Identification of Aberrant Type I Chain Elongation and Branching on Lactotetrasylceramides from Colon Carcinoma by Mass Spectrometry Analysis
Date Issued
2006
Date
2006
Author(s)
Fan, Yao-Yun
DOI
en-US
Abstract
Tumor associated antigens arising from aberrant glycosylation is a well recognized phenomenon in cancer biology. Among the more notable structural features is the rare occurrence of extended type 1 chain on the lactosylceramides of the human colonic adenocarcinoma cell line, Colo205, concomitant with multiple fucosylation to give multimeric Lewisb/a terminal epitopes. While a dimeric Leb/a-Lea determinant has been well characterized previously, a trimeric stretch of type 1 units, -3Galb1-3GlcNAcb1-, and its fucosylated variants have not been unequivocally demonstrated. In a broader context of developing facile mass spectrometry (MS) methodologies for the sequencing of linear and branched poly-N-acetyl-lactosamine (polyLacNAc), this thesis demonstrates that the type 1 unit can be not only linearly extended to three or more tandem repeats, but also branched at C6 of 3-linked Gal in a manner similar to its type 2 polyLacNAc counterparts. A combination of chemical and enzymatic approaches, HPLC purification, and both low and high energy CID tandem mass spectrometry (MS/MS) analyses showed that the glycan chains carried on the lactosylceramides of Colo205 comprise a complex mixture of type 1 and 2 hybrids as well as those exclusively of extended type 1 chain. Complementary fragmentation characteristics were established to allow discrimination of linkage specific branching versus linear extension.
Identification of these novel structures further prompted an investigation into the candidate glycosyltransferases that may be responsible for mediating their aberrant synthesis. Based on in vitro enzymatic synthesis systems, linear extended type 1 and 2 chains and their hybrids were first synthesized which serve as authentic standards to validate MS/MS fragmentation pattern established; as well as substrates for further enzymatic (a-fucosylation and to assay for the specific activities of various I branching enzyme, (b6-N-acetyl-glucosaminyltransferases (IGnT or(b6GnT). The former allows a reconstitution of authentic oligomeric Lewis X and A antigens to be used in conjunction with other analytical methods to define the specificity of a monoclonal antibody which apparently recognizes only the GSLs from Colo205 of a size larger than those carrying a dimeric Lewis A epitopes. Variably fucosylated hybrid type 1 and 2 chains with at least an internal Lewis X epitope were established as being the most likely carrier of the epitope recognized. The latter led to a successful demonstration that extended type 1 and the hybrid chains can be branched in a manner similar to type 2 polyLacNAc chains by all three available human IGnTs albeit at a lower reactivity. IGnT2 is the only transcript expressed at any significant level in Colo205 and the most active in branching an extended type 1 chain with the branched Gal position critically established by further MS/MS analysis of the synthesized products.
In short, this work has unambiguously identified the presence of linear and branched extended type 1 chains on the GSLs of a colonic adenocarcinoma cell line based on advanced MS/MS analysis. With the following up in vitro enzymatic synthesis studies coupled with further MS/MS analysis of the products made, the implicated glycosyltransferases were shown to be capable of making the novel tumor-associated structures and the derived synthetic glycan epitopes were further used successfully to define the specificity of a monoclonal antibody raised against the tumor-associated antigens on GSLs, as well as to validate the MS/MS methodologies developed.
Subjects
延伸性和分支狀第一型醣鍊
不正常醣化結構
Tumor associated antigens
human colonic adenocarcinoma
branched / extended type 1 chain
SDGs
Type
other
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