Glycobiology and Extracellular Matrices
The stem region of α1,6-fucosyltransferase FUT8 is required for multimer formation but not catalytic activity
Alpha-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.Glycosyltransferases EXTL2 and EXTL3 cellular balance dictates heparan sulfate biosynthesis and shapes gastric cancer cell motility and invasion
Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells’ glycocalyx and extracellular matrix. By acting as scaffolds for protein–protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell–extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood.Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b
The N-glycans attached to proteins contain various GlcNAc branches, the aberrant formation of which correlates with various diseases. N-Acetylglucosaminyltransferase-IVa (GnT-IVa or MGAT4A) and Gnt-IVb (or MGAT4B) are isoenzymes that catalyze the formation of the β1,4-GlcNAc branch in N-glycans. However, the functional differences between these isozymes remain unresolved. Here, using cellular and UDP-Glo enzyme assays, we discovered that GnT-IVa and GnT-IVb have distinct glycoprotein preferences both in cells and in vitro.Incorporation of fucose into glycans independent of the GDP-fucose transporter SLC35C1 preferentially utilizes salvaged over de novo GDP-fucose
Mutations 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.Fringe GlcNAc-transferases differentially extend O-fucose on endogenous NOTCH1 in mouse activated T cells
NOTCH1 is a transmembrane receptor that initiates a cell–cell signaling pathway controlling various cell fate specifications in metazoans. The addition of O-fucose by protein O-fucosyltransferase 1 (POFUT1) to epidermal growth factor-like (EGF) repeats in the NOTCH1 extracellular domain is essential for NOTCH1 function, and modification of O-fucose with GlcNAc by the Fringe family of glycosyltransferases modulates Notch activity. Prior cell-based studies showed that POFUT1 modifies EGF repeats containing the appropriate consensus sequence at high stoichiometry, while Fringe GlcNAc-transferases (LFNG, MFNG, and RFNG) modify O-fucose on only a subset of NOTCH1 EGF repeats.POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1
Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein–protein interactions with latent transforming growth factor (TGF) β-binding protein 1 (LTBP1), microfibrils regulate TGF-β signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-β signaling.Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation
Glycosylation, 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.Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells
Sialic acid (Sia)-binding immunoglobulin-like lectin 7 (Siglec-7) is an inhibitory receptor primarily expressed on natural killer (NK) cells and monocytes. Siglec-7 is known to negatively regulate the innate immune system through Sia binding to distinguish self and nonself; however, a counter-receptor bearing its natural ligand remains largely unclear. Here, we identified a counter-receptor of Siglec-7 using K562 hematopoietic carcinoma cells presenting cell surface ligands for Siglec-7. We affinity-purified the ligands using Fc-ligated recombinant Siglec-7 and diSia-dextran polymer, a strong inhibitor for Siglec-7.Collagen IVα345 dysfunction in glomerular basement membrane diseases. III. A functional framework for α345 hexamer assembly
We identified a genetic variant, an 8-residue appendage, of the α345 hexamer of collagen IV present in patients with glomerular basement membrane diseases, Goodpasture’s disease and Alport syndrome, and determined the long-awaited crystal structure of the hexamer. We sought to elucidate how variants cause glomerular basement membrane disease by exploring the mechanism of the hexamer assembly. Chloride ions induced in vitro hexamer assembly in a composition-specific manner in the presence of equimolar concentrations of α3, α4, and α5 NC1 monomers.
