Protein O-fucosyltransferases Research Articles

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs.

Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.

An Essential Fringe‐Like Protein in the Plasmodium falciparum Asexual Life Cycle

Malaria is a deadly disease caused by the apicomplexan parasite Plasmodium. Plasmodium asexual replication occurs in the red blood cell (RBC) and is what causes clinical symptoms of disease. In the RBC, Plasmodium moves through 3 developmental stages ending with schizogony and creating 16–32 daughter merozoites. At the end of schizogony, merozoites egress the host cell by breaking out of two membranes, the parasitophorous vacuole membrane and the RBC membrane. Schizogony and successful egress allows for exponential Plasmodium replication and RBC infection. Our lab recently identified a B3GLCT‐like protein in the Plasmodium ER (PfB3ER) that interacts with an essential ER chaperone and whose expression profile is similar to that of proteins required for egress. B3GLCT‐like proteins are known to act as glucosyltransferases and work in concert with protein O‐fucosyltransferases (POFUT2) to modify thrombospondin‐like repeats in mammalian cells. Surprisingly, the P. falciparum POFUT2 has been shown to be non‐essential while PfB3ER is predicted to be essential in the asexual stages. Based on PfB3ER homology to the mammalian B3GLCT and its predicted expression profile, we hypothesize that the O‐glycosylation function of PfB3ER is required for efficient egress of P. falciparum from RBCs. To test this, we have employed CRISPR/Cas9 gene editing to HA‐tag PfB3ER and create conditional mutants utilizing the TetR‐DOZI‐aptamer system. We show that PfB3ER localizes to the ER, unlike previous published results that suggest that PfB3ER is exported to the host RBC, and that PfB3ER is primarily expressed during schizogony. Further, we show that PfB3ER is essential for the asexual replication of P. falciparum. Future studies will focus on the specific stage of the intraerythrocytic lifecycle that are affected by PfB3ER knockdown and test the requirement of its glycosyltransferase activity. Together, these data have uncovered an essential function for a putative glycosyltransferase in the intraerythrocytic lifecycle of P. falciparum.Support or Funding Information R01 AI130139/AI/NIAID NIH HHS/United States

How does GDP‐fucose get into the Endoplasmic Reticulum for O‐fucosylation of EGF Repeats and TSRs?

Fucose is a common monosaccharide found in glycan chains of all mammalian cells. Fucose is transferred from GDP‐fucose to glycoproteins by fucosyltransferases, resulting in terminal fucosylation of N‐linked glycans or mucin‐type O‐glycans, or direct modification of serine or threonine residues by Protein O‐fucosyltransferases (POFUT) 1 or 2. Defects in fucosylation can lead to developmental disorders or cancer. In particular, POFUT1 adds O‐fucose to Epidermal Growth Factor‐like (EGF) repeats of Notch receptors, while POFUT2 adds O‐fucose to Thrombospondin type 1 Repeats (TSRs) found in multiple cell‐surface and secreted proteins, including the A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) family of extracellular proteases. Elimination of Pofut1 or Pofut2 results in early embryonic lethality in mice, indicating these two fucosylation pathways play essential roles in mouse development. Both POFUT1 and POFUT2 are located in the endoplasmic reticulum (ER), while fucosyltransferases involved in terminal fucosyaltion are localized in the trans‐Golgi. GDP‐fucose is synthesized in the cytoplasm either through a de novo or salvage pathway, and the GDP‐fucose is then transported into the lumen of the ER or Golgi for use by the fucosyltransferases. Currently two GDP‐fucose transporters, SLC35C1 and SLC35C2, have been identified, both of which are localized to the Golgi. It is unknown how GDP‐fucose is transported into the ER where Notch receptors and ADAMTS proteins are fucosylated. In order to uncover where the GDP‐fucose for POFUT1 and POFUT2 comes from, we have generated CRISPR‐Cas9 mutant cell lines in which fucosylation is impaired in two ways: 1. Inactivating GDP‐fucose biosynthetic enzymes from the salvage or de novo pathways; 2. Inactivating the GDP‐fucose transporters. HEK293T cells have proven to be an excellent model for fucosylation studies. All the fucose biosynthesis genes are normally expressed, and impaired fucosylation can be expected in knockout mutant cell lines. Using CRISPR‐Cas9 technology, the following genes have been knocked out: GMDS and TSTA3 of the de novo pathway, FUK and GFPP of the salvage pathway, and the GDP‐fucose transporters SLC35C1 and SLC35C2. O‐Fucosylation of EGF repeats and TSRs and terminal fucosylation of N‐linked glycans is being analyzed using Mass Spectrometry and lectin binding. Notch function is also being evaluated. These results should reveal whether a preferred biosynthetic pathway or transporter for different forms of fucosylation exists.Support or Funding InformationSupported by NIH grant GM061126.

A potential role for protein O-fucosylation during pollen-pistil interactions

ABSTRACTPutative protein O-fucosyltransferases (POFTs) represent a large family of Glycosyl Transferase family 65 domain-containing proteins in land plants, with at least 39 proposed members in the Arabidopsis thaliana genome alone. We recently identified a member of this family, AtOFT1 (At3g05320), in which loss-of-function mutants display impaired sexual reproduction that was linked to a defective male gamete. Specifically, oft1 mutant pollen tubes are ineffective at penetrating the stigma-style interface leading to a drastic reduction in seed set and a nearly 2000-fold reduction in pollen transmission. Our findings establish that AtOFT1 plays a critical role in pollen tube penetration through the stigma/style in Arabidopsis and further suggest an important role for protein O-glycosylation events that potentially influence pollen tube mechanical strength or the ability to respond to positional guidance cues during the process of tube growth and fertilization.

A Putative Protein O-Fucosyltransferase Facilitates Pollen Tube Penetration through the Stigma-Style Interface.

During pollen-pistil interactions in angiosperms, the male gametophyte (pollen) germinates to produce a pollen tube. To fertilize ovules located within the female pistil, the pollen tube must physically penetrate specialized tissues. Whereas the process of pollen tube penetration through the pistil has been anatomically well described, the genetic regulation remains poorly understood. In this study, we identify a novel Arabidopsis (Arabidopsis thaliana) gene, O-FUCOSYLTRANSFERASE1 (AtOFT1), which plays a key role in pollen tube penetration through the stigma-style interface. Semi-in vivo growth assays demonstrate that oft1 mutant pollen tubes have a reduced ability to penetrate the stigma-style interface, leading to a nearly 2,000-fold decrease in oft1 pollen transmission efficiency and a 5- to 10-fold decreased seed set. We also demonstrate that AtOFT1 is localized to the Golgi apparatus, indicating its potential role in cellular glycosylation events. Finally, we demonstrate that AtOFT1 and other similar Arabidopsis genes represent a novel clade of sequences related to metazoan protein O-fucosyltransferases and that mutation of residues that are important for O-fucosyltransferase activity compromises AtOFT1 function in vivo. The results of this study elucidate a physiological function for AtOFT1 in pollen tube penetration through the stigma-style interface and highlight the potential importance of protein O-glycosylation events in pollen-pistil interactions.

LIF upregulates poFUT1 expression and promotes trophoblast cell migration and invasion at the fetal-maternal interface.

Trophoblast cell migration and invasion are crucial for the establishment of a successful pregnancy. Protein O-fucosyltransferases, such as poFUT1 and poFUT2, catalyze the O-fucosylation of proteins and have important roles in embryonic development. Leukemia inhibitory factor (LIF) is a critical cytokine in the regulation of embryonic development and implantation. However, the exact roles of poFUTs in embryo migration and invasion and the effects of LIF on the expression of poFUTs have not been studied in detail. In the current study, we showed that poFUT1 and LIF were highly expressed in human trophoblast cells and in the serum of women during the first trimester of a normal pregnancy. However, in patients with threatened abortion, poFUT1 and LIF levels were found to be reduced. There were no significant differences in the expression levels of poFUT2 between the two groups. The migration and invasion potential of trophoblasts in an explant culture and in an in vitro implantation model was decreased or increased upon altering poFUT1 expression levels by siRNA or cDNA transfection. Our results also revealed that LIF upregulated the expression of poFUT1. The upregulation of poFUT1 by LIF promoted trophoblast cell migration and invasion at the fetal–maternal interface by activating the PI3K/Akt signaling pathway. Taken together, these study findings suggest that poFUT1 may be used as a marker of embryo implantation.

6-Alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin Type-1 repeats by protein O-fucosyltransferases 1 and 2

Protein O-fucosyltransferase 1 (Pofut1) and protein O-fucosyltransferase 2 (Pofut2) add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O-fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O-fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O-fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O-fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O-fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O-fucose, providing evidence that 6AF is a significant new tool in the study of protein O-fucosylation.

Structures of NodZ α1,6-fucosyltransferase in complex with GDP and GDP-fucose

Rhizobial NodZ α1,6-fucosyltransferase (α1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5'-diphosphate-β-L-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signalling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two α1,6-FucT structures have been determined, both without any donor or acceptor molecule that couldhighlight the structural background of the catalytic mechanism. Here, the first crystal structures of α1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which isabyproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 Å resolution. The fucose residue is exposed to solvent and is disordered. The enzyme-product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-L-glucosamine (penta-NAG). The structure has been determined at 1.98 Å resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among α1,2-, α1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand βC2 and helix αC3. In addition, there is a shift of the αC3 helix itself upon GDP-Fuc binding.

Crystal structure of mammalian α1,6-fucosyltransferase, FUT8

Mammalian alpha1,6-fucosyltransferase (FUT8) catalyses the transfer of a fucose residue from a donor substrate, guanosine 5'-diphosphate-beta-L-fucose to the reducing terminal N-acetylglucosamine (GlcNAc) of the core structure of an asparagine-linked oligosaccharide. Alpha1,6-fucosylation, also referred to as core fucosylation, plays an essential role in various pathophysiological events. Our group reported that FUT8 null mice showed severe growth retardation and emphysema-like lung-destruction as a result of the dysfunction of epidermal growth factor and transforming growth factor-beta receptors. To elucidate the molecular basis of FUT8 with respect to pathophysiology, the crystal structure of human FUT8 was determined at 2.6 A resolution. The overall structure of FUT8 was found to consist of three domains: an N-terminal coiled-coil domain, a catalytic domain, and a C-terminal SH3 domain. The catalytic region appears to be similar to GT-B glycosyltransferases rather than GT-A. The C-terminal part of the catalytic domain of FUT8 includes a Rossmann fold with three regions that are conserved in alpha1,6-, alpha1,2-, and protein O-fucosyltransferases. The SH3 domain of FUT8 is similar to other SH3 domain-containing proteins, although the significance of this domain remains to be elucidated. The present findings of FUT8 suggest that the conserved residues in the three conserved regions participate in the Rossmann fold and act as the donor binding site, or in catalysis, thus playing key roles in the fucose-transferring reaction.

Reaction mechanism and substrate specificity for nucleotide sugar of mammalian α1,6-fucosyltransferase—a large-scale preparation and characterization of recombinant human FUT8

FUT8, mammalian alpha1,6-fucosyltransferase, catalyzes the transfer of a fucose residue from the donor substrate, guanosine 5'-diphosphate (GDP)-beta-L-fucose, to the reducing terminal GlcNAc of the core structure of asparagine-linked oligosaccharide via an alpha1,6-linkage. FUT8 is a typical type II membrane protein, which is localized in the Golgi apparatus. We have previously shown that two neighboring arginine residues that are conserved among alpha1,2-, alpha1,6-, and protein O-fucosyltransferases play an important role in donor substrate binding. However, details of the catalytic and reaction mechanisms and the ternary structure of FUT8 are not understood except for the substrate specificity of the acceptor. To develop a better understanding of FUT8, we established a large-scale production system for recombinant human FUT8, in which the enzyme is produced in soluble form by baculovirus-infected insect cells. Kinetic analyses and inhibition studies using derivatives of GDP-beta-L-fucose revealed that FUT8 catalyzes the reaction which depends on a rapid equilibrium random mechanism and strongly recognizes the base portion and diphosphoryl group of GDP-beta-L-fucose. These results may also be applicable to other fucosyltransferases and glycosyltransferases.

CigA, a light-inducible gene involved in vegetative growth in Mucor circinelloides is regulated by the carotenogenic repressor crgA

A strategy based on the differential-display technique has been applied to identify and to isolate potential target genes controlled by crgA, a negative regulator of the light-inducible carotenogenesis in the fungus Mucor circinelloides. This approach resulted in the identification and cloning of the gene cigA, whose expression is repressed by the crgA gene. After a light pulse, there is an immediate though transient accumulation of transcripts of cigA gene. A similar result was obtained when mycelia were exposed to continuous illumination, suggesting the existence of a photoadaptation mechanism in M. circinelloides. The protein sequence deduced from the nucleotide sequence of cigA shows homology to the mouse and human GDP-fucose protein O-fucosyltransferases. Null cigA mutants were generated by gene replacement. Lack of cigA function does not affect carotenogenesis but provokes a slight reduction in vegetative growth that is independent of light. Introduction of wild-type cigA alleles into a cigA − strain restored the wild-type phenotype for vegetative growth. These results suggest that cigA is involved in vegetative growth and, moreover, that crgA controls cellular processes other than carotenogenesis.