Biosynthetic processing of the oligosaccharide chains of cellular fibronectin

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-³H]mannose or [³⁵S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-β-N-acetylglucosaminidase H, which specifically cleaves the ‘high-mannose’ class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing.This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a ‘high-mannose’ oligosaccharide precursor and a ‘complex’ carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (M_r 2100) comparable to (Man)₉GlcNAc (M_r 2080), and was sensitive to endo-β-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistent to endo-β-N-acetylglucosaminadase H cleavage. The final ‘complex’ or processed oligosaccharide structure contained approximately two-thirds less associated with the mature glycoprotein. They also indicate that the ‘complex’ structure is synthesized as a ‘high-mannose’ intermediate which is processed by the removal of mannose.     

A systematic study of site-specific GalNAc-type O-glycosylation modulating proprotein convertase processing

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.     

Mannose 6-phosphate-independent membrane association of cathepsin D, glucocerebrosidase, and sphingolipid-activating protein in HepG2 cells.

The membrane association of the lysosomal enzymes cathepsin D and glucocerebrosidase and its naturally occurring sphingolipid activating protein was studied in HepG2 cells. We differentially permeabilized cells with low concentrations of saponin, at which secretory proteins rinsed out completely, whereas integral membrane proteins were not released. All relevant intracellular compartments were shown to be permeabilized by saponin. Metabolic labeling showed that early precursors of cathepsin D, sphingolipid activating protein, and glucocerebrosidase were completely released from the cells, whereas more than 80% of the high molecular mass intermediates were retained by the cells. Treatment of permeabilized cells with 10 mM mannose 6-phosphate released only 50% of the cell-associated cathepsin D. Glucocerebrosidase remained membrane-associated, but cathepsin D and sphingolipid activating protein were released from the cells after proteolytic processing. Sphingolipid activating proteins and cathepsin D behaved similarly during biosynthesis and showed similar sensitivity to mannose 6-phosphate. The membrane association of the intermediate form of cathepsin D was independent of the presence of N-linked oligosaccharides. Subcellular fractionation on sucrose gradients showed that the lysosomal proteins became membrane-associated probably in the Golgi complex, and that both mannose 6-phosphate-dependent and mannose 6-phosphate-independent membrane association occur in the same compartments. We conclude that, in HepG2 cells, cathepsin D, sphingolipid activating protein, and glucocerebrosidase exhibit MPR-independent membrane association which is acquired in the same compartments beyond the rough endoplasmic reticulum.     

Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.     

Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro

Abstract: With [³H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.     

D-mannose transport and metabolism in isolated enterocytes

D-mannose transport and metabolism has been studied in enterocytes isolated from chicken small intestine. In the presence of Na(+), the mannose taken up by the cells either remains free, is phosphorylated, is catabolized to H(2)O, or becomes part of membrane components. The mannose remaining free in the cytosol is released when the cells are transferred to an ice bath. The Na(+)-dependent D-mannose transport is electrogenic and inhibited by ouabain and dinitrophenol; its substrate specificity differs from SGLT-1 transporter. The Glut2 transporter inhibitors phloretin and cytochalasin B added following 30-min mannose uptake reduced the previously accumulated D-mannose, whereas these two agents increased the cell to external medium 3-O-methyl-glucose (3-OMG) concentration ratio. D-mannose efflux rate from preloaded D-[2-(3)H]-mannose enterocytes is Na(+)-independent. Phloretin did not affect D-mannose efflux rate, whereas it inhibited that of 3-OMG. Neither mannose uptake nor efflux rate were affected by fructose. It is concluded that part of the mannose taken up by the enterocytes is rapidly metabolized and that enterocytes have two D- mannose transport systems: one is concentrative and Na(+)-dependent and the other is Na(+)-independent and passive.     

Eukaryotic Oligosaccharyltransferase Generates Free Oligosaccharides during N-Glycosylation

Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.     

Studies on the glycosylation of wild-type and mutant forms of Aspergillus niger pectin methylesterase

Pectin methylesterase (PME) is one of a number of enzymes released by the fungus Aspergillus niger that are involved in the degradation of specific plant cell-wall structures. PME is a glycoprotein with three potential sites for N-linked glycosylation. The glycosylation may affect the hydrolytic activity or the substrate specificity of PME. In this work, we investigate first the structures and the attachment sites of the glycans present on recombinant wild-type PME. Further, a series of PME mutants was created in which the three potential N-linked glycosylation sites were eliminated in all possible combinations. The glycosylation of the mutants and their activities were then studied. Mass spectrometric techniques tailored for carbohydrate analysis were applied to both characterize the glycan structures and to determine the specific sites of attachment. High mannose structures with variable numbers of mannose were found on the wild-type, as well as the mutant forms. Studies using the mutants suggest that glycosylation does not strongly influence the activity. Whether it may affect the substrate specify of the enzyme is unknown, and that aspect will be explored in future work.     

Molecular Characterization of Arylsulfatase G: EXPRESSION,PROCESSING, GLYCOSYLATION,TRANSPORT, AND ACTIVITY*

Arylsulfatase G (ARSG) is a recently identified lysosomal sulfatase that was shown to be responsible for the degradation of 3-O-sulfated N-sulfoglucosamine residues of heparan sulfate glycosaminoglycans. Deficiency of ARSG leads to a new type of mucopolysaccharidosis, as described in a mouse model. Here, we provide a detailed molecular characterization of the endogenous murine enzyme. ARSG is expressed and proteolytically processed in a tissue-specific manner. The 63-kDa single-chain precursor protein localizes to pre-lysosomal compartments and tightly associates with organelle membranes, most likely the endoplasmic reticulum. In contrast, proteolytically processed ARSG fragments of 34-, 18-, and 10-kDa were found in lysosomal fractions and lost their membrane association. The processing sites and a disulfide bridge between the 18- and 10-kDa chains could be roughly mapped. Proteases participating in the processing were identified as cathepsins B and L. Proteolytic processing is dispensable for hydrolytic sulfatase activity in vitro. Lysosomal transport of ARSG in the liver is independent of mannose 6-phosphate, sortilin, and Limp2. However, mutation of glycosylation site N-497 abrogates transport of ARSG to lysosomes in human fibrosarcoma cells, due to impaired mannose 6-phosphate modification.     

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts.  Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies.       

Effects of Mannose on Pathogenesis of Acanthamoeba castellanii

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.     

A novel cell penetrating aspartic protease inhibitor blocks processing and presentation of tetanus toxoid more efficiently than pepstatin A

Selective inhibition of enzymes involved in antigen processing such as cathepsin E and cathepsin D is a valuable tool for investigating the roles of these enzymes in the processing pathway. However, the aspartic protease inhibitors, including the highly potent pepstatin A (PepA), are inefficiently transported across the cell membrane and thus have limited access to antigen processing compartments. Previously described mannose-pepstatin conjugates were efficiently taken up by the cells via receptor mediated uptake. However, cells without mannose receptors are unable to take up these conjugates efficiently. The aim of the present study was to synthesize new cell-permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell penetrating peptides (CPPs). To achieve this, the most commonly used CPPs namely pAntp_(43-58) (penetratin), Tat_(49-60), and 9-mer of l-arginine (R9), were synthesized and coupled to pepstatin. The enzyme inhibitory properties of these bioconjugates and their cellular uptake into MCF7 (human breast cancer cell line), Boleths (EBV-transformed B-cell line) and dendritic cells (DC) were the focus of our study. We found that the bioconjugate PepA-penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor tested, and was more efficient than unconjugated PepA. Additionally, we found that PepA-P efficiently inhibited the tetanus toxoid C-fragment processing in peripheral blood mononuclear cells (PBMC), primary DC and in primary B cells. Therefore, PepA-P can be used in studying the role of intracellular aspartic proteases in the MHC class II antigen processing pathway. Moreover, inhibition of tetanus toxoid C-fragment processing by PepA-P clearly implicates the role of aspartic proteinases in antigen processing.     

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.     

Phosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deficient fibroblasts

Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos.     

Immunomodulatory effects of IL-12 released from poly-N-acetyl glucosamine gel matrix during schistosomiasis infection

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35 days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.     

N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.     

Paracrine and Transpresentation Functions of IL-15 Are Mediated by Diverse Splice Versions of IL-15Rα in Human Monocytes and Dendritic Cells

Species-specific differences of post-translational modifications suggested the existence of human IL-15Rα isoforms. We identified eight new isoforms that are predicted to modify the intracellular C termini of IL-15Rα, and another N-terminal exon “Ex2A” that was consistently present in all but one of the C-terminal isoforms. Ex2A encodes a 49-amino acid domain that allowed the transfer of IL-15/IL-15Rα complex to the cell surface but prevented its cleavage from cell membranes and its secretion thus facilitating the transpresentation of IL-15 as part of the immunological synapse. The Ex2A domain also affected the O-glycosylation of IL-15Rα that explained the species-specific differences. The Ex2A domain appeared to be removed from major IL-15Rα species during protein maturation, but both Ex2A and IL-15Rα appeared on the surface of monocytic cells upon activation. The membrane-associated form of the only C-terminal isoform that lacked Ex2A (IC3) was retained inside the cell, but soluble IL-15/IL-15Rα complexes were readily released from cells that expressed IL-15/IL-15Rα-IC3 thus limiting this IL-15/IL-15Rα isoform to act as a secreted molecule. These data suggest that splice versions of IL-15Rα determine the range of IL-15 activities.     

The soluble form of rat liver alpha-mannosidase is immunologically related to the endoplasmic reticulum membrane alpha-mannosidase

The soluble alpha-mannosidase of rat liver, originally described as a cytoplasmic alpha-mannosidase, has been purified to homogeneity by conventional techniques. The purified enzyme has an apparent molecular weight of 350,000 and is composed of 107-kDa subunits. The soluble alpha-mannosidase has the same enzymatic properties as the endoplasmic reticulum (ER) membrane alpha-mannosidase of rat liver (Bischoff, J., and Kornfeld, R. (1983) J. Biol. Chem. 258, 7909-7910) which is believed to play a role in oligosaccharide processing in the rough ER. Like the membrane-bound ER alpha-mannosidase, the soluble alpha-mannosidase can hydrolyze alpha-linked mannose from both p-nitrophenyl alpha-mannoside (Km = 0.14 mM) and high mannose oligosaccharides, is not inhibited by the mannose analogues swainsonine and 1-deoxymannojirimycin, is stabilized by MnCl2 or CoCl2, and does not bind to concanavalin A-Sepharose. A goat polyclonal antibody raised against the purified soluble alpha-mannosidase specifically recognizes the rat liver membrane-bound ER alpha-mannosidase, leading us to propose that they are two forms of the same enzyme and that the soluble form is derived from the ER membrane alpha-mannosidase by proteolysis. The antibody also cross-reacts with both the soluble and membrane-bound forms of ER alpha-mannosidase activity in cultured Chinese hamster ovary cells and rat H35 hepatoma cells. Since the ER alpha-mannosidase is presumed to be involved in the early steps of oligosaccharide processing, the action of the purified soluble form of the enzyme on high mannose oligosaccharides was examined. Surprisingly, the enzyme released free mannose from oligosaccharides ranging in size from Glc1Man9GlcNAc to Man5GlcNAc with almost equal efficiency. However, a long term incubation of the enzyme with Man9GlcNAc led to the accumulation of Man7GlcNAc and produced only small amounts of Man6GlcNAc and Man5GlcNAc. Structural analysis of these reaction products indicated that the purified soluble form of ER alpha-mannosidase shows little specificity for which mannose residues it removes from Man9GlcNAc. In contrast, as shown in the accompanying paper, the intracellular action of ER alpha-mannosidase on glycoprotein-bound Man9GlcNAc2 is highly specific.     

Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains

The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor. In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant [rho degrees ] with EDTA and two non-ionic surfactants (Hecameg and HTAC). The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives. However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells. The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity. The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the [rho degrees ] mutant. Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE. Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase. Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively. The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.     

Shunting of insulin from a retroendocytotic pathway to a degradative pathway by sodium vanadate

Adipocytes route internalized insulin through two major pathways, a degradative pathway and a retroendocytotic pathway. To examine whether sorting of incoming insulin-receptor complexes can be altered, we assessed the effect of vanadate on the intracellular processing of both insulin and insulin receptors. After cells were pretreated with vanadate (1 mM for 30 min at 37 degrees C), 125I-insulin was loaded into the cell interior. When the net efflux of insulin from cells into the medium was then monitored, vanadate was found to slow the efflux of insulin from a t1/2 of 6.2 min (controls) to 11 min. Since efflux reflects both the rapid extrusion of intact insulin and the slower release of degradative products, we proposed that vanadate diverts more insulin into the degradative pathway. Further evidence in support of this idea included the following: 1) when intracellular degradation of insulin was impaired by chloroquine, undegraded insulin accumulated faster within vanadate-treated cells, consistent with greater flux through a degradative pathway; 2) vanadate increased the percentage of degraded insulin released from cells from 61 and 72%; and 3) under steady-state binding conditions, more insulin resided in the cell interior of vanadate-treated cells (44.8% versus 34.5%), and the time required for the intracellular pool to reach equilibrium was prolonged (t1/2 of 5.5 min versus 4.0). Neither insulin internalization nor degradation was impaired by vanadate alone. In related studies Tris was found to inhibit insulin-mediated receptor recycling by only 10%, whereas in the presence of vanadate (plus Tris) almost all incoming insulin receptors were prevented from recycling. Vanadate alone had no effect on the ability of insulin receptors to recycle. Based on these results we conclude that: 1) vanadate shunts incoming insulin from a more rapid retroendocytotic pathway to a slower degradative pathway and diverts insulin receptors from a Tris-insensitive recycling pathway to one that can be completely inhibited by Tris; 2) these effects are selective, in that vanadate impairs neither insulin degradation nor receptor uptake and recycling. Considered together, these findings support the idea that a sorting mechanism exists for the intracellular routing of incoming insulin-receptor complexes.