Glycopeptide-albumin derivative: it preparation and histochemical ligand properties.
Carrier-immobilized mono- or disaccharides and other carbohydrate structures, derived by custom-made chemical synthesis, have already proven to be valuable ligands for localizing carbohydrate-binding proteins in tissue sections. Defined purified glycopeptides, as components of neoglycoproteins, offer the possibility of increasing their structural complexity and, thereby, their receptor selectivity. To test the feasibility of this approach, the glycopeptide man6-glcNAc2-asparagine derived from ovalbumin was purified after pronase digestion. It was coupled to bovine serum albumin as carrier protein with the homobifunctional linking agent bis-(sulphosuccinimidyl)suberate to yield the diglycosylated concanavalin A-reactive product. Following biotinylation, it was used to detect mannose-specific binding sites in fixed cells of seven human leukemia or lymphoma lines and in fixed, paraffin-embedded sections of human breast cancer. In comparison to chemically mannosylated bovine serum albumin with ten sites of glycosylation or to ovalbumin, this derivative produced a similar pattern of reaction with a quantitatively lower extent of staining in most cases. Remarkably, the presence of potential endogenous ligands for the detected receptor sites was ascertained using the plant lectin concanavalin A. Thus, the conjugation of a purified, deliberately selected glycopeptide to a suitable carrier produces a histochemical tool for detecting glycopeptide-specific binding sites.
Gabius
H J
HJ
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, Germany.
Brinck
U
U
Lüsebrink
T
T
Ciesiolka
T
T
Gabius
S
S
D001216:Asparagine / Q000031:analogs & derivatives* / Q000737:chemistry; D001402:B-Lymphocytes / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D001943:Breast Neoplasms / Q000737:chemistry; D002460:Cell Line; D003208:Concanavalin A / Q000737:chemistry; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006651:Histocytochemistry; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D008358:Mannose / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry*; D011402:Pronase / Q000378:metabolism; D012709:Serum Albumin; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry; D013601:T-Lymphocytes / Q000737:chemistry; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D009844:Oligosaccharides; D012709:Serum Albumin; D013388:Succinimides; C023840:mannose-bovine serum albumin conjugate; D003208:Concanavalin A; D012710:Serum Albumin, Bovine; C072827:(mannose)6-(N-acetylglucosamine)2-asparagine; D001710:Biotin; D001216:Asparagine; C035760:bis(sulfosuccinimidyl)suberate; D011402:Pronase; D008358:Mannose