Structural and Functional Glycosphingolipidomics by Glycoblotting with an Aminooxy-Functionalized Gold Nanoparticle

Abstract

The first paragraph (144 words).Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. Standardized method allowing for high throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian genomics and proteomics. Here we communicate such an approach to a novel "omics", namely glycosphingolipidomics based on the glycoblotting method.The method, which involves selective ozonolysis of the C-C double bond in ceramide moiety and subsequent enrichment of generated GSL-aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for structural identification and functional characterization of whole GSLs present in the living cells/tissues. 3 Main text (1424 words).Cell surface GSLs exhibit various and crucial functions in cell growth, differentiation, adhesion, and malignant alteration 1 . It has been documented that dramatic changes in GSL composition and metabolism are strongly associated with oncogenic transformation 2,3 . Therefore, identification of disease-related structural alteration of cell surface GSLs will become a key to develop diagnostic biomarkers and therapeutic anticancer vaccines. However, purification and structural characterization of cellular GSLs have not been routinely possible to date, typically requiring tedious and time-consuming extraction/purification steps of GSLs of interest from extremely complex mixtures before analysis. In general, protocols for the isolation of GSLs would vary depending upon the analytical methods as well as chemical properties of individual GSLs and would need specialized expertise at step-by-step/case-by-case handling for separation. These crucial problems in the enrichment of whole GSLs make it difficult to achieve high throughput structural and functional analyses of biologically importantGSLs. In addition, we should pay attention to the fact that cell surface GSLs often exhibit specific biological functions through dynamic mechanism such as clustering or To illustrate the new method, crude lipids fraction 24 of C57BL/6JJcl adult (7 weeks) and embryonic (13.5 days) mouse brains were subjected directly to "ozonolysis" and "glycoblotting" protocol. Reaction of GSL-aldehydes with aoGNPs proceeded smoothly under mild condition without any special reagent. After simple purificationGSLs transferred onto aoGNPs (GSLs-GNPs) were employed directly for further structural characterization. The merit of this method is evident because whole procedure from GSLs enrichment to mass spectrometry-based structural profiling can be performed within 3~4 hours by employing approximately 100 mg (wet w...

Synthesis and Cytotoxicity of Ring C‐Functionalized Derivatives of the Marine Natural Product (–)‐Dibromophakellstatin

“…[16] Azide 30 was purified by filtration through a pad of silica gel [CHCl 3 /MeOH (9:1) + 1 % aqueous ammonia] but was unstable and used immediately in the subsequent click reaction. As an alternative coupling partner for the terminal alkyne 23, aminoazide 32 was prepared starting from hexaethylene glycol (31) in three steps by double tosylation, conversion to the diazide [17] and mono-Staudinger reduction (Scheme 5b).…”

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

“…A high-throughput analysis of GSLs was conducted using oligosaccharides obtained by the ozonolysis of GSLs ( 44 ). However, the chemical method resulted in a low yield of GSL-oligosaccharides ( 6 ).…”

Fluorescence polarization-based assay using N-glycan-conjugated quantum dots for screening in hemagglutinin blockers for influenza A viruses

“…Egg yolk sialylated glycopeptide (A2 glycopeptide) was purchased from TCI (Tokyo, Japan). 11-Mercaptoundecylphosphorylcholine (PC-SH) and 11,11'-dithio bis [undec-11-yl 12-(aminooxyacetyl) amino hexa (ethyleneglycol) ] (ao-SH) were synthesized by the procedures reported previously (Holmlin RE et al, 2001;Nagahori et al, 2009;Ohyanagi et al, 2011). The ao-PC-QDs were prepared from the above Qdot 545 ITK, trioctylphosphine oxide (TOPO)-coated QDs (100 L of 1 M solution, 5.8 nm, CdSe/ZnS, λ em = 545 nm), by a general protocol for coating with ao-SH (50%) and PC-SH (50%).…”

“…Sialylated biantennary N-glycan (A2) prepared by treating A2 glycopeptide with PNGase F (Roche Diagnostics Japan, Tokyo, Japan) was subjected to coupling with ao-PC-QDs by the "glycoblotting" (Nishimura et al, 2005;Ohyanagi et al, 2011) reaction and gave PC-QDs displaying A2 glycans (A2-PC-QDs, Figure 1). Characterization of A2-PC-QDs was performed by 5 direct MALDI-TOFMS (Nagahori et al, 2009;Ohyanagi et al, 2011) and the results showed the advent of an expected molecular ion signal at m/z 3155.7 corresponding to the heterodisulfide A2-S-S-PC instead of disappearance of the signal at m/z 908.7 due to ao-S-S-PC corresponding to free aminooxy-functional groups. As standards or simple model compounds in the competitive binding assay, 2-aminobenzamide (2AB) labeled A2 (2AB-A2, 1) (Dell et al, 2008), and two known sialosides, methyl 5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosonic acid (2) (Sauter et al, 1992) and octyl 5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosonic acid (3) (Crich and Li, 2007), were also used.…”