Evaluation of laser microdissection as a tool in cancer glycomic studies

Laser microdissection (LMD) is a recent development that enables the isolation of specific cell populations from tissue sections. This study focuses on the potential of LMD as a tool in cancer glycomics using colon cancer as a model. LMD was performed on hematoxylin and eosin stained frozen tissue sections. Tumor cells and normal epithelial cells were selectively microdissected. N-Glycans from the LMD- and the bulk tissue-derived samples were liberated by hydrazinolysis and then labeled with 2-aminopyridine. After sialidase digestion, the resulting asialo-N-glycans were analyzed by normal and reversed phase HPLC combined with mass spectrometry. Comparison of the various N-glycan profiles with the aid of LMD identified seven characteristic N-glycans with significantly different expression profiles between normal and cancerous cells that could not be detected by conventional analysis. Thus, LMD is a potent and useful tool for analyzing variations in the expression of N-glycans by overcoming the problem of tissue sample heterogeneity.     

Novel fucogangliosides found in human colon adenocarcinoma tissues by means of glycomic analysis

The structures of acidic glycosphingolipids in colon adenocarcinoma have been analyzed extensively using a number of conventional methods, such as thin-layer chromatography and methylation analysis, and a variety of acidic glycosphingolipids present in the tissues have been reported. However, because of a number of limitations in the techniques used in previous studies in terms of resolution, quantification, and sensitivity, we employed a different method that could be applied to small amounts of tissue. In this technique, the carbohydrate moieties of acidic glycosphingolipids from approximately 20mg of colon adenocarcinoma were released by endoglycoceramidase II and were labeled by pyridylamination. They were separated and structurally characterized by a two-dimensional HPLC mapping technique, electrospray ionization tandem mass spectrometry (ESI-MS/MS), and enzymatic cleavage. A total of 22 major acidic glycosphingolipid structures were identified, and their relative quantities were revealed in detail. They are composed of 1 sulfated (SM3), 1 lacto-series (SLe(a)), 6 kinds of ganglio-series, and 14 kinds of neolacto-series glycosphingolipids. They include most of the acidic glycosphingolipids previously reported to be present in the tissues and two previously unknown fucogangliosides sharing the same terminal structure: NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc, and NeuAcalpha2-6(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3-Galbeta1-4Glc. Thus, this highly sensitive, high-resolution analysis enabled the identification of novel structures of acidic glycosphingolipids from small amounts of already comprehensively studied cancerous tissues. This method is a powerful tool for microanalysis of glycosphingolipid structures from small quantities of cancerous tissues and should be applicable to different types of malignant tissues.     

Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions.