Molecular basis of anti-horseradish peroxidase staining in Caenorhabditis elegans.
Cross-reactivity with anti-horseradish peroxidase antiserum is a feature of many glycoproteins from plants and invertebrates; indeed staining with this reagent has been used to track neurons in Drosophila melanogaster and Caenorhabditis elegans. Although in insects the evidence indicates that the cross-reaction results from the presence of core alpha1,3-fucosylated N-glycans, the molecular basis for anti-horseradish peroxidase staining in nematodes has been unresolved to date. By using Western blots of wild-type and mutant C. elegans extracts in conjunction with specific inhibitors, we show that the cross-reaction is due to core alpha1,3-fucosylation. Of the various mutants examined, one with a deletion of the fut-1 (K08F8.3) gene showed no reaction to anti-horseradish peroxidase; the molecular phenotype was rescued by injection of either the K08F8 cosmid or the fut-1 open reading frame under control of the let-858 promoter. Furthermore, expression of fut-1 cDNA in Pichia and insect cells in conjunction with antibody staining, high pressure liquid chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses showed that FUT-1 is a core alpha1,3-fucosyltransferase with an unusual substrate specificity. It is the only core fucosyltransferase in plants and animals described to date that does not require the prior action of N-acetylglucosaminyltransferase I.
Paschinger
Katharina
K
Department für Chemie, Universität für Bodenkultur, A-1190 Wien, Austria.
Rendic
Dubravko
D
Lochnit
Günter
G
Jantsch
Verena
V
Wilson
Iain B H
IB
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000276:immunology*; D029742:Caenorhabditis elegans Proteins / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000276:immunology; D007106:Immune Sera / Q000276:immunology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000276:immunology; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
D029742:Caenorhabditis elegans Proteins; D007106:Immune Sera; D011134:Polysaccharides; D006735:Horseradish Peroxidase; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase