Phosphatidylglucoside exists as a single molecular species with saturated fatty acyl chains in developing astroglial membranes.
We previously found that phosphatidylglucoside (PtdGlc), a novel glycolipid expressed in HL60 cells, plays a role in forming signaling microdomains involved in cellular differentiation. Because cells contain minute levels of PtdGlc, pure PtdGlc is very difficult to isolate. Thus, its complete structure has never been assessed. To aid in analyzing PtdGlc, we generated a PtdGlc-specific monoclonal antibody, DIM21, by immunizing mice with detergent-insoluble membranes isolated from HL60 cells [Yamazaki, Y., et al. (2006) J. Immunol. Methods 311, 106-116]. DIM21 immunostaining of murine CNS tissues revealed stage- and cell type-specific localization of the DIM21 antigen during development, with especially high levels of expression in radial glia/astroglia. DIM21 immunostained cultured hippocampal astroglia in a punctate fashion. To characterize the structure of PtdGlc, we isolated DIM21 antigen from fetal brains. Using successive column chromatography, we purified two previously unrecognized glycolipids, PGX-1 and PGX-2, from embryonic day 21 rat brains. DIM21 reacted more strongly to PGX-2 than to PGX-1. Structural analyses with 600 MHz (1)H NMR, FT-ICR mass spectrometry, and GC revealed that PGX-1 is phosphatidyl beta-d-(6-O-acetyl)glucopyranoside and PGX-2 is phosphatidyl beta-d-glucopyranoside. The yields of PGX-1 and PGX-2 were approximately 250 +/- 150 and 440 +/- 270 nmol/g of dried brains, respectively. Surprisingly, both glycolipids were composed exclusively of C18:0 at the C1 position and C20:0 at the C2 position of the glycerol backbone. This saturated fatty acyl chain composition comprising a single molecular species rarely occurs in known mammalian lipids and provides a molecular basis for why PtdGlc resides in raftlike lipid microdomains.
Nagatsuka
Yasuko
Y
Hirabayashi Research Unit, Neuronal Circuit Mechanisms Research Group, and Laboratory for Neuronal Architecture, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.
Horibata
Yasuhiro
Y
Yamazaki
Yasuhiro
Y
Kinoshita
Masami
M
Shinoda
Yoko
Y
Hashikawa
Tsutomu
T
Koshino
Hiroyuki
H
Nakamura
Takemichi
T
Hirabayashi
Yoshio
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001253:Astrocytes / Q000737:chemistry*; D001921:Brain / Q000196:embryology; D005260:Female; D020404:Glycerophospholipids; D013058:Mass Spectrometry; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D011247:Pregnancy; D051381:Rats
D000911:Antibodies, Monoclonal; D020404:Glycerophospholipids; C429823:phosphatidylglucose