title: alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.
abstract: Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0624.
authors.forename: J
authors.initials: J
authors.lastname: Petryniak
authors.forename: T K
authors.initials: TK
authors.lastname: Huard
authors.forename: I J
authors.initials: IJ
authors.lastname: Goldstein
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000032:analysis*; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006026:Glycoside Hydrolases; D007106:Immune Sera; D007457:Iodine Radioisotopes; D037102:Lectins; D008262:Macrophage Activation; D008264:Macrophages / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009439:Neuraminidase; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007106:Immune Sera; D007457:Iodine Radioisotopes; D037102:Lectins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; D009439:Neuraminidase; D005690:Galactose