Elderberry bark lectin--gold techniques for the detection of Neu5Ac (alpha 2,6) Gal/GalNAc sequences: applications and limitations.
The lectin from the elderberry (Sambucus nigra L.) bark, shown to recognize the sequence neuraminic acid (alpha 2,6) galactose/N-acetylgalactosamine, was applied for detecting binding sites in Lowicryl K4M sections by light and electron microscopy. The lectin was used either directly complexed to colloidal gold or in a two-step cytochemical affinity technique. The lectin-gold complex proved to be superior and thus was extensively tested on rat liver, kidney and hepatoma cells as well as on sheep and bovine submandibular glands. Controls to establish specificity of lectin-gold binding included sugar and glycoprotein inhibition tests and enzymic removal of sialic acid. In agreement with biochemical data demonstrating the potentiating effect of sialic acid on the binding of the lectin to oligosaccharides, enzymic removal of sialic acid from liver sections resulted in abolition of lectin staining. However, in the submandibular glands, neuraminidase pretreatment of the sections had no effect on the subsequent lectin-gold binding. In rat kidney some structures became negative while others retained the lectin-gold staining due to binding to penultimate N-acetylgalactosamine exposed after sialic acid removal. In line with this, spot blot analysis demonstrated that the lectin-gold complex reacted with both fetuin and asialofetuin. Taken together, these results suggest that, for cytochemical staining, the sialic acid and the galactose/N-acetylgalactosamine lectin combining subsites of Sambucus nigra L. lectin are equally reactive with cellular glycoconjugates and that neuraminidase predigestion of tissue sections is of utmost importance to ensure specificity of staining for the sequence neuraminic acid (alpha 2,6) galactose/N-acetylgalactosamine.
Taatjes
D J
DJ
Interdepartmental Electron Microscopy, Biocenter, University of Basle, Switzerland.
Roth
J
J
Peumans
W
W
Goldstein
I J
IJ
D000116:Acetylgalactosamine / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004052:Diethylnitrosamine / Q000494:pharmacology; D005688:Galactosamine / Q000031:analogs & derivatives*; D005690:Galactose / Q000032:analysis* / Q000378:metabolism; D006046:Gold; D006651:Histocytochemistry / Q000379:methods*; D037102:Lectins; D008114:Liver Neoplasms, Experimental / Q000032:analysis / Q000139:chemically induced; D008854:Microscopy, Electron / Q000379:methods; D009438:Neuraminic Acids / Q000032:analysis* / Q000378:metabolism; D037121:Plant Lectins; D051381:Rats; D054788:Ribosome Inactivating Proteins; D012756:Sheep; D013194:Staining and Labeling / Q000379:methods*; D013363:Submandibular Gland / Q000032:analysis; D000509:alpha-Fetoproteins
D037102:Lectins; D009438:Neuraminic Acids; D037121:Plant Lectins; C050600:Sambucus nigra lectins; D000509:alpha-Fetoproteins; D004052:Diethylnitrosamine; D006046:Gold; D005688:Galactosamine; D054788:Ribosome Inactivating Proteins; D000116:Acetylgalactosamine; D005690:Galactose