"Structural analysis of cytokine signaling modulators" by Cristina Ileana Silvescu

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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