SPECIFIC AIMS of current NIH RO1 grant (2001-2005)

A. SPECIFIC AIMS

The long-term objective of this program is to understand how carbohydrate modification of cell-surface and secreted proteins influences processes fundamental to development. We are specifically interested in addressing how mucin-type O-linked sugars are important for proper organization, positioning, attachment and fusion of epidermal cells in a model organism that is amenable to genetic manipulation. While mucin-type glycoproteins are ubiquitous constituents of the extracellular environment and their side chains undergo dynamic, tissue-specific post-translational carbohydrate modifications during development, it is not fully understood how the O-linked sugar components change, which proteins are O-glycosylated, and if O-glycans are involved in the organization, attachment and maintenance of the epithelia and their associated tissues.
     Mucin-type O-glycosylation is regulated by a repertoire of ppGaNTase enzymes, encoded by a multi-gene family. We are dividing our research plan into two parts. First, we will develop our focus, by identifying the ppGaNTase isozymes that are most relevant to epidermal (hypodermal) cells in C. elegans, only during embryogenesis. In the second part of this research program, we will select the most critical ppGaNTases to conduct an in-depth analysis of the role of mucin-type O-glycosylation in epidermal cells in development.

Part one. (Analysis of the complete ppGaNTase family)
We hypothesize that the O-glycosylation machinery or O-glycosylation potential differs from cell to cell and is regulated by differential spatial and temporal expression of specific ppGaNTase isoforms during development. To test this hypothesis, our:
first specific aim is to map, at single cell resolution, the gene expression pattern of those ppGaNTase genes that are expressed in the hypodermis of C. elegans during embryogenesis, using GFP reporter constructs and anti-ppGaNTase antibodies. Expression pattern maps will be superimposed upon the embryonic cell lineage map to display timing and cell identity of ppGaNTase gene activation.
     Screens for C. elegans mutants with hypodermal cell defects have identified extracellular matrix components, including some glycoproteins that are rich in potential mucin-type O-glycosylation sites. We hypothesize that the O-linked sugars that modify such secreted or cell-surface polypeptides have critical functions in development.  To test this hypothesis, our:
second specific aim is to identify the ppGaNTase genes that are critical for hypodermal cells during development. To conduct a rapid loss-of-function screen, the expression of ppGaNTases will be inhibited by double stranded RNA interference (RNAi) and also by creating transgenic nematodes designed to express specific antisense RNAs, using hypodermal cell tissue-specific promoters.
     These first two aims will allow us to identify the most important glycosyltransferases for an in-depth analysis of hypodermal cell development.

Part two. (Analysis of only the most critical ppGaNTases)

To determine if the spatial, temporal, and specificity controls of O-glycosylation are critical for hypodermal cell development, our:
third specific aim is to isolate worms with deletions in the ppGaNTase genes and to over-express ppGaNTase enzymes. To determine if isoform-specificity is important for development, we will attempt to rescue the ppGaNTase gene ablation phenotype with transgenes expressing different members of the ppGaNTase family. To examine the need for controlling the timing of O-glycosylation, we will over-express the critical ppGaNTase(s) early in development.
     To identify the downstream targets of glycosyltransferases that are most important for hypodermal cell development, our:
fourth specific aim will be screen a cDNA expression library for proteins that are recognized and glycosylated by this enzyme. We will perform in situ hybridization screening to determine which proteins with mucin domains are expressed in hypodermal tissue. As above, we will use RNAi on this group of proteins to identify which target protein has a loss-of-function phenotype that exhibits a hypodermal cell defect. The protein identified in this screen will be mapped for O-glycosylation sites and the role of the mucin domain in hypodermal cell development will be genetically dissected.