n-acetylglucosamine-6-sulfate and galactose-6-sulfate

Cystic fibrosis (CF) is the most lethal genetic disorder in Caucasians and is characterized by the production of excessive amounts of viscous mucus secretions in the airways of patients, leading to airway obstruction, chronic bacterial infections, and respiratory failure. Previous studies indicate that CF-derived airway mucins are glycosylated and sulfated differently compared with mucins from nondiseased (ND) individuals. To address unresolved questions about mucin glycosylation and sulfation, we examined O-glycan structures in mucins purified from mucus secretions of two CF donors versus two ND donors. All mucins contained galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac). However, CF mucins had higher sugar content and more O-glycans compared with ND mucins. Both ND and CF mucins contained GlcNAc-6-sulfate (GlcNAc-6-Sul), Gal-6-Sul, and Gal-3-Sul, but CF mucins had higher amounts of the 6-sulfated species. O-glycans were released from CF and ND mucins and derivatized with 2-aminobenzamide (2-AB), separated by ion exchange chromatography, and quantified by fluorescence. There was nearly a two-fold increase in sulfation and sialylation in CF compared with ND mucin. High performance liquid chromatography (HPLC) profiles of glycans showed differences between the two CF samples compared with the two ND samples. Glycan compositions were defined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Unexpectedly, 260 compositional types of O-glycans were identified, and CF mucins contained a higher proportion of sialylated and sulfated O-glycans compared with ND mucins. These profound structural differences in mucin glycosylation in CF patients may contribute to inflammatory responses and increased pathogenesis by Pseudomonas aeruginosa.

Topics: Acetylgalactosamine; Acetylglucosamine; Carbohydrate Sequence; Case-Control Studies; Chromatography, High Pressure Liquid; Cystic Fibrosis; Fucose; Galactose; Gas Chromatography-Mass Spectrometry; Glycosylation; Humans; Molecular Sequence Data; Mucins; N-Acetylneuraminic Acid; Oligosaccharides; Respiratory Mucosa; Spectrometry, Mass, Fast Atom Bombardment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfates; Sulfuric Acid Esters

L-Selectin, a receptor bearing a C-type lectin domain, mediates the initial attachment of lymphocytes to high endothelial venules of lymph nodes. One of the endothelial-derived ligands for L-selectin is GlyCAM-1 (previously known as Sgp50), a mucin-like glycoprotein with sulfated, sialylated, and fucosylated O-linked oligosaccharide chains. Sialylation, sulfation, and fucosylation appear to be required for the avid interaction of this ligand with L-selectin, but the exact carbohydrate structures involved in recognition remain undefined. In this study, we examine the nature of the sulfate-modified carbohydrates of GlyCAM-1. GlyCAM-1 was metabolically labeled in lymph node organ culture with 35SO4 and a panel of tritiated carbohydrate precursors. Mild hydrolysis conditions were established that released sulfated oligosaccharides without cleavage of sulfate esters. Low molecular weight and singly charged fragments, obtained by a combination of gel filtration and anion-exchange chromatography, were analyzed. The structural identification of the fragments relied on the use of a variety of radiolabeled sugar precursors, further chemical and enzymatic hydrolysis, and high-pH anion-exchange chromatography analysis. Sulfated constituents of GlyCAM-1 were identified as Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. In the accompanying paper [Hemmerich, S., & Rosen, S.D. (1994) Biochemistry 33, 4830-4835] evidence is presented that (SO4-6)Gal beta 1-->4GlcNAc forms the core of a sulfated sialyl Lewis x structure that may comprise a recognition determinant on GlyCAM-1.

Topics: Acetylglucosamine; Amino Acid Sequence; Animals; Carbohydrate Sequence; Cell Adhesion Molecules; Chromatography, Liquid; Galactose; Hydrolysis; L-Selectin; Lymph Nodes; Mice; Mice, Inbred ICR; Molecular Sequence Data; Mucins