Anti-mouse GPI-PLD antisera highlight structural differences between murine and bovine GPI-PLDs.
Despite its well characterised biochemistry, the physiological role of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is unknown. Most of the previous studies investigating the distribution of GPI-PLD have focused on the human and bovine forms of the enzyme. Studies on mouse GPI-PLD are rare, partly due to the lack of a specific anti-mouse GPI-PLD antibody, but also due to the apparent low reactivity of existing antibodies to rodent GPI-PLDs. Here we describe the isolation of a mouse liver cDNA, the construction and expression of a recombinant enzyme and the generation of an affinity-purified rabbit anti-mouse GPI-PLD antiserum. The antibody shows good reactivity to partially purified murine and purified bovine GPI-PLD. In contrast, a rat anti-bovine GPI-PLD antibody shows no reactivity with the mouse enzyme and the two antibodies recognise different proteolytic fragments of the bovine enzyme. Comparison between the rodent, bovine and human enzymes indicates that small changes in the amino acid sequence of a short peptide in the mouse and bovine GPI-PLDs may contribute to the different reactivities of the two antisera. We discuss the implications of these results and stress the importance of antibody selection while investigating GPI-PLD in the mouse.
Gregory
Patrick
P
Institute of Biochemistry and Molecular Biology, University of Bern, Bühlstrasse 28, CH-3012 Bern, Switzerland.
Ziemiecki
Andrew
A
Zürcher
Gisela
G
Brodbeck
Urs
U
Bütikofer
Peter
P
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000138:chemical synthesis; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000737:chemistry / Q000276:immunology; D008099:Liver / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology; D010739:Phospholipase D / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism; D014611:Vaccination
D000906:Antibodies; D018076:DNA, Complementary; D000939:Epitopes; D007106:Immune Sera; D010446:Peptide Fragments; D011994:Recombinant Proteins; D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D014357:Trypsin