CHO) cells and a murine lymphoma cell line to study the recycling of the 215-kD and the 46-kD mannose 6-phosphate receptors to various regions of the Golgi to determine the site where the receptors first encounter newly synthesized lysosomal enzymes. For assessing return to the trans-most Golgi compartments containing sialyltransferase (trans-cisternae and trans-Golgi network), the oligosaccharides of receptor molecules on the cell surface were labeled with [3H]galactose at 4 degrees C. Upon warming to 37 degrees C, the [3H]galactose residues on both receptors were substituted with sialic acid with a t1/2 approximately 3 hrs. Other glycoproteins acquired sialic acid at least 8-10 times slower. Return of the receptors to the trans-Golgi cisternae containing galactosyltransferase could not be detected. Return to the cis/middle Golgi cisternae containing alpha-mannosidase I was measured by adding deoxymannojirimycin, a mannosidase I inhibitor, during the initial posttranslational passage of [3H]mannose-labeled glycoproteins through the Golgi, thereby preserving oligosaccharides which would be substrates for alpha-mannosidase I. After removal of the inhibitor, return to the early Golgi with subsequent passage through the Golgi complex was measured by determining the conversion of the oligosaccharides from high mannose to complex-type units. This conversion was very slow for the receptors and other glycoproteins (t1/2 approximately 20 h). Exposure of the receptors and other glycoproteins to the dMM-sensitive alpha-mannosidase without movement through the Golgi apparatus was determined by measuring the loss of mannose residues from these proteins. This loss was also slow. These results indicate that both Man-6-P receptors routinely return to the Golgi compartment which contains sialyltransferase and recycle through other regions of the Golgi region less frequently. We infer that the trans-Golgi network is the major site for lysosomal enzyme sorting in CHO and murine lymphoma cells.     

Intact Golgi synthesize complex branched O-linked chains on glycoside primers: evidence for the functional continuity of seven glycosyltransferases and three sugar nucleotide transporters

We examined the functional co-localization and continuity of glycosyltransferases and sugar nucleotide transporters in the Golgi of two Chinese hamster ovary (CHO) cell lines that synthesize different types of O-linked oligosaccharides. CHO cells normally synthesize primarily Sia2,3Gal1,3GalNAc- on glycoproteins. CHO cells transfected with core-2 GlcNAc transferase (Core 2) can synthesize glycoproteins containing branched O-linked oligosaccharides with poly-N-acetyllactosamines. CHO lines incubated with [³H]galactose and GalNAc--phenyl (GAP) as a primer, synthesize labeled glycoside products that faithfully resemble those found on the endogenous acceptors: CHO cells make Sia2,3[³H]Gal1,3GAP, while CHO Core2 cells synthesize GAPs with complex branched chains including poly-N-acetyllactosamines. To determine if isolated Golgi preparations make similar products, we prepared Golgi by established homogenization methods, documented their intactness, and added tracer UDP-[³H]Gal, unlabeled sugar nucleotides, and GAP. CHO Golgi preparations synthesized only Sia2,3[³H]Gal1,3GAP. CHO Core2, also made this product and a small amount of Core-2 GlcNAc transferase-dependent products. No endogenous glycoproteins were labeled. However, when either cell line was gently permeabilized with streptolysin-O or given hypo-osmotic shock, both GAP and endogenous acceptors were efficiently glycosylated within an intact functional Golgi lumen and remained there. Significantly, Golgi from CHO Core2 cells made mostly branched GAP products including some with poly-N-acetyllactosamines as complex as those made and secreted by living cells incubated with GAP. These results suggest that the lumen of the Golgi apparatus is functionally continuous or interconnected. Once glycosides diffuse into the Golgi lumen, they have access to all the sugar nucleotide transporters and glycosyltransferases used for complex GAP-based products without requiring metabolic energy or inter-vesicular transport. Glycosylation of artificial acceptors could be used to track the functional continuity or co-localization of multiple glycosyltransferases and transporters under conditions where Golgi morphology disintegrates and/or reappears.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.     

An intrinsic membrane glycoprotein of the golgi apparatus with O-linked N-acetylglucosamine facing the cytosol

We have recently described the occurrence of integral membrane glycoproteins in rat liver smooth and rough endoplasmic reticulum with O-N-acetylglucosamine facing the cytosolic and luminal sides of the membrane (Abeijon, C., and Hirschberg, C. B. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1010-1014). We now report that integral membrane glycoproteins with cytosolic facing O-N-acetylglucosamine also occur in membranes of rat liver Golgi apparatus. This was determined following incubation of vesicles from the Golgi apparatus, which were sealed and of the same membrane topographical orientation as in vivo, with UDP-[14C]galactose and saturating amounts of bovine milk galactosyltransferase. This enzyme does not enter the lumen of the vesicles and specifically catalyzes the addition of galactose, in a beta 1-4 linkage, to terminal N-acetylglucosamine. Under these conditions, galactose was transferred to a glycoprotein of molecular mass of 92 kDa. This protein was insoluble in sodium carbonate, pH 11.5, conditions under which integral membrane proteins remain membrane bound and was insensitive to treatment with peptide:N-glycosidase F. beta Elimination and chromatography showed that radiolabeled galactose was part of a disaccharide which was characterized as Gal beta 1-4GlcNAcitol. This glycoprotein is specific of the Golgi apparatus membrane. Intrinsic membrane glycoproteins with this unusual carbohydrate membrane orientation thus occur in the endoplasmic reticulum and Golgi apparatus of rat liver.     

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.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

Intracellular transport of membrane glycoproteins: two closely related histocompatibility antigens differ in their rates of transit to the cell surface

The intracellular transport of two closely related membrane glycoproteins was studied in the murine B cell lymphoma line, AKTB-1b. Using pulse-chase radiolabeling, the kinetics of appearance of the class I histocompatibility antigens, H-2Kk and H-2Dk, at the cell surface were compared and found to be remarkably different. Newly synthesized H-2Kk is transported rapidly such that all radiolabeled molecules reach the surface within 1 h. In contrast, the H-2Dk antigen is transported slowly with a half-time of 4-5 h. The rates of surface appearance for the two antigens closely resemble the rates at which their Asn-linked oligosaccharides mature from endoglucosaminidase H (endo H)-sensitive to endo H-resistant forms, a process that occurs in the Golgi apparatus. This suggests that the rate-limiting step in the transport of H-2Dk to the cell surface occurs before the formation of endo H-resistant oligosaccharides in the Golgi apparatus. Subcellular fractionation experiments confirmed this conclusion by identifying the endoplasmic reticulum (ER) as the site where the H-2Dk antigen accumulates. The retention of this glycoprotein in the ER does not appear to be due to a lack of solubility or an inability of the H-2Dk heavy chain to associate with beta 2-microglobulin. Our data is inconsistent with a passive membrane flow mechanism for the intracellular transport of membrane glycoproteins. Rather, it suggests that one or more receptors localized to the ER membrane may mediate the selective transport of membrane glycoproteins out of the ER to the Golgi apparatus. The fact that H-2Kk and H-2Dk are highly homologous (greater than or equal to 80%) indicates that this process can be strongly influenced by limited alterations in protein structure.     

Glycolipid and glycoprotein transport through the Golgi complex are similar biochemically and kinetically. Reconstitution of glycolipid transport in a cell free system

Glycolipid transport between compartments of the Golgi apparatus has been reconstituted in a cell free system. Transport of lactosylceramide (galactose beta 1-4-glucose-ceramide) was followed from a donor to an acceptor Golgi population. The major glycolipid in CHO cells is GM3 (sialic acid alpha 2-3 galactose beta 1-4-glucose-ceramide). Donor membranes were derived from a Chinese hamster ovary (CHO) cell mutant (Lec2) deficient in the Golgi CMP-sialic acid transporter, and therefore contained lactosylceramide as the predominant glycolipid. Acceptor Golgi apparatus was prepared from another mutant, Lec8, which is defective in UDP-Gal transport. Thus, glucosylceramide is the major glycolipid in Lec8 cells. Transport was measured by the incorporation of labeled sialic acid into lactosylceramide (present originally in the donor) by transport to acceptor membranes, forming GM3. This incorporation was dependent on ATP, cytosolic components, intact membranes, and elevated temperature. Donor membranes were prepared from Lec2 cells infected with vesicular stomatitus virus (VSV). These membranes therefore contain the VSV membrane glycoprotein, G protein. Donor membranes derived from VSV-infected cells could then be used to monitor both glycolipid and glycoprotein transport. Transport of these two types of molecules between Golgi compartments was compared biochemically and kinetically. Glycolipid transport required the N- ethylmaleimide sensitive factor previously shown to act in glycoprotein transport (Glick, B. S., and J. E. Rothman. 1987. Nature [Lond.]. 326:309-312; Rothman, J. E. 1987. J. Biol. Chem. 262:12502-12510). GTP gamma S inhibited glycolipid and glycoprotein transport similarly. The kinetics of transport of glycolipid and glycoprotein were also compared. The kinetics of transport to the end of the pathway were similar, as were the kinetics of movement into a defined transport intermediate. It is concluded that glycolipid and glycoprotein transport through the Golgi occur by similar if not identical mechanisms.     

Schistosoma mansoni synthesizes glycoproteins containing terminal O-linked N-acetylglucosamine residues

In this report, we describe our studies on the structures of the O-linked oligosaccharides in glycoproteins synthesized by the human blood fluke Schistosoma mansoni. Adult male schistosomes were incubated with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel newly synthesized glycoproteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were labeled by each of the radioactive precursors. Glycopeptides were prepared from radiolabeled glycoproteins by pronase treatment and fractionated on columns of concanavalin A-Sepharose and pea lectin-agarose. The O-linked oligosaccharides were released from glycopeptides by treatment with mild base/borohydride. All O-linked material was found in glycopeptides not bound by either of the immobilized lectins. The structures of the released chains were then analyzed by a variety of techniques. Our results demonstrate that the schistosomes synthesize glycoproteins containing two major types of simple O-linked sugar chains. One type, which represents a minor fraction of the O-linked oligosaccharides, contains N-acetylgalactosamine linked to peptide. These O-linked chains occur as terminal O-linked N-acetylgalactosamine and the O-linked disaccharide, galactose----N-acetylgalactosamine. Sialic acid was not present in either of these O-linked chains or in any other glycopeptides derived from adult male schistosomes. However, the major type of O-linked chain in glycoproteins synthesized by adult schistosomes is an unusual terminal O-linked N-acetylglucosamine linked to peptide. This latter structure represents approximately 10% of the total radioactive N-acetylglucosamine recovered in all glycopeptides. Our results also suggest the possibility that the O-linked oligosaccharides are highly clustered on the glycopeptides.     

Recycling cell surface glycoproteins undergo limited oligosaccharide reprocessing in LEC1 mutant Chinese hamster ovary cells

The ability of particular cell surface glycoproteins to recycle and become exposed to individual Golgi enzymes has been demonstrated. This study was designed to determine whether endocytic trafficking includes significant reentry into the overall oligosaccharide processing pathway. The Lec1 mutant of Chinese hamster ovary (CHO) cells lack N - acetylglucosaminyltransferase I (GlcNAc-TI) activity resulting in surface expression of incompletely processed Man5GlcNAc2 N -linked oligosaccharides. An oligosaccharide tracer was created by exoglycosylation of cell surface glycoproteins with purified porcine GlcNAc-TI and UDP-[3H]GlcNAc. Upon reculturing, all cell surface glycoproteins that acquired [3H]GlcNAc were acted upon by intracellular mannosidase II, the next enzyme in the Golgi processing pathway of complex N -linked oligosaccharides (t1/2= 3-4 h). That all radiolabeled cell surface glycoproteins were included in this endocytic pathway indicates a common intracellular compartment into which endocytosed cell surface glycoproteins return. Significantly, no evidence was found for continued oligosaccharide processing consistent with transit through the latter cisternae of the Golgi apparatus. These data indicate that, although recycling plasma membrane glycoproteins can be reexposed to individual Golgi-derived enzymes, significant reentry into the overall contiguous processing pathway is not evident.      

Biosynthesis of the glycoproteins present in plasma membrane, lysosomes and secretory materials, as visualized by radioautography

Synopsis The three major types of glycoproteins present in animal cells, that is, the secretory, lysosomal and plasma membrane glycoproteins, were examined with regard to the sites of synthesis of their carbohydrate side chains and to their subsequent migration within cells.The site at which a monosaccharide is added to a growing glycoprotein depends on the position of that monosaccharide in the carbohydrate side-chain. Thus, radiauutography of thyroid cells within minutes of the intravenous injection of labelled mannose, a sugar located near the base of the larger side-chains, reveals that it is incorporated in rough endoplasmic reticulum, whereas the more distally located galactose and fucose are incorporated in the Golgi apparatus. Recently [³H]N-acetylmannosamine, a specific precursor for the terminally located sialic acid residues, was shown to be also added in the Golgi apparatus. Presumably synthesis of glycoproteins is completed in this organelle.Radioautographs of animals sacrificed a few hours after injection of [³H]N-acetylmannosamine show that, in many secretory cells, labelled glycoproteins pass into secretory products. In these cells, as well as in non-secretory cells, the label may also appear within lysosomes and at the cell surface. In the latter site, it is presumably included within the plasma membrane glycoproteins whose carbohydrate side-chains form the cell coat. The continual migration of glycoproteins from Golgi apparatus to cell surface implies turnover of plasma membrane glycoproteins. Radioautographic quantitation of [³H]fucose label at the surface of proximal tubule cells in the kidney of singly-injected adult mice have shown that, after an initial peak, cell surface labelling decreases at a rate indicating a half-life of plasma membrane glycoproteins of about three days.     

Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.     

Evidence that all newly synthesized proteins destined for fast axonal transport pass through the Golgi apparatus

Effects of the sodium ionophore, monensin, were examined on the passage from neuronal cell body to axon of materials undergoing fast intracellular transport. In vitro exposure of bullfrog dorsal root ganglia to concentrations of drug less than 1.0 micron led to a dose-dependent depression in the amount of fast-transported [3H]leucine- or [3H]glycerol-labeled material appearing in the nerve trunk. Incorporation of either precursor was unaffected. Exposure of a desheathed nerve trunk to similar concentrations of monensin, while ganglia were incubated in drug-free medium, had no effect on transport. With [3H]fucose as precursor, fast transport of labeled glycoproteins was depressed to the same extent as with [3H]leucine; synthesis, again, was unaffected. By contrast, with [3H]galactose as precursor, an apparent reduction in transport of labeled glycoproteins was accounted for by a marked depression in incorporation. The inference from these findings, that monensin acts to block fast transport at the level of the Golgi apparatus, was supported by ultrastructural examination of the drug-treated neurons. An extensive and selective disruption of Golgi saccules was observed, accompanied by an accumulation of clumped smooth membranous cisternae. Quantitative analyses of 48 individual fast-transported protein species, after separation by two-dimensional gel electrophoresis, revealed that monensin depresses all proteins to a similar extent. These results indicate that passage through the Golgi apparatus is an obligatory step in the intracellular routing of materials destined for fast axonal transport.     

Schistosoma mansoni synthesizes novel biantennary Asn-linked oligosaccharides containing terminal beta-linked N-acetylgalactosamine.

This report describes the structure of novel complex-type Asn-linked oligosaccharides in glycoproteins synthesized by the human blood fluke, Schistosoma mansoni. Adult schistosome worm pairs (male and female) isolated from infected hamsters were metabolically radiolabelled with either [3H]glucosamine, [3H]mannose or [3H]galactose. The glycopeptides prepared by pronase digestion of the total glycoprotein fraction were isolated by affinity chromatography on columns of immobilized Concanavalin A (Con A) and Wisteria floribunda agglutinin (WFA). A subset of glycopeptides, designated IIb, that bound to both Con A and WFA was isolated. WFA has been shown to have affinity for oligosaccharides containing beta 1,4-linked N-acetylgalactosamine (GalNAc) at their non-reducing termini. Compositional analysis of IIb glycopeptides demonstrated that they contained N-acetylglucosamine (GlcNAc), GalNAc, mannose (Man) and fucose (Fuc), but no galactose (Gal) or N-acetylneuraminic acid (NeuAc). Methylation analyses and exoglycosidase digestions indicated that IIb glycopeptides were complex-type biantennary structures with branches containing the sequence GalNAc beta 1-4-[+/- Fuc alpha 1-3]GlcNAc beta 1-2Man alpha 1-R. The discovery of these unusual oligosaccharides synthesized by a human parasite, which appear to be similar to some newly discovered mammalian cell-derived oligosaccharides, may shed light on future studies related to the role oligosaccharides may play in host-parasite interactions.     

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.     

In vitro synthesis and secretion of glycoprotein by human mammary tissue

Summary Organ cultures of human surgical specimens can be used to investigate glycoprotein production in vitro under conditions in which three-dimensional tissue structures and cell-cell interactions resemble those present in vivo. In this report, an organ-culture system is used to investigate the synthesis, transport and release of glycoprotein by normal and benign hyperplastic human mammary epithelium. Autoradiography of explants pulse-labeled with individual glycoprotein precursors ([³H]glucosamine, [³H]fucose, [³H]acetylmanosamine) and maintained in organ culture for intervals up to 72hr revealed that glycoprotein is synthesized and then secreted by mammary epithelium. Incorporation of each isotope took place in the Golgi apparatus. Most of the newly synthesized glycoprotein, labeled with each of the three precursors, then was transported to apical cell surfaces and secreted into gland lumina. Observations were indistinguishable in normal and benign hyperplastic glands. Thus nonlactating human mammary epithelium exhibits a glycoprotein secretory activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [³H]glucosamine-labeled macromolecules released into the medium showed a group of glycoproteins with a molecular weight of 48,000±6,000 daltons plus high-molecular-weight glycosylated components at the top of gels. The nature of gp48 is not known, but similar molecular-weight glycoproteins also are released by surgical specimens of human mammary cancer maintained in organ culture. Z. A. T. received support from NCI Grant No. CA-14089.