Form Of Protein Glycosylation Research Articles

Updated restraint dictionaries for carbohydrates in the pyranose form.

Restraint dictionaries are used during macromolecular structure refinement to encapsulate intramolecular connectivity and geometric information. These dictionaries allow previously determined `ideal' values of features such as bond lengths, angles and torsions to be used as restraint targets. During refinement, restraints influence the model to adopt a conformation that agrees with prior observation. This is especially important when refining crystal structures of glycosylated proteins, as their resolutions tend to be worse than those of nonglycosylated proteins. Pyranosides, the overwhelming majority component in all forms of protein glycosylation, often display conformational errors in crystal structures. Whilst many of these flaws usually relate to model building, refinement issues may also have their root in suboptimal restraint dictionaries. In order to avoid subsequent misinterpretation and to improve the quality of allpyranose monosaccharide entries in the CCP4 Monomer Library, new dictionaries with improved ring torsion restraints, coordinates reflecting the lowest-energy ring pucker and updated geometry have been produced and evaluated. These new dictionaries are now part of the CCP4 Monomer Library and will be released with CCP4 version 8.0.

Abstract P2-04-03: The roles of fucosylation in promoting tumorigenic signaling in breast cancer

Abstract Fucosylation is an important form of protein glycosylation involved in the progression of cancer. Aberrant fucose and fucosylated serum protein/oligosaccharide levels have been previously described in breast cancer (BC). Fucosylation has been reported impact proliferation and key signaling pathways (e.g., ERK1/2, p38 MAPK, and E-selectin) in human breast invasive ductal carcinoma. However, precise roles and functions of fucosylated proteins function in BC remain largely unclear. In our preliminary analyses, we performed immunofluorescent lectin staining of BC tissue microarrays and cell lines grown in vitro and found that fucosylation is increased in BC vs. normal breast tissues. To study the functional contributions of fucosylation to BC biology, we either pharmacologically or genetically suppressed fucosylation in BC cell lines. The treated or modified cell lines were characterized for their ability to grow/proliferate in 2D or 3D, as well as on adhesion to extracellular matrix (ECM). We observed that inhibiting fucosylation blunted BC cell proliferation, particularly in 3D, and attenuated adhesive capacity. To determine the mechanism(s) underlying the biological functions of fucosylation in BC cells, we performed parallel fucosylated and phosphoproteomic mass spectrometric analyses on BC cells that were either pharmacologically or genetically inhibited for fucosylation to identify: (i) all fucosylated proteins in BC cells and (ii) signaling pathways that comprise directly fucosylated proteins and that are significantly altered in activity (phosphorylation). All filtered hits were subject to Ingenuity Pathway Analysis. Thirteen signaling pathways were identified in common amongst both pharmacologically or genetically modulated BC groups. These pathways clustered into the following 3 major functional groups: cell-cell/cell-matrix contact, RNA processing, and endosomal/phagosomal dynamics. The most fucosylated proteins included integrins. We validated the direct fucosylation of integrins and further dissected the molecular and functional contributions of fucosylation to integrin signaling in vitro and in vivo in mouse models of breast cancer. Our data identify new roles for fucosylation in regulating key signal transduction pathways in BC. How integrin signaling and BC cell biology is regulated by fucosylation, as well as potential clinical utility of fucosylated integrins for BC, will be discussed. Citation Format: Kara Snyder, Hui Ren, Daniel Lester, Bin Fang, John Koomen, Eric Lau. H. The roles of fucosylation in promoting tumorigenic signaling in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-04-03.

The tonoplast--where sweetness is dispensable.

The proteolytic machinery of certain cellular organelles is potentially harmful to resident proteins. In mammals, the large luminal domains of integral lysosomal proteins are heavily glycosylated to protect them from degradation. In this issue of Journal of Experimental Botany (pages 1769–1781) Pedrazzini and co-workers reveal that plants are very different. Glycosylation is one of the most common protein modifications in all eukaryotes. Resident proteins of different intracellular organelles including the endoplasmic reticulum (ER), Golgi apparatus and hydrolysing compartments are often glycosylated at multiple sites with oligosaccharide chains of varying length and composition. The attached carbohydrates modulate protein stability, activity and trafficking and mediate protein–protein interactions. N-glycosylation, the most prevalent form of protein glycosylation in eukaryotes, is defined by the covalent linkage of an oligosaccharide (N-glycan) to selected asparagine residues of newly synthesized secretory proteins as they are translocated into the ER. Once attached to the polypeptide chain, the N-glycans play crucial roles in protein folding and quality control processes (Helenius and Aebi, 2001) – for example, they regulate a specific cell death event in Arabidopsis (de Oliveira et al., 2016). Immediately after the transfer to the protein, N-glycans are subjected to step-wise processing reactions that alter the composition of the N-glycan and by doing so control the fate of glycoproteins. As a consequence, the exposure of a defined glyco-code can promote protein folding or alternatively serve as a signal for the degradation of terminally misfolded or orphan glycoproteins. In principle, these essential N-glycan-dependent processes look highly conserved between animals and plants (Hong et al., 2012; Huttner and Strasser, 2012). By contrast, in the Golgi apparatus where the N-glycans acquire their final composition (referred to as complex N-glycans) N-glycan processing steps differ substantially in eukaryotes. A large variety of structurally and functionally diverse complex oligosaccharides are generated in the Golgi in mammals. In different plant species, on the other hand, only a limited number of complex N-glycan structures have been reported so far (Wilson et al., 2001). An outstanding question is therefore whether the reduced structural diversity of Golgi-processed N-glycans in plants also leads to fundamental differences in the biological role of these protein-linked carbohydrates. While most of the machinery and pathways involved in biosynthesis and processing of N-glycan structures have been identified, the study of the biological role associated with distinct plant N-glycan structures on individual proteins has proved challenging so far (Strasser, 2014). More detailed and comprehensive (glyco)proteomic approaches of different organelles are needed for understanding the structure and function of glycoproteins in a specific cellular environment. Now Pedrazzini et al. (2016) reveal that the Arabidopsis tonoplast is almost completely lacking glycoproteins with complex N-glycans and even proteins with ER-derived N-glycans are highly underrepresented.

Secretome Analysis Identifies Novel Signal Peptide Peptidase-Like 3 (SPPL3) Substrates and Reveals a Role of SPPL3 in Multiple Golgi Glycosylation Pathways*

Signal peptide peptidase-like 3 (Sppl3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, Sppl3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological Sppl3 substrates. Sppl3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are Sppl3 substrates or whether Sppl3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify Sppl3 substrates we used Sppl3-deficient and Sppl3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics "secretome protein enrichment with click sugars (SPECS)" method. SPECS analysis identified numerous additional new Sppl3 candidate glycoprotein substrates, several of which were biochemically validated as Sppl3 substrates. All novel Sppl3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel Sppl3 substrates catalyze the modification of N-linked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that Sppl3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, Sppl3 emerges as a crucial player of Golgi function and the newly identified Sppl3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of Sppl3 in the Golgi network and in vivo. Data are available via ProteomeXchange with identifier PXD001672.

Novel forms of protein glycosylation

A large number of new glycans, derived from glycoproteins, has been characterized in the past few years. O-linked fucose was found in epidermal growth factor-like domains of several proteins. For the N-linked glycans of Helix pomatia hemocyanin, novel types of antennae were identified. The positions of noncarbohydrate substituents were established in N-glycans. C-mannosylation of a tryptophan residue was discovered in human ribonuclease 2 and is the first example of C-glycosylation in glycoproteins.

Purification and Structural Characterization of a Filamentous, Mucin-like Proteophosphoglycan Secreted by Leishmania Parasites

Parasitic protozoa of the genus Leishmania secrete a filamentous macromolecule that forms networks and appears to be associated with cell aggregation. We report here the purification of this parasite antigen from Leishmania major culture supernatant and its compositional (75.6% carbohydrate, 20% phosphate, 4.4% amino acids, w/w), structural, and ultrastructural characterization as a highly unusual proteophosphoglycan (PPG). Mild acid hydrolysis, which cleaves preferentially hexose 1-phosphate bonds, releases the PPG glycans. Their structures are Galbeta1-4Man, Manalpha1-2Man, Galbeta1-3Galbeta1-4Man, PO4-6(Galbeta1-3)0-2Galbeta1-4Man, and PO4-6(Arabeta1-2Galbeta1-3)Galbeta1-4Man. These glycans are also components of the parasite glycolipid lipophosphoglycan, but their relative abundance and structural organization in PPG are different. Some of them represent novel forms of protein glycosylation. 31P NMR on native PPG demonstrates that phosphate is exclusively in phosphodiester bonds and that the basic structure R-Manalpha1-PO4-6-Gal-R connects the glycans. A phosphodiester linkage to phosphoserine (most likely R-Manalpha1-PO4-Ser) anchors the PPG oligosaccharides to the polypeptide. PPG has a unique amino acid composition; glycosylated phosphoserine (>43 mol %), serine, alanine, and proline account for more than 87 mol % and appear to be clustered in large proteinase-resistant domains. Electron microscopy of purified PPG reveals cable-like, flexible, long (to 6 microm), and unbranched filaments. The overall structure of PPG shows many similarities to mammalian mucins. Potential functions of this novel mucin-like molecule for the parasites are discussed.

Novel forms of protein glycosylation

Glycosylation plays several important roles in glycoprotein function. Some of these roles, such as protein folding, protein stability and efficient intracellular transport, are more dependent simply on the presence of a saccharide, rather than on its exact structure. Other functions are directly related to specific saccharide structures. This article focuses on the occurrence and potential functions of such novel and specific glycosylations as O-N-acetylglucosamine, glucose-phosphate-man nose, N-acetylgalactosamine-bearing N-linked oligosaccharides, glucosylasparagine and glycosylations of epidermal growth factor modules.

RNA polymerase II is a glycoprotein. Modification of the COOH-terminal domain by O-GlcNAc

The largest subunit of mammalian RNA polymerase II (RNAP II) contains at its carboxyl terminus an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain, designated the COOH-terminal domain (CTD), is essential for viability and is extensively phosphorylated during the transition from preinitiation complex assembly to elongation (1). Indeed, phosphorylation of the CTD may play an important regulatory role in this transition. We show here that the CTD is also modified by a novel form of protein glycosylation, O-GlcNAc. This modification has been found on numerous transcription factors and other nuclear and cytosolic proteins (2). Glycopeptides obtained by proteolytic digestion of the CTD were purified by reverse-phase high performance liquid chromatography and sequenced. Results from such experiments suggest that glycosylation occurs at multiple sites throughout the CTD, similar to the phosphorylation of this domain. The carbohydrate, however, is not detectable on the phosphorylated form of the enzyme. This observation is consistent with the idea that phosphorylation and glycosylation are mutually exclusive modifications. The CTD of RNAP II, therefore, appears to exist in three distinct conformational states: unmodified, phosphorylated, and glycosylated. The differential modification of the CTD may play an important role in the regulated expression of genes transcribed by RNA polymerase II.

Glycosylation of nuclear and cytoplasmic proteins is ubiquitous and dynamic.

Introduction We have described a major new form of protein glycosylation (termed 0-GlcNAc) that is found in all eukaryotes from yeast to man. 0-GlcNAc is distinct from other types of protein-bound saccharides in several respects (for review see [ l ] ) : (1) 0-ClcNAc is a simple, unmodified monosaccharide moiety glycosidically linked to the side-chain hydroxyls of serine or threonine, often occurring at multiple sites on the same protein. (2) Virtually all 0-GlcNAc is found within the nucleoplasmic and cytoplasmic compartments of cells. (3) 04;lcNAc appears to be highly dynamic and responsive to cellular stimuli in a fashion analogous to phosphorylation. In this short review, we will first outline evidence localizing 0-GlcNAc to the nucleoplasmic and cytoplasmic cellular compartments and discuss proteins that bear 0-GlcNAc. Subsequently, we will illustrate what is known about the sequence localization of 0-GlcNAc attachment sites and the enzymes that specifically attach or remove the saccharide. Finally, we will describe our working hypotheses about the functions of this ubiquitous eukaryotic protein modification.

Nuclear and cytoplasmic glycosylation: novel saccharide linkages in unexpected places.

Abstract In contrast to all other known forms of protein glycosylation, certain recently described protein-saccharide structures are highly localized to the nucleoplasmic and cytoplasmic compartments of the cell. The presence of these novel saccharides on proteins of the nuclear pore, chromatin and cytoskeleton poses fascinating questions concerning their possible functions, modes of biosynthesis, and potential roles in intracellular transport.