Glycobiology and Extracellular Matrices
The stem region of α1,6-fucosyltransferase FUT8 is required for multimer formation but not catalytic activityAlpha-1,6-fucosyltransferase (FUT8) synthesizes core fucose in N-glycans, which plays critical roles in various physiological processes. FUT8, as with many other glycosyltransferases, is a type-II membrane protein, and its large C-terminal catalytic domain is linked to the FUT8 stem region, which comprises two α-helices. Although the stem regions of several glycosyltransferases are involved in the regulation of Golgi localization, the functions of the FUT8 stem region have not been clarified as yet.
N-glycosylation of mannose receptor (CD206) regulates glycan binding by C-type lectin domainsThe macrophage mannose receptor (MR, CD206) is a transmembrane endocytic lectin receptor, expressed in selected immune and endothelial cells, and is involved in immunity and maintaining homeostasis. Eight of the ten extracellular domains of the MR are C-type lectin domains (CTLDs) which mediate the binding of mannose, fucose, and GlcNAc in a calcium-dependent manner. Previous studies indicated that self-glycosylation of MR regulates its glycan binding. To further explore this structure–function relationship, we studied herein a recombinant version of mouse MR CTLD4-7 fused to human Fc-portion of IgG (MR-Fc).
Incorporation of fucose into glycans independent of the GDP-fucose transporter SLC35C1 preferentially utilizes salvaged over de novo GDP-fucoseMutations in the SLC35C1 gene encoding the Golgi GDP-fucose transporter are known to cause leukocyte adhesion deficiency II. However, improvement of fucosylation in leukocyte adhesion deficiency II patients treated with exogenous fucose suggests the existence of an SLC35C1-independent route of GDP-fucose transport, which remains a mystery. To investigate this phenomenon, we developed and characterized a human cell–based model deficient in SLC35C1 activity. The resulting cells were cultured in the presence/absence of exogenous fucose and mannose, followed by examination of fucosylation potential and nucleotide sugar levels.
Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylationGlycosylation, the most common posttranslational modification of proteins, is a stepwise process that relies on tight regulation of subcellular glycosyltransferase location to control the addition of each monosaccharide. Glycosyltransferases primarily reside and function in the endoplasmic reticulum (ER) and the Golgi apparatus; whether and how they traffic beyond the Golgi, how this trafficking is controlled, and how it impacts glycosylation remain unclear. Our previous work identified a connection between N-glycosylation and Rab11, a key player in the post-Golgi transport that connects recycling endosomes and other compartments.
N-acetylglucosamine drives myelination by triggering oligodendrocyte precursor cell differentiationMyelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis.
Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferasesMammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality.
Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferaseOligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the WT OST to support growth.
Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partnersS-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L.
Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machineryProtein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called “targeting by timing.” Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis.
N-Glycans on EGF domain-specific O-GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localizationEpidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions.
The SH3 domain in the fucosyltransferase FUT8 controls FUT8 activity and localization and is essential for core fucosylationCore fucose is an N-glycan structure synthesized by α1,6-fucosyltransferase 8 (FUT8) localized to the Golgi apparatus and critically regulates the functions of various glycoproteins. However, how FUT8 activity is regulated in cells remains largely unclear. At the luminal side and uncommon for Golgi proteins, FUT8 has an Src homology 3 (SH3) domain, which is usually found in cytosolic signal transduction molecules and generally mediates protein-protein interactions in the cytosol. However, the SH3 domain has not been identified in other glycosyltransferases, suggesting that FUT8's functions are selectively regulated by this domain.
Structural basis of substrate recognition and catalysis by fucosyltransferase 8Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown.
Steric constraints control processing of glycosylphosphatidylinositol anchors in Trypanosoma bruceiThe transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site–associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by the addition of 4–6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown transport-dependent increases in E6 size consistent with post-glycan processing in the endoplasmic reticulum.
Glycosylation at an evolutionary nexus: the brittle star Ophiactis savignyi expresses both vertebrate and invertebrate N-glycomic featuresEchinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea.
Sulfated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogenyAmong the earliest deuterostomes, the echinoderms are an evolutionary important group of ancient marine animals. Within this phylum, the holothuroids (sea cucumbers) are known to produce a wide range of glycoconjugate biopolymers with apparent benefits to health; therefore, they are of economic and culinary interest throughout the world. Other than their highly modified glycosaminoglycans (e.g. fucosylated chondroitin sulfate and fucoidan), nothing is known about their protein-linked glycosylation.
N-Glycosylation regulates ligand-dependent activation and signaling of vascular endothelial growth factor receptor 2 (VEGFR2)The tumor microenvironment and proinflammatory signals significantly alter glycosylation of cell-surface proteins on endothelial cells. By altering the N-glycosylation machinery in the endoplasmic reticulum and Golgi, proinflammatory cytokines promote the modification of endothelial glycoproteins such as vascular endothelial growth factor receptor 2 (VEGFR2) with sialic acid–capped N-glycans. VEGFR2 is a highly N-glycosylated receptor tyrosine kinase involved in pro-angiogenic signaling in physiological and pathological contexts, including cancer.
Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2The type-I LacdiNAc (LDN; GalNAcβ1–3GlcNAc) has rarely been observed in mammalian cells except in the O-glycan of α-dystroglycan, in contrast to type-II LDN structures (GalNAcβ1–4GlcNAc) in N- and O-glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. Although a β1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2; type-I LDN synthase) has been cloned, the function of type-I LDN in mammalian cells is still unclear, as its carrier protein(s) has not been identified.
The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferasesThe chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis.
Comprehensive analysis of N-glycans in IgG purified from ferrets with or without influenza A virus infectionInfluenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, N-glycosylation of IgGs plays a critical role in antigen binding and effector functions. The ferret is the most commonly used animal model for studying influenza pathogenesis, virus transmission, and vaccine development, but its IgG structure and functions remain largely undefined.
CD16a with oligomannose-type N-glycans is the only “low-affinity” Fc γ receptor that binds the IgG crystallizable fragment with high affinity in vitroFc γ receptors (FcγRs) bind circulating IgG (IgG1) at the surface of leukocytes. Antibodies clustered at the surface of a targeted particle trigger a protective immune response through activating FcγRs. Three recent reports indicate that the composition of the asparagine-linked carbohydrate chains (N-glycans) of FcγRIIIa/CD16a impacted IgG1-binding affinity. Here we determined how N-glycan composition affected the affinity of the “low-affinity” FcγRs for six homogeneous IgG1 Fc N-glycoforms (G0, G0F, G2, G2F, A2G2, and A2G2F).
Metabolic labeling of HIV-1 envelope glycoprotein gp120 to elucidate the effect of gp120 glycosylation on antigen uptakeThe glycan shield on the envelope glycoprotein gp120 of HIV-1 has drawn immense attention as a vulnerable site for broadly neutralizing antibodies and for its significant impact on host adaptive immune response to HIV-1. Glycosylation sites and glycan composition/structure at each site on gp120 along with the interactions of gp120 glycan shield with broadly neutralizing antibodies have been extensively studied. However, a method for directly and selectively tracking gp120 glycans has been lacking.
Identification of multiple isomeric core chitobiose–modified high-mannose and paucimannose N-glycans in the planarian Schmidtea mediterraneaCell surface–associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type N-glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of N-linked oligosaccharides from the planarian Schmidtea mediterranea revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated (n = 3–5) core fucose structures; the latter structures have not been reported in other systems.
Single-subunit oligosaccharyltransferases of Trypanosoma brucei display different and predictable peptide acceptor specificitiesTrypanosoma brucei causes African trypanosomiasis and contains three full-length oligosaccharyltransferase (OST) genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. These OSTs have different peptide acceptor and lipid-linked oligosaccharide donor specificities, and trypanosomes do not follow many of the canonical rules developed for other eukaryotic N-glycosylation pathways, raising questions as to the basic architecture and detailed function of trypanosome OSTs.
Analysis of substrate specificity of Trypanosoma brucei oligosaccharyltransferases (OSTs) by functional expression of domain-swapped chimeras in yeastN-Linked protein glycosylation is an essential and highly conserved post-translational modification in eukaryotes. The transfer of a glycan from a lipid-linked oligosaccharide (LLO) donor to the asparagine residue of a nascent polypeptide chain is catalyzed by an oligosaccharyltransferase (OST) in the lumen of the endoplasmic reticulum (ER). Trypanosoma brucei encodes three paralogue single-protein OSTs called TbSTT3A, TbSTT3B, and TbSTT3C that can functionally complement the Saccharomyces cerevisiae OST, making it an ideal experimental system to study the fundamental properties of OST activity.
The glycosyltransferase GnT-III activates Notch signaling and drives stem cell expansion to promote the growth and invasion of ovarian cancerGlycosylation changes associated with cellular transformation can facilitate the growth and progression of tumors. Previously we discovered that the gene Mgat3 encoding the glycosyltransferase GnT-III is elevated in epithelial ovarian carcinomas (EOCs) and leads to the production of abnormal truncated N-linked glycan structures instead of the typical bisected forms. In this study, we are interested in discovering how these abnormal glycans impact the growth and progression of ovarian cancer. We have discovered using stable shRNA gene suppression that GnT-III expression controls the expansion of side-population cells, also known as cancer stem cells.