Synthesis of N-glycan oxazolines: donors for endohexosaminidase catalysed glycosylation

Oxazoline mono-, di-, tri- and hexasaccharides, corresponding to the core components of N-linked glycoprotein high mannose glycans, are synthesised as potential glycosyl donors for endohexosaminidase catalysed glycosylation of glycopeptides and glycoprotein remodelling. The crucial beta-D-Manp-(1-->4)-D-GlcpNAc linkage is synthesised via epimerisation of gluco disaccharide substrates by sequential triflation and nucleophilic substitution. Oxazolines are formed directly from the anomeric OPMP protected N-acetyl glucosamine derivatives. Efficient endohexosaminidase catalysed glycosylation of a synthetic beta-D-GlcpNAcAsn glycosyl amino acid is demonstrated with the trisaccharide oxazoline donor.     

Megalomycin C, a macrolide antibiotic that blocks protein glycosylation and shows antiviral activity

Megalomycin C, a natural macrolide antibiotic showed a potent antiherpetic activity. At concentrations that efficiently prevented HSV-1 multiplication, the compound had no cytotoxic or antiproliferative effects. Viral DNA and protein synthesis took place at normal levels in the presence of the antibiotic, suggesting that neither the translation of viral mRNA, nor the synthesis of viral nucleic acids was affected. The incorporation of mannose and galactosamine into viral proteins was blocked and precursor, but not mature, HSV-1 glycoproteins were detected in the presence of megalomycin C. Non-infectious HSV-1 viral particles were formed when the compound was present, but their glycoproteins were not properly glycosylated.     

Dictyostelium calcium-binding protein 4a interacts with nucleomorphin, a BRCT-domain protein that regulates nuclear number

Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin-binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium-binding protein 4a (CBP4a) in a Ca(2+)-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with nucleomorphin requires the DEED domain, this suggests that CBP4a may respond to Ca(2+)-signalling through modulating factors that might function in concert to regulate nuclear number.     

The hypothetical protein Atu4866 from Agrobacterium tumefaciens adopts a streptavidin-like fold

Atu4866 is a 79-residue conserved hypothetical protein of unknown function from Agrobacterium tumefaciens. Protein sequence alignments show that it shares > or =60% sequence identity with 20 other hypothetical proteins of bacterial origin. However, the structures and functions of these proteins remain unknown so far. To gain insight into the function of this family of proteins, we have determined the structure of Atu4866 as a target of a structural genomics project using solution NMR spectroscopy. Our results reveal that Atu4866 adopts a streptavidin-like fold featuring a beta-barrel/sandwich formed by eight antiparallel beta-strands. Further structural analysis identified a continuous patch of conserved residues on the surface of Atu4866 that may constitute a potential ligand-binding site.     

Metabolic alteration of the N-glycan structure of a protein from patients with a heterozygous protein deficiency

Glycosylation, an important post-translation modification, could alter biological activity or influence the clearance rates of glycoproteins. We report here the first example of a heterozygous protein deficiency leading to metabolic alteration of N-glycan structures in residual secreted protein. Analysis of C1 esterase inhibitor (C1INH) glycans from normal individuals and patients with hereditary deficiency of C1INH demonstrated identical O-glycan structures but the N-glycans of patients with a heterozygous genetic deficiency were small, highly charged and lacked sialidase releasable N-acetylneuraminic acid. Structural studies indicate that the charge character of these aberrant N-glycan structures may result from the presence of mannose-6-phosphate residues. These residues might facilitate secretion of C1INH through an alternate lysosomal pathway, possibly serving as a compensatory mechanism to enhance plasma levels of C1INH in these deficient patients.     

Crystal structure of a conserved hypothetical protein from Escherichia coli

The crystal structure of a conserved hypothetical protein from Escherichia coli has been determined using X-ray crystallography. The protein belongs to the Cluster of Orthologous Group COG1553 (National Center for Biotechnology Information database, NLM, NIH), for which there was no structural information available until now. Structural homology search with DALI algorism indicated that this protein has a new fold with no obvious similarity to those of other proteins with known three-dimensional structures. The protein quaternary structure consists of a dimer of trimers, which makes a characteristic cylinder shape. There is a large closed cavity with approximate dimensions of 16 Å × 16 Å × 20 Å in the center of the hexameric structure. Six putative active sites are positioned along the equatorial surface of the hexamer. There are several highly conserved residues including two possible functional cysteines in the putative active site. The possible molecular function of the protein is discussed.     

The promise of protein glycosylation for personalised medicine

BackgroundComplex diseases such as cancer are a consequence of numerous causes. State of the art personalised medicine approaches are mostly based on evaluating patients' individual genetic background. Despite the advances of genomics it fails to take individual dynamic influences into account that contribute to the individual and unique glycomic and glycoproteomic “configurations” of every living being.Scope of reviewGlycomic and glycoproteomic-based personalised medicine diagnostics are still in their infancies, however some initial success stories indicate that these fields are highly promising to mediate novel early diagnosis and disease stratification markers, subsequently resulting in improved patient well-being and reduced treatment costs. In this review we not only summarise current protein glycosylation based examples that substantially improve or possess great potential for personalised medicine, but also describe current limitations as well as future perspectives and challenges associated with establishing protein glycosylation aspects for this purpose.Major conclusionsMany protein biomarkers currently in clinical use are glycoproteins, however, their glycosylation status is seldom evaluated in a clinical context. To date just few examples have already been successfully translated into clinical practice, making protein glycosylation a highly promising diagnostic target with humongous potential for personalised medicine.General significanceThere is an urgent need for markers that enable the establishment of an individualised and optimised patient treatment at the earliest disease stage possible. The glycosylation status of a patient and/or specific marker proteins can provide important clues that result in improved patient management. This article is part of a Special Issue entitled “Glycans in personalised medicine” Guest Editor: Professor Gordan Lauc.     

Mammalian cell gene expression: protein glycosylation.

Considerable advances have been made in identifying the factors determining the glycosylation pattern of glycoproteins secreted by mammalian cells. This has allowed a greater appreciation of the way in which recombinant proteins may be glycosylated after expression in a heterologous system. The studies reviewed herein extend the wider view that glycosylation of native and recombinant proteins is a complex event dependent on the protein moiety, the host cell, and also the environment in which transfected cells are cultured. The details of the way in which these factors combine to establish the glycosylation pattern of a secreted protein are now beginning to be unravelled.     

The crystal structure of hypothetical protein MTH1491 from Methanobacterium thermoautotrophicum

As part of our structural proteomics initiative, we have determined the crystal structure of MTH1491, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum. MTH1491 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized proteins. It belongs to a family of proteins whose biological function is not known. The crystal structure of MTH1491, the first structure for this family of proteins, consists of an overall five-stranded parallel beta-sheet with strand order 51234 and flanking helices. The oligomeric form of this molecule is a trimer as seen from both crystal contacts and gel filtration studies. Analysis revealed that the structure of MTH1491 is similar to that of dehydrogenases, amidohydrolases, and oxidoreductases. Using a combination of sequence and structural analyses, we showed that MTH1491 does not belong to either the dehydrogenase or the amidohydrolase superfamilies of proteins.     

Thy-1 is a differentiation antigen that characterizes immature murine erythroid and myeloid hematopoietic progenitors

To determine the role of Thy-1 antigen in murine hematopoietic differentiation, bone marrow was treated with anti-Thy-1.2 antibody and complement or complement alone. Growth of immature hematopoietic progenitors, erythroid burst-forming units (BFU-E), and granulocyte/macrophage colony-forming units (CFU-GM) was greatly reduced following antibody and complement treatment and was not restored by mitogen-stimulated spleen cell supernatants. In contrast, more mature erythroid and myeloid progenitors, the erythroid colony-forming unit (CFU-E) and the macrophage progenitor stimulated by L-cell-conditioned media (LCM), were spared by anti-Thy-1.2 antibody and complement treatment. Here, to separate the effects of anti-Thy-1.2 antibody treatment on accessory cells from those on progenitors, splenic T cells and thymocytes were added to treated marrow at ratios of up to 200%. Growth of BFU-E and CFU-GM was not restored. To more precisely replace required accessory cells, male complement-treated marrow was cocultured with female anti-Thy-1.2 antibody and complement-treated marrow. Even marrow cells failed to restore female BFU-E and CFU-GM growth. Fluorescent-activated cell sorting (FACS) and immune sheep red cell rosetting with anti-Thy-1.2-labeled marrow were then performed to determine if immature hematopoietic progenitors bear Thy-1.2. These techniques revealed enrichment of BFU-E and CFU-GM in the Thy-1.2-positive fraction, demonstrating the presence of Thy-1.2 on early murine hematopoietic progenitors. CFU-E and CFU-M were present in the Thy-1.2-negative fraction following FACS separation. These data demonstrate that Thy-1.2 is a differentiation antigen, present on at least some murine BFU-E and CFU-GM and lost as they mature to CFU-E and CFU-M.     

Sweet to the extreme: protein glycosylation in Archaea

Post-translational modifications account for much of the biological diversity generated at the proteome level. Of these, glycosylation is the most prevalent. Long thought to be unique to Eukarya, it is now clear that both Bacteria and Archaea are also capable of N-glycosylation, namely the covalent linkage of oligosaccharides to select target asparagine residues. However, while the eukaryal and bacterial N-glycosylation pathways are relatively well defined, little is known of the parallel process in Archaea. Of late, however, major advances have been made in describing the process of archaeal N-glycosylation. Such efforts have shown, as is often the case in archaeal biology, that protein N-glycosylation in Archaea combines particular aspects of the eukaryal and bacterial pathways along with traits unique to this life form. For instance, while the oligosaccharides of archaeal glycoproteins include nucleotide-activated sugars formed by bacterial pathways, the lipid carrier on which such oligosaccharides are assembled is the same as used in eukaryal N-glycosylation. By contrast, transfer of assembled oligosaccharides to their protein targets shows Archaea-specific properties. Finally, addressing N-glycosylation from an archaeal perspective is providing new general insight into this event, as exemplified by the solution of the first crystal structure of an oligosaccharide transferase from an archaeal source.