A beta-N-acetylglucosaminyl phosphate diester residue is attached to the glycosylphosphatidylinositol anchor of human placental alkaline phosphatase: a target of the channel-forming toxin aerolysin.
Glycosylphosphatidylinositol (GPI)-anchored proteins are ubiquitous in eukaryotes. The minimum conserved GPI core structure of all GPI-anchored glycans has been determined as EtN-PO4-6Manalpha1-2Manalpha1-6Manalpha1-4GlcN-myo-inositol-PO3H. Human placental alkaline phosphatase (AP) has been reported to be a GPI-anchored membrane protein. AP carries one N-glycan, (NeuAcalpha2-->3)2Gal2GlcNAc2Man3GlcNAc(+/-Fuc)GlcNAc, and a GPI anchor, which contains an ethanolamine phosphate diester group, as a side chain. However, we found that both sialidase-treated soluble AP (sAP) and its GPI-anchored glycan bound to a Psathyrella velutina lectin (PVL)-Sepharose column, which binds beta-GlcNAc residues. PVL binding of asialo-sAP and its GPI-anchored glycan was diminished by digestion with diplococcal beta-N-acetylhexosaminidase or by mild acid treatment. After sequential digestion of asialo-sAP with beta-N-acetylhexosaminidase and acid phosphatase, the elution patterns on chromatofocusing gels were changed in accordance with the negative charges of phosphate residues. Trypsin-digested sAP was analyzed by liquid chromatography/electrospray ionization mass spectrometry, and the structures of two glycopeptides with GPI-anchored glycans were confirmed as peptide-EtN-PO4-6Manalpha1-->2(GlcNAcbeta1-PO4-->6)Manalpha1-6(+/-EtN-PO4-->)Manalpha1-->4GlcN, which may be produced by endo-alpha-glucosaminidase. In addition to AP, GPI-anchored carcinoembryonic antigen, cholinesterase, and Tamm-Horsfall glycoprotein also bound to a PVL-Sepharose column, suggesting that the beta-N-acetylglucosaminyl phosphate diester residue is widely distributed in human GPI-anchored glycans. Furthermore, we found that the beta-N-acetylglucosaminyl phosphate diester residue is important for GPI anchor recognition of aerolysin, a channel-forming toxin derived from Aeromonas hydrophila.
Fukushima
Keiko
K
Department of Biochemistry, Sasaki Institute, 2-2 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Ikehara
Yukio
Y
Kanai
Michiko
M
Kochibe
Naohisa
N
Kuroki
Masahide
M
Yamashita
Katsuko
K
D000333:Aeromonas / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry*; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry*; D002240:Carbohydrate Sequence; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004952:Esters; D004983:Ethanolamines / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006596:Hexosaminidases / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry; D009439:Neuraminidase / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010920:Placenta / Q000201:enzymology*; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D012685:Sepharose / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D001427:Bacterial Toxins; D004952:Esters; D004983:Ethanolamines; D017261:Glycosylphosphatidylinositols; D009005:Monosaccharides; D010455:Peptides; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin; D012685:Sepharose; D000469:Alkaline Phosphatase; D006596:Hexosaminidases; D009439:Neuraminidase; D014357:Trypsin; D010100:Oxygen