α‐d‐Galactose‐bearing glycoproteins on the surface of stimulated murine peritoneal macrophages

Abstract

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Gr@~bniu simplicifoliu I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 1251. The labeled glycoproteins migrated as broad bands of molecular mass 92 -109 kDa and 11 5 -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 11 5-kDa glycoproteins had p l 5.2 -5.4 and p l < 4, respectively. Digestion of both glycoproteins with agalactosidase released 23% oftheir 3H content and abolished their ability to bind to the G. simpliciJ?olia I lectin, showing that they contain terminal cc-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 11 5-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 highmannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Duturu strumonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-Pgalactosidase 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 0-glycanase did not alter their mobility in SDSIPAGE, suggesting a lack or low content of 0-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Samhucus nigra and Mauckiu umurensis immobilized lectins, indicating the presence of sialic acid linked x2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and a2,3 to N-acetyllactosamine residues, respectively.

?-d-Galactosylation of surface fucoglycoconjugate(s) upon stimulation/activation of murine peritoneal macrophages

“…Macrophage membrane molecules undergo changes during the process of stimulation/activation [1,2] a process that involves glycoconjugate molecules [3][4][5][6][7][8]. The question arises as to whether these differentiation-related changes in the expression of carbohydrates on the macrophage surface are of biological significance, and whether these carbohydrates play a role in some of the biological functions of these cells, e.g., in cell-cell and cell-milieu interactions.…”