Enhanced Cross-Presentation and Improved CD8+ T Cell Responses after Mannosylation of Synthetic Long Peptides in Mice

The use of synthetic long peptides (SLP) has been proven to be a promising approach to induce adaptive immune responses in vaccination strategies. Here, we analyzed whether the efficiency to activate cytotoxic T cells by SLP-based vaccinations can be increased by conjugating SLPs to mannose residues. We could demonstrate that mannosylation of SLPs results in increased internalization by the mannose receptor (MR) on murine antigen-presenting cells. MR-mediated internalization targeted the mannosylated SLPs into early endosomes, from where they were cross-presented very efficiently compared to non-mannosylated SLPs. The influence of SLP mannosylation was specific for cross-presentation, as no influence on MHC II-restricted presentation was observed. Additionally, we showed that vaccination of mice with mannosylated SLPs containing epitopes from either ovalbumin or HPV E7 resulted in enhanced proliferation and activation of antigen-specific CD8⁺ T cells. These findings demonstrate that mannosylation of SLPs augments the induction of a cytotoxic T cell response in vitro and in vivo and might be a promising approach to induce cytotoxic T cell responses in e.g. cancer therapy and anti-viral immunity.     

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

Mannosylation of glycoproteins and dolichol derivatives in fibroblasts from patients with cystic fibrosis

Increased incorporation of mannose into endogenous glycoprotein fractions has been found in whole cell lysates and crude membrane preparations of cultured skin fibroblasts from patients with cystic fibrosis (1.3–2.3-times normal) when GDP[¹⁴C]mannose served as the mannosyl donor. In contrast, the incorporation of mannose from GDPmannose into lipid fractions containing dolichol phosphate and dolichol pyrophosphate oligosaccharides as well as the incorporation of mannose from dolichol phospho[³H]mannose into both glycoproteins and dolichol derivatives were not significantly different among cell preparations from patients with cystic fibrosis and normal controls. Mannosyltransferase activity toward exogenous glycoproteins as well as the activities of soluble and membranous α-mannosidase and β-mannosidase appeared to be normal and could not account for the observed differences. The altered incorporation of mannose into endogenous glycoprotein may reflect changes in glycosylation processes other than mannosylation.     

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.     

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C²-Man-)Trp.     

Sensitivity of mannosyl transferases from Salmonella serotype E1 and B to modification of the donor heterocyclic base

A series of synthetic nucleoside diphosphate mannoses with different heterocyclic bases were tested as GDP-Man analogues in enzymatic mannosylation during assembly of O-antigen repeating units of Salmonella anatum and Salmonella typhimurium. The substrate efficiency was found to depend strongly on oxygen atom presence at C6 of the purine residue, the H2N-C2-N1-H grouping of the heterocyclic nucleus being less important. UDP-Man proved to be an efficient substrate for the mannosylation reactions.     

Synthesis of retinylphosphate mannose in yeast and its possible involvement in lipid-linked oligosaccharide formation

A membrane fraction from Saccharomyces cerevisiae as well as a mannosyltransferase purified therefrom was shown to catalyze the transfer of mannose from GDPmannose to retinyl phosphate. The product formed has chromatographic and chemical properties characteristic for retinylphosphate mannose. The enzyme requires divalent cations. Mg2+ is more effective than Mn2+ with an optimum concentration around 25 mM. Amphomycin at a concentration of 0.1 mg/ml inhibits the reaction to 50%. Glycosyl transfer was specific for mannose residues from GDPmannose and did not occur with dolichylphosphate mannose nor with UDP galactose; UDPglucose is a poor donor. Formation of retinylphosphate mannose is inhibited by dolichyl phosphate. This observation as well as similarities between retinylphosphate mannose and dolichylphosphate mannose synthesis in respect to ion requirement, inhibition by amphomycin are suggestive that both reactions are catalyzed by one and the same enzyme. In experiments studying the glycosyl donor specificity in the assembly of lipid-linked oligosaccharide intermediates involved in N-glycosylation of proteins, it could be demonstrated that retinylphosphate mannose can replace dolichylphosphate mannose in the final steps of mannosylation.     

The fibrinogen C-terminal domain is seldom C-mannosylated but its C-mannosylation is important for the secretion of microfibril-associated glycoprotein 4

BackgroundC-mannosylation is the one of glycosylations. Microfibril-associated glycoprotein 4 (MFAP4), an important protein for tissue homeostasis and cell adhesion, contains a consensus sequence of C-mannosylation in its fibrinogen C-terminal domain. In this study, we sought to demonstrate that fibrinogen C-terminal domain is a new substrate domain for C-mannosylation.MethodsWe established an MFAP4-overexpresssing HT1080 cell line and purified recombinant MFAP4 protein from the conditioned medium for LC-MS/MS analysis. Subcellular localization of MFAP4 was observed under confocal fluorescence microscope.ResultsWe found that MFAP4 is C-mannosylated at Trp²³⁵ in the fibrinogen C-terminal domain by LC-MS/MS. To determine the functions of the C-mannosylation of MFAP4, we established a C-mannosylation-defective mutant MFAP4-overexpresssing HT1080 cell line and measured its secretion of MFAP4. The secretion of MFAP4 decreased significantly in the C-mannosylation-defective mutant MFAP4-overexpresssing cell line versus wild-type cells. Moreover, co-transfection experiments indicated that C-mannosylated MFAP4 accelerated its secretion.ConclusionsOur results demonstrate that the fibrinogen C-terminal domain is a novel C-mannosylation domain and that the C-mannosylation of MFAP4 is important for its secretion.General significanceThese results suggest that C-mannosylation has a role for dominant effect for MFAP4 secretion.     

Regulation of N-glycosylation and secretion of Isthmin-1 by its C-mannosylation

BackgroundC-mannosylation is a type of protein glycosylation. Human Isthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C-mannosylation sequences at Trp²²³ and Trp²²⁶. In this study, we sought to examine the role of C-mannosylation in the secretion of ISM1.MethodsWe established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy.ResultsWe found that ISM1 is C-mannosylated at Trp²²³ and Trp²²⁶ in the TSR domain. To determine the functions of the C-mannosylation of ISM1, we established a C-mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N-glycosylated only in these C-mannosylation-defective cells.ConclusionsISM1 is C-mannosylated in its TSR domain, and the status of the C-mannosylation of ISM1 affects its N-glycosylation.General significanceThe C-mannosylation of ISM1 regulates its N-glycosylation status.     

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4)

BackgroundC-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear.MethodsWe established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan.ResultsWe identified that ADAMTS4 is C-mannosylated at Trp⁴⁰⁴ in the metalloprotease domain and at Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ in the thrombospondin type 1 repeat (TSR). The replacement of Trp⁴⁰⁴ with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp⁵²³, Trp⁵²⁶, and Trp⁵²⁹ with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity.ConclusionsOur results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity.General significanceBecause C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.     

Identification of DPY19L3 as the C-mannosyltransferase of R-spondin1 in human cells

R-spondin1 (Rspo1) is a secreted protein that enhances Wnt signaling, which has crucial functions in embryonic development and several cancers. C-mannosylation is a rare type of glycosylation and might regulate secretion, protein–protein interactions, and enzymatic activity. Although human Rspo1 contains 2 predicted C-mannosylation sites, C-mannosylation of Rspo1 has not been reported, nor have its functional effects on this protein. In this study, we demonstrate by mass spectrometry that Rspo1 is C-mannosylated at W¹⁵³ and W¹⁵⁶. Using Lec15.2 cells, which lack dolichol-phosphate-mannose synthesis activity, and mutant Rspo1-expressing cells that replace W¹⁵³ and W¹⁵⁶ by alanine residues, we observed that C-mannosylation of Rspo1 is required for its secretion. Further, the enhancement of canonical Wnt signaling by Rspo1 is regulated by C-mannosylation. Recently DPY19 was reported to be a C-mannosyltransferase in Caenorhabditis elegans, but no C-mannosyltransferases have been identified in any other organism. In gain- and loss-of-function experiments, human DPY19L3 selectively modified Rspo1 at W¹⁵⁶ but not W¹⁵³ based on mass spectrometry. Moreover, knockdown of DPY19L3 inhibited the secretion of Rspo1. In conclusion, we identified DPY19L3 as the C-mannosyltransferase of Rspo1 at W¹⁵⁶ and found that DPY19L3-mediated C-mannosylation of Rspo1 at W¹⁵⁶ is required for its secretion.     

Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.Protein glycosylation, which is a major posttranslational modification, plays essential roles in eukaryotic cells from fungi to mammals (19). N-linked oligosaccharides in glycoproteins that share relatively common structures are structurally classified into high-mannose, complex, and hybrid types (3). O-linked oligosaccharides in glycoproteins are diverse with respect to their sugar components and the mode of sugar linkages among the eukaryotic organisms (8, 19). O mannosylation, which is commonly found in the glycoproteins of fungi, has been extensively studied in the budding yeast Saccharomyces cerevisiae (4, 21, 35). The initial reaction of mannose transfer to serine and threonine residues in proteins is catalyzed by protein O-d-mannosyltransferase (Pmt) in the endoplasmic reticulum (ER), where dolichyl phosphate-mannose is required as an immediate sugar donor (4). In the Golgi complex, O mannosylation in S. cerevisiae is linearly elongated by up to five mannose residues by mannosyltransferases (Mnt) that utilize GDP-mannose as the mannosyl donor. At least six Pmt-encoding genes (PMT1 to -6), three α-1,2-Mnt-encoding genes (KRE2, KTR1, and KTR3), and three α-1,3-Mnt-encoding genes (MNN1, MNT2, and MNT3) are known to be involved in O mannosylation in S. cerevisiae (21, 31, 45).The Pmt family of proteins can be classified into the PMT1, PMT2, and PMT4 subfamilies based on phylogeny (6). Proteins of the PMT1 subfamily form a heteromeric complex with proteins belonging to the PMT2 subfamily, and PMT4 subfamily proteins form a homomeric complex (7). Simultaneous disruptions of three different types of PMT genes were lethal (4), suggesting that each class provided a unique function for O mannosylation. Yeasts other than S. cerevisiae, such as Schizosaccharomyces pombe (38, 41), Candida albicans (29), and Cryptococcus neoformans (28), possess three to five pmt genes, which have been characterized. Several studies provide evidence that protein O mannosylation modulates the functions and stability of secretory proteins and thereby affects the growth and morphology of these yeasts. O mannosylation by Pmt2 in S. cerevisiae (ScPmt2) provides protection from ER-associated degradation and also functions as a fail-safe mechanism for ER-associated degradation (11, 13, 23). Likewise, in C. albicans, CaPmt1- and CaPmt4-mediated O mannosylation specifically protects CaSec20 from proteolytic degradation in the ER (40). Cell wall integrity is maintained in S. cerevisiae by increased stabilization and correct localization of the sensor proteins ScWsc and ScMid2 due to O mannosylation by ScPmt2 and ScPmt4 (20). Similarly, the stability and localization to the plasma membrane of axial budding factor ScAxl2/Bud10 is enhanced by ScPmt4-mediated O mannosylation, increasing its activity (32). ScPmt4-mediated O glycosylation also functions as a sorting determinant for cell surface delivery of ScFus1 (30). CaPmt4-mediated O glycosylation is required for environment-specific morphogenetic signaling and for the full virulence of C. albicans (29).With respect to filamentous fungi like Aspergillus that develop hyphae in a highly ordered manner, which then differentiate to form conidiospores, little is known about the function and synthetic pathway of the O-mannose-type oligosaccharides. O-Glycans in glycoproteins of Aspergillus include sugars other than mannose, and their structures have been determined (8). The initial mannosylation catalyzed by Pmts is found in Aspergillus and occurs as in yeasts (8).We characterized the pmtA gene of Aspergillus nidulans (AnpmtA), belonging to the PMT2 subfamily, and found that the mutant exhibited a fragile cell wall phenotype and alteration in the carbohydrate composition, with a reduction in the amount of skeletal polysaccharides in the cell wall (26, 33). Recently, the Afpmt1 gene belonging to the PMT1 family of Aspergillus fumigatus, a human pathogen, was characterized. AfPmt1 is crucial for cell wall integrity and conidium morphology (46).In this study, we characterize the pmtB and pmtC genes of A. nidulans to understand their contribution to the cell morphology of this filamentous fungus. We also demonstrate that the PmtA, PmtB, and PmtC proteins have distinct specificities for protein substrates and function differently during cell growth of filamentous fungi.     

O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.     

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949–56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326–31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19–48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60–81 pmol/mg protein in spleen and lung and 6.9–18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.     

From the freezer to the clinic: Antifreeze proteins in the preservation of cells,tissues, and organs

Understanding the mechanisms by which natural anti‐freeze proteins protect cells and tissues from cold could help to improve the availability of donor organs for transplantation.

The first successful organ transplant in humans was performed in 1954 by Joseph Murray, who used a patient’s twin as a kidney donor. Murrays’ breakthrough paved the way for organ transplantation and the number of transplanted organs has grown ever since. For example, in 2017, a total of 139.024 solid organs—mostly kidney, liver, heart, lung, pancreas, and small bowel—were transplanted (Fig 1A). But this number only reflects 10% of the worldwide need; many patients still die of end‐stage organ failure while on a waiting list. The limited number of donor organs contributes only partially to this shortage. Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime. In fact, the percentage of donor organs that are unused is estimated to range from around 25% for kidneys and livers up to 70–80% for hearts and lungs (Giwa et al, 2017); Fig 1B).Open in a separate windowFigure 1Organ transplantation and preservability statusStatistics show a positive correlation between the duration of ex vivo preservation and the number of organ transplants. Number of solid organs transplanted in 2017 (A). Percentage of organs failed to be transplanted (B). Duration of solid organ ex vivo preservation in static cold storage (C). Sources: Data from the Global Observatory on Donation and Transplantation and (Parsons et al, 2014), (Guibert et al, 2011) and (Editorial: Buying time for transplants (2017))

Many donor organs are not transplanted eventually owing to inefficient preservation techniques that shorten their extracorporeal lifetime.

To address the shortage of donor organs and decrease the number of organs that go to waste, biobanks could efficiently store viable tissues and organs until transplantation. Yet, the current standard for ex vivo preservation of donor organs is static cold storage (4–8°C) which, depending on the organ, ensures viable conservation for only some hours; hearts are typically viable for a maximum of only 4 h (Fig 1C). In addition, this approach leads to hypothermic damage and to ischemia/reperfusion injury.Hence, there is an urgent need for strategies that prolong the viable preservation of donor organs. Two main strategies have emerged for cryopreservation and subzero storage, both of which cool tissues below the freezing point. While subzero storage just below 0°C may suffice for short‐term preservation, cryopreservation at −80°C or even lower temperatures is required for long‐term storage in biobanks. A proof‐of‐principle study already demonstrated that subzero preservation extended the preservation of rat hearts up to 24 h after collection (Amir et al, 2004); cryopreservation of whole hearts is currently not possible. The main reason is that lowering the temperature below the freezing point of water leads to ice formation, which causes cell damage and destroys tissues. One of the main challenges in biomedical research for organ transplantation is therefore finding non‐toxic and biocompatible antifreeze compounds that enable subzero storage and cryopreservation without causing tissue damage. An additional benefit is a larger time window to perform evaluation in terms of organ size and human leukocyte antigens matching and preparing the recipient patient to increase the chance of a successful transplantation.     

Direct utilization of mannose for mammalian glycoprotein biosynthesis

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2- 3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic- derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.      

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

O-Mannosylation and N-glycosylation are essential protein modifications that are initiated in the endoplasmic reticulum (ER). Protein translocation across the ER membrane and N-glycosylation are highly coordinated processes that take place at the translocon-oligosaccharyltransferase (OST) complex. In analogy, it was assumed that protein O-mannosyltransferases (PMTs) also act at the translocon, however, in recent years it turned out that prolonged ER residence allows O-mannosylation of un-/misfolded proteins or slow folding intermediates by Pmt1-Pmt2 complexes. Here, we reinvestigate protein O-mannosylation in the context of protein translocation. We demonstrate the association of Pmt1-Pmt2 with the OST, the trimeric Sec61, and the tetrameric Sec63 complex in vivo by co-immunoprecipitation. The coordinated interplay between PMTs and OST in vivo is further shown by a comprehensive mass spectrometry-based analysis of N-glycosylation site occupancy in pmtΔ mutants. In addition, we established a microsomal translation/translocation/O-mannosylation system. Using the serine/threonine-rich cell wall protein Ccw5 as a model, we show that PMTs efficiently mannosylate proteins during their translocation into microsomes. This in vitro system will help to unravel mechanistic differences between co- and post-translocational O-mannosylation.     

In vitro biosynthesis of glycosylphosphatidylinositol in Aspergillus fumigatus

Glycosylphosphatidylinositol (GPI) represents a mechanism for the attachment of proteins to the plasma membrane found in all eukaryotic cells. GPI biosynthesis has been mainly studied in parasites, yeast, and mammalian cells. Aspergillus fumigatus, a filamentous fungus, produces GPI-anchored molecules, some of them being essential in the construction of the cell wall. An in vitro assay was used to study the GPI biosynthesis in the mycelium form of this organism. In the presence of UDP-GlcNAc and coenzyme A, the cell-free system produces the initial intermediates of the GPI biosynthesis: GlcNAc-PI, GlcN-PI, and GlcN-(acyl)PI. Using GDP-Man, two types of mannosylation are observed. First, one or two mannose residues are added to GlcN-PI. This mannosylation, never described in fungi, does not require dolichol phosphomannoside (Dol-P-Man) as the monosaccharide donor. Second, one to five mannose residues are added to GlcN-(acyl)PI using Dol-P-Man as the mannose donor. The addition of ethanolamine phosphate groups to the first, second, and third mannose residue is also observed. This latter series of GPI intermediates identified in the A. fumigatus cell-free system indicates that GPI biosynthesis in this filamentous fungus is similar to the mammalian or yeast systems. Thus, these biochemical data are in agreement with a comparative genome analysis that shows that all but 3 of the 21 genes described in the Saccharomyces cerevisiae GPI pathways are found in A. fumigatus.     

Correlation between number and position of floral organs in Arabidopsis

Background and Aims

The study of variation in number, position and type of floral organs may serve as a key to understanding the mechanisms underlying their variation, and will make it possible to improve the analysis of gene function in model plant species by means of a more accurate characterization of mutant phenotypes. The present analysis was carried out in order to understand the correlation between number and position of floral organs in Arabidopsis thaliana.

Methods

An analysis of number and position of organs in flowers of wild type as well as in a series of mutations with floral organ position alterations was carried out, using light and electron microscopy. Variation common to different genotypes was analysed by means of individual diagrams, upon which generalized diagrams depicting variation in number and position of organs, were built by superimposition.

Key Results and Conclusions

It is shown that in the Arabidopsis flower a correlation exists between positions of petals and sepals, as well as between positions of stamens and carpels, whereas the position of carpels does not seem to depend on number and position of petals and stamens. This suggests that the position of organs in the basal (sepals) and apical (carpels) parts of the flower are determined before that in the intermediate zone. This assumption is consistent with the results of mathematical modelling and is supposed to be the consequence of stem-cell activity in the flower.     

Structure–activity relationship studies on acremomannolipin A,the potent calcium signal modulator with a novel glycolipid structure 2: Role of the alditol side chain stereochemistry

Five alditol analogs 1b–1f of a novel glycolipid acremomannolipin A (1a), the potential Ca²⁺ signal modulator isolated from Acremonium strictum, were synthesized by employing a stereoselective β-mannosylation of appropriately protected mannose with five hexitols with different stereochemistry, and their potential on modulating Ca²⁺ signaling were evaluated. All these analogs were more potent compared to the original compound 1a, and proved that mannitol stereochemistry of 1a was not critical for the potent calcium signal modulating.