Christopher M. West

Current ideas on the significance of protein glycosylation

Carbohydrate has been removed from a number of glycoproteins without major effect on the structure or enzyme activity of the protein. Thus carbohydrate has been suggested to underly a non-primary function for proteins, such as in relatively non-specific interactions with other carbohydrates or macromolecules, stabilization of protein conformation, or protection from proteolysis. This non-specific concept is consistent with both the general similarity in carbohydrate structure on very diverse glycoproteins and the frequent structural microheterogeneity of carbohydrate chains at given sites. The concept is supported in a general sense by the viability of cells whose glycosylation processes have been globally disrupted by mutation or pharmacological inhibitors.In contrast to the above observations, other studies have revealed the existence of specific, selective receptors for discrete oligosaccharide structures on glycoproteins which seem to be important for compartmentalization of the glycoprotein, or the positioning of cells on which the glycoprotein is concentrated. Sometimes multivalency in the carbohydrate-receptor interaction is crucial. There are additional possible roles for carbohydrate in the transduction of information upon binding to a receptor. The possibility of specific roles for carbohydrate is supported by the existence of numerous unique carbohydrate structures, many of which have been detected as glycoantigens by monoclonal antibodies, with unique distributions in developing and differentiated cells.This article attempts to summarize and rationalize the contradictory results. It appears that in general carbohydrate does in fact underlie only roles secondary to a proteins primary function. These secondary roles are simple non-specific ones of protection and stabilization, but often also satisfy the more sophisticated needs of spatial position control and compartmentalization in multicellular eukaryotic organisms. It is suggested that there are advantages, evolutionarily speaking, for the shared use of carbohydrate for non-specific roles and for specific roles primarily as luxury functions to be executed during the processes of cell differentiation and morphogenesis.

The cytoplasmic F-box binding protein SKP1 contains a novel pentasaccharide linked to hydroxyproline in Dictyostelium.

SKP1 is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an oligosaccharide containing Fuc and Gal, which is unusual for a cytoplasmic or nuclear protein. To establish how it is glycosylated, SKP1 labeled with [3H]Fuc was purified to homogeneity and digested with endo-Lys-C. A single radioactive peptide was found after two-dimensional high performance liquid chromatography. Analysis in a quadrupole time-of-flight mass spectrometer revealed a predominant ion with a novel mass. Tandem mass spectrometry analysis yielded a set of daughter ions which identified the peptide and showed that it was modified at Pro-143. A second series of daughter ions showed that Pro-143 was hydroxylated and derivatized with a potentially linear pentasaccharide, Hex→Hex→Fuc→Hex→HexNAc→(HyPro). The attachment site was confirmed by Edman degradation. Gas chromatography-mass spectrometry analysis of trimethylsilyl-derivatives of overexpressed SKP1 after methanolysis showed the HexNAc to be GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1 and from a fucosylation mutant, followed by matrix-assisted laser desorption time-of-flight mass spectrometry analysis, showed that the sugar chain consisted ofd-Galpα1→6-d-Galpα1→l-Fucpα1→2-d-Galpβ1→3GlcNAc. Matrix-assisted laser-desorption time-of-flight mass spectrometry analysis of all SKP1 peptides resolved by reversed phase-high performance liquid chromatography showed that SKP1 was only partially hydroxylated at Pro-143 and that all hydroxylated SKP1 was completely glycosylated. Thus SKP1 is variably modified by an unusual linear pentasaccharide, suggesting the localization of a novel glycosylation pathway in the cytoplasm.

Glutamine supports recovery from loss of transepithelial resistance and increase of permeability induced by media change in Caco-2 cells1

Recent evidence suggests that the conditionally essential amino acid glutamine is important for intestinal barrier function. However, the mechanism remains undefined. To determine the effects of glutamine on permeability of intestinal epithelial cell monolayers, Caco-2 cells were grown on membrane filters and exposed to 4 mmol/L sodium butyrate in order to rapidly achieve high levels of alkaline phosphatase and high transepithelial resistance as seen in functionally mature enterocytes. A standard method of medium exchange consisting of removal and replacement resulted in a catastrophic loss of transepithelial resistance and increase of mannitol and dextran fluxes that required 2-4 hrs and protein synthesis to recover. The effect was attributed to exposure of the upper monolayer surface to atmosphere and could be avoided by refeeding by incremental perfusion. Spontaneously-differentiated Caco-2 monolayers were resistant to this stress. This novel stress test was employed as a sensitive assay for the requirement of glutamine for monolayer transepithelial resistance and mannitol permeability. Pre-stress glutamine availability was more important than Gln-availability during the recovery phase. Thus the transepithelial resistance and permeability of butyrate-induced monolayers is dynamically-regulated in response to atmospheric exposure, by a mechanism that depends on threshold levels of glutamine availability.

Comparative analysis of spore coat Formation, structure, and function in Dictyostelium

Dictyostelium produces spores at the end of its developmental cycle to propagate the lineage. The spore coat is an essential feature of spore biology contributing a semipermeable chemical and physical barrier to protect the enclosed amoeba. The coat is assembled from secreted proteins and a polysaccharide, and from cellulose produced at the cell surface. They are organized into a polarized molecular sandwich with proteins forming layers surrounding the microfibrillar cellulose core. Genetic and biochemical studies are beginning to provide insight into how the deliveries of protein and cellulose to the cell surface are coordinated and how cysteine-rich domains of the proteins interact to form the layers. A multidomain inner layer protein, SP85/PsB, seems to have a central role in regulating coat assembly and contributing to a core structural module that bridges proteins to cellulose. Coat formation and structure have many parallels in walls from plant, algal, yeast, protist, and animal cells.

The spore coat of a fucosylation mutant in Dictyostelium discoideum

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.

Formation and organization of the spore coat ofDictyostelium discoideum

Abstract Macromolecular components of the spore coat of Dictyostelium discoideum have been localized by gold-labeled affinity cytochemistry. The outer electron-dense layer is the residence of three prominent glycoproteins that express a fucose-dependent epitope, whereas the inner electron-dense layer includes SP85 and the galactose/ N -acetylgalactosamine-containing polysaccharide (GPS). The cellulosic layers are interposed between them. The outer-layer glycoproteins and the GPS also can be found in the interspore fluid, which is usually lost during collection of the spores. Assembly of the spore coat, examined over time, showed that all components, except for the cellulose, are found in an internal secretory vesicle population. All components are found in each vesicle but are not uniformly intermixed within them. Cellulose does not appear until after the outer electron-dense layer of the spore coat has been organized following secretion. The GPS is excluded from the outer dense layer and largely from the cellulosic layer, being more concentrated in the inner layer. SP85 remains localized in the inner dense layer near the cell surface with a circumferentially focal distribution. The distinct distributions of these macromolecular species in the mature spore coat are foreshadowed by their mosaic distribution in the prespore vesicles from which they originate.

Identification of a UDP-GlcNAc:Skp1-Hydroxyproline GlcNAc-transferase in the Cytoplasm of Dictyostelium

Skp1 is a cytoplasmic and nuclear protein required for the ubiquitination of cell cycle regulatory proteins and transcriptional factors. In Dictyostelium, Skp1 is modified by a linear pentasaccharide, Galα1–6Galα1-Fucα1–2Galβ1–3GlcNAc, attached to a hydroxyproline (HyPro) residue at position 143. To study the formation of the GlcNAc-HyPro linkage, an assay was developed for the transfer of [3H]GlcNAc from UDP-[3H]GlcNAc to Skp1-HyPro-143 or a synthetic Skp1 4-HyPro peptide. The cytosolic but not the particulate fraction of the cell mediated transfer in a time-, concentration-, and HyPro-dependent fashion. Incorporated radioactivity was alkali-resistant and was recovered as GlcNH2 after acid hydrolysis, consistent with linkage of GlcNAc to HyPro. The GlcNAc-transferase activity was purified 130,000-fold as a single component with a recovery of 5%. Key to the purification was the synthesis of a novel affinity resin linking UDP-GlcNAc at its 5-uridyl position. The purified activity had an apparent M r of ∼45,000 by gel filtration, required dithiothreitol and a divalent cation, and consisted predominantly of a M r 51,000 band after SDS-polyacrylamide gel electrophoresis that was photoaffinity labeled with 5-125I-[3-(p-azidosalicylamido)-1-propenyl-UDP-GlcNAc in a UDP-GlcNAc-sensitive fashion. Its apparent K m values for UDP-GlcNAc and Skp1 were submicromolar. The presence of the enzyme in the cytosolic fraction, its dependence on a reducing environment, and its high affinity for UDP-GlcNAc strongly suggest that Skp1 is glycosylated by a HyPro GlcNAc-transferase that resides in the cytoplasm.

Purification and Characterization of an 1,2-L-Fucosyltransferase, Which Modifies the Cytosolic Protein FP21, from the Cytosol of Dictyostelium

A novel fucosyltransferase (cFTase) activity has been enriched over 106-fold from the cytosolic compartment of Dictyostelium based on transfer of [3H]fucose from GDP-[3H]fucose to Galβ1,3GlcNAcβ-paranitrophenyl (paranitrophenyl-lacto-N-bioside or pNP-LNB). The activity behaved as a single component during purification over DEAE-, phenyl-, Reactive Blue-4-, GDP-adipate-, GDP-hexanolamine-, and Superdex gel filtration resins. The purified activity possessed an apparent M of 95 × 103, was Mg-dependent with a neutral pH optimum, and exhibited a K for GDP-fucose of 0.34 μM, a K for pNP-LNB of 0.6 mM, and a V for pNP-LNB of 620 nmol/min/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the Superdex elution profile identified a polypeptide with an apparent M of 85 × 103, which coeluted with the cFTase activity and could be specifically photolabeled with the donor substrate inhibitor GDP-hexanolaminyl-azido-I-salicylate. Based on substate analogue studies, exoglycosidase digestions, and co-chromatography with fucosylated standards, the product of the reaction with pNP-LNB was Fucα1, 2Galβ1,3GlcNAcβ-pNP. The cFTase preferred substrates with a Galβ1,3 linkage, and thus its acceptor substrate specificity resembles the human Secretor-type α1,2-FTase. Afucosyl isoforms of the FP21 glycoprotein, GP21-I and GP21-II, were purified from the cytosol of a Dictyostelium mutant and found to be substrates for the cFTase, which exhibited an apparent K of 0.21 μM and an apparent V of 460 nmol/min/mg protein toward GP21-II. The highly purified cFTase was inhibited by the reaction products Fucα1,2Galβ1,3GlcNAcβ-pNP and FP21-II. FP21-I and recombinant FP21 were not inhibitory, suggesting that acceptor substrate specificity is based primarily on carbohydrate recognition. A cytosolic location for this step of FP21 glycosylation is implied by the isolation of the cFTase from the cytosolic fraction, its high affinity for its substrates, and its failure to be detected in crude membrane preparations.

Immunochemical, genetic and morphological comparison of fucosylation mutants of Dictyostelium discoideum

Mutations in three loci in Dictyostelium discoideum which affect fucosylation are described. Mutations in two of these loci resulted in the simultaneous loss of two separate carbohydrate epitopes. The GA-X epitope, which was competed by L-fucose, was absent in strains carrying a modC354, modD352 or modE353 mutation. These strains exposed a new carbohydrate epitope, competed by N-acetylglucosamine, and the size of several glycoproteins was reduced. A second epitope (GA-XII) was also absent in strains carrying the modC354 or modE353 mutations, reducing the size of the glycoprotein which normally expresses it. Fucose content was reduced in the three mutants, suggesting that each mutation affected a separate step in fucosylation. The lesions did not appear to inhibit synthesis of the underlying carbohydrate, because detergent extracts of mutant vesicles were more active than normal vesicles at transferring [14C]fucose from GDP-[14C]fucose to endogenous acceptor species. The modD352 and modE353 mutant strains incorporated exogenous [3H]fucose poorly, suggesting that lesions in the modD and modE genes interfere with the biosynthesis of fucoconjugates downstream from the previously described GDP-fucose synthesis defect of the modC mutation. Intact modE353 mutant vesicles were relatively inefficient in in vitro assays, suggesting a global fucosylation defect (which is consistent with the loss of both glycoantigens, GA-X and GA-XII, in this mutant). Finally, the modC354 mutation led to delayed accumulation of slime sheath in vitro. The three genetic loci define a fucosylation pathway in D. discoideum comprising defined biochemical steps which contribute to multicellular morphogenesis in this organism.

Glycoantigen expression is regulated both temporally and spatially during development in the cellular slime molds Dictyostelium discoideum and D. mucoroides

Six monoclonal antibodies were isolated which react with common antigens shared by multiple glycoconjugate species in the cellular slime mold Dictyostelium discoideum. Based on competition of antibody binding by glycopeptides and simple sugars, and inhibition of antibody binding by antigen pretreatment with Na periodate, it is argued that at least five of the six antibodies recognize epitopes which contain carbohydrate. These epitopes are consequently referred to as glycoantigens (GAs).Three of the GAs are expressed during growth and throughout the developmental cycle, but are eventually enriched in prestalk and stalk cells. The remaining three are expressed only during and/or after aggregation and are exclusively expressed or highly enriched in prespore cells and spores. These conclusions are derived from Western blot immunoanalysis of purified cell types, immunofluorescence, and EM immunocytochemistry.The two GAs found only in prespore cells appear to be exclusively enclosed within prespore vesicles. The third GA of this type, which is only enriched in prespore cells compared to prestalk cells, is also found in other vesicle types as well as on the cell surface.Two of the GAs enriched in prestalk cells are initially found in all cells of the slug. They are undetectable in spores and prominent in stalk cells. The third GA, though found in the interiors of both prestalk and prespore cells, is enriched on the cell surface of prestalk cells.The chief characteristics of expression of four of these GAs are conserved in the related species D. mucoroides. This species is characterized by continuous trans differentiation of prespore cells into prestalk cells. This shows that the prespore cells maintain specific mechanisms for turning over their cell type specific GAs and that prestalk cells express a specific mechanism for inducing at least one of their cell-type specific GAs.These observations identify specific carbohydrate structures (as GAs) whose synthesis, subsequent localization and turnover are developmentally regulated. The exclusive association of two GAs with prespore vesicles identifies these GAs as markers for this organelle and raises questions regarding the functional significance of this association. The restricted cell surface localization of the other four GAs, together with data from cell adhesion studies, suggest the possibility of a potential role for these GAs in intercellular recognition leading to cell sorting.