Functional Glycomics of Multi-fucosylated Extended Type 1 Chain: Regulated Expression of A Serum Mannose-Binding Protein Ligand in Colonic Adenocarcinomas

多重岩藻醣化之延伸性第一型醣鏈的功能性醣質體學

林祈宏(Chi-Hung Lin)

指導教授 ： 邱繼輝 ； 呂平江

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2008年)

摘要

Galβ1-4GlcNAc, known as LacNAc or type 2 chain, is an essential building block of protein N- and O-glycans as well as lacto-series glycosphinglipids (GSL). Further extension and/or branching of type 2 chain gives the polyLacNAc chains, which are carriers of glycotopes functionally implicated in many cellular communications including metastasis. Type 1 chain, Galβ1-3GlcNAc, on the other hand, is generally identified as terminal epitopes such as Lewis a (Lea) or sialyl Lewis a (sLea) and not further extended. One well-documented exception is the occurrence of multi-fucosylated extended type 1 chains in the form of Lea/b-Lea on GSL of Colo205. These rarely reported extended type 1 chains represent an analytical challenge for their unambiguous identification, as well as a target for functional glycomic studies. Using Colo205 as our experimental model system, concerted mass spectrometry (MS) techniques were first developed to enable facile discrimination of type 1 versus type 2 chain, which led to the identification of extended type 1 chains on N- and O-glycans of Colo205 and trimeric type 1 chain in either linear or branched form on GSL. With these analytical tools in place, the focus of this thesis work was turned to identify biosynthetic basis and functional implications.
A structural informative enzyme activity assay was established to study the kinetics and subcellular localization of endogenous β3GalT. To attribute the observed activity to one of many family members, a workflow of minimum biochemical enrichment steps was developed based on microsome/Golgi membrane preparation and one-step affinity column to allow proteomic identification of β3GalT5 as the major source of β3GalT activity in Colo205, consistent with previous mRNA transcript analysis. This proteomic approach was further applied to identify FucT3 and FucT6 as the fucosyltransferases for Lewis antigen biosynthesis in Colo205.
To ascertain if the expression level of β3GalT5 actually correlates with that of extended type 1 chain, comparative glycomic analysis across colonic cancer cell lines known to express multi-fucosylated extended type 1 chain and/or binding to MBP were undertaken. Both mRNA expression profiling and activity assays demonstrated that the expression levels and activities of β3GalT5 varied greatly in these three cell lines, which correlated positively with the ease in identifying extended type 1 chain on their GSL by glycomic sequencing. Synthesis of extended type 1 chain was shown to be induced by transfection and over-expression of β3GalT5 in DLD-1, which otherwise does not carry this structure on its GSL. These results led to a proposed model that elevated β3GalT5 activity is sufficient to induce biosynthesis of extended type 1 chain on GSLs without necessarily evoking a specific β3GnT.
Extending the comparative glycomic analysis to the glycoproteins, extended type 1 chain was shown to be present on DLD-1 only after β3GalT5 transfection. Sequential enrichment by amine column and mAb affinity would improve the detection limits for low abundant glycan chains but only through a targeted approach using endo-β-galacosidase could extended type 1 chain fragments be released for direct detection. MBP affinity was shown to enrich large complex type N-glycans from Colo205 and DLD3GT5 but not DLD-1, thereby consistent with previous identification of multi-fucosylated extended type 1 chain as MBP ligand. Further glycomic mapping of N-glycans released from MBP bound glycoproteins and glycopeptides suggested that multimeric Lea is required for MBP binding but not necessary in linear form. Based on proteomic analysis, several glycoproteins bearing MBP ligands including CD98, were identified.

關鍵字

醣質體學 ； 質譜 ； 醣蛋白質體學 ； 醣轉移酵素

並列摘要

無資料

並列關鍵字

Glycomics ； mass spectrometry ； glycoproteomics ； glycosyltransferase

參考文獻

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