Date of Award

Spring 2006

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Vernon Reinhold

Abstract

The N-linked glycans attached to cytokine receptors modulate signal transduction by interacting with galectin-3 and generating a cell surface lattice. This opposes constitutive endocytosis and reduces the thresholds for cytokine signaling. The preferred ligands for galectin-3 are the poly-N-acetyl lactosaminylated tetra-antennary glycans which are synthesized by Mgat 5 and are present at high levels in tumor cells. A null mutation in Mgat 5 inhibits lattice formation and cancer progression in cells with an oncogenic background. GlcNAc feeding reestablishes lattice formation, cytokine signaling and tumorigenesis.

A MALDI-TOF analysis was employed to identify the N-glycans involved in modulating signal transduction in wild type and null Mgat 5 cells grown with different GlcNAc supplements. Native N-glycan profiling showed alterations in N glycosylation generated by GlcNAc supplements through stimulation of hexosamine pathway. After methylation the tetra-antennary and poly-N-acetyl lactosaminylated glycans were identified only in Mgat 5 wild type cells either supplemented or not with GlcNAc. These structures were absent from the signaling deficient Mgat 5 null cells. Although lattice formation and tumor progression are reestablished after GlcNAc feeding, the preferred galectin-3 ligands were absent from Mgat 5 null cells. Collision Induced Dissociation in a MALDI-IT-TOF instrument was used for N-glycan structural determination. For monitoring the alterations in N-glycosylation, null and wild type Mgat 5 cells were grown in medium supplemented with increasing GlcNAc concentrations. The N-glycans were extracted and subjected to neuraminic acid release to facilitate quantitative estimation. Oligosaccharide profiles for the Mgat 5 wild type cells showed little variation in the high mannose glycan series and a slight increase in the relative abundances of complex type glycans. In the Mgat 5 null cells the increments in GlcNAc concentration determine increasing levels of bi- and triantennary glycans and decreasing levels of high mannose glycans. These suggest that present in a higher number the glycans with lower LacNAc multiplicity are able to establish lattice interactions with adequate avidity to stimulate cytokine signaling and tumor progression in a Mgat 5 null background.

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