The aim of this project is to define new biological functions for specific glycan determinants in mammals. The applicant's strategy is to use glycosylation mutants of CHO cells, embryonic stem (ES) cells and mice to identify glycan functions. In previous studies, the applicant has shown that Mgat1-/- mice lacking complex and hybrid N-glycans die at mid-gestation, though Mgat1-/- blastocysts are rescued by maternal Mgat1 transcripts through implantation. In chimeras, Mgat1-/- embryonic stem (ES) cells develop beyond E9.5 and contribute to essentially all tissues.
In Specific Aim 1, the applicant will use Mgat1-/-, Mgat1+/-, and Mgat1+/+ ES cells to generate chimeras to address two questions: 1) Are complex or hybrid N-glycans required for the development of a functional immune system?; and 2) Are complex or hybrid N-glycans required for early embryogenesis and implantation? If a requirement is demonstrated in either case, the stage at which N-glycans are necessary will be established. The applicant has isolated several gain-of-function Chinese hamster ovary (CHO) glycosylation mutants and shown that all of them express a developmentally regulated glycosyltransferase that is not expressed in wild-type CHO cells. In LEC11B cells Fuc-TVI is up-regulated due to the loss of a trans-acting negative regulatory activity.
In Specific Aim 2, the applicant will isolate this trans-activating activity. She will also determine the mechanisms by which expression of GlcNAc-TIII are up-regulated in certain human cancers. The CHO gain-of-function mutants provide access to novel molecules that control glycosyltransferase expression in mammals. Targeted mutations of glycosyltransferase genes in the mouse have precisely defined many biological functions that are mediated by specific glycan determinants. Glycosyltransferases that transfer a single sugar residue to N-glycans and thereby change their recognition by lectins are likely to mediate functional cell-cell interactions.
In Specific Aim 3, the applicant proposes to identify biological functions for sugar residues that modify the core of N-glycans by characterizing mice that lack one or more of the glycosyltransferases GlcNAc-TIII, Fuc-TVIII, GlcNAc-TVII and/or GlcNAc-TVIII.
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