Phosphatidylglucoside Exists as a Single Molecular Species with Saturated Fatty Acyl Chains in Developing Astroglial Membranes

Abstract

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.

New insights on glucosylated lipids: Metabolism and functions

“…Acetylated PtdGlc (1-stearyl-2-arachidoyl-sn-glycerol-3-phosphoryl β-D-(6-O-acetyl)glucopyranoside) also exists in fetal rat brain [134] (Fig. 4).…”

Glycerophospholipid regulation of modality-specific sensory axon guidance in the spinal cord

“…Phosphatidyl-b-D-glucoside {PtdGlc [2-O-arachidoyl-1-O-(1-stearoyl)-sn-glycer-3-yl]-b-D-glucopyranosyl phosphate; fig. S1A} is a membrane glycerophospholipid (3) that is localized to radial glia and nascent astrocytes (4,5). We showed that PtdGlc and its hydrolytic derivative lyso-phosphatidyl-b-D-glucoside (LysoPtdGlc; fig.…”

The Novel Neutrophil Differentiation Marker Phosphatidylglucoside Mediates Neutrophil Apoptosis

“…Mouse anti-PtdGlc IgM DIM21 and mouse anti-LacCer IgM T5A7 were prepared as described (5,17,19). Mouse anti-human Fas (CD95) IgM CH-11 and IgG ZB4 were obtained from Medical and Biological Laboratories (Nagoya, Japan).…”

“…Reduction of endogenous cholesterol with methyl-b-cyclodextrin suppresses anti-PtdGlc Ab-stimulated tyrosine phosphorylation, resulting in the up-and downregulation of CD38 and c-Myc expression, respectively (16). Therefore, PtdGlc may form functional raft-like domains on the plasma membranes of HL-60 cells, although it does not have C24 fatty acid chains (17). PtdGlc forms distinct lipid domains from sphingolipids on the outer leaflet of the plasma membrane of HL-60 cells (18).…”