A structure-function analysis of Nudel, a protein required for Drosophila dorsoventral polarity.

다국어 초록 (Multilingual Abstract)

Dorsoventral patterning of the Drosophila embryo requires Nudel, a large mosaic protein with a protease domain. Nudel also contains other recognizable motifs including multiple copies of the protein-protein binding LDL-A module, an RGD integrin binding motif, a C-terminal protease-like domain and several regions suitable for both N- and O-linked glycosylation including glycosaminoglycan (GAG) addition. Previous studies have implicated Nudel's protease domain as the trigger of a proteolytic cascade that activates the Toll signaling pathway to establish dorsoventral polarity in the embryo. However, the function of other regions of Nudel has been unclear.
Mutant <italic>nudel</italic> alleles fall into 2 different phenotypic categories: class I alleles produce embryos that arrest early in development within fragile eggshells and class II alleles produce dorsalized embryos that lack all ventral and lateral structures. Previous sequencing of the protease domain of the class II <italic>nudel</italic> mutant alleles revealed that all but one, <italic>ndl<super>9</super></italic>, contain molecular lesions in conserved regions of the protease domain. Upon sequencing of the <italic> ndl<super>9</super></italic> allele, I detected an alteration of a cysteine just N-terminal to the protease domain. In other serine proteases, an analogous cysteine is involved in a crucial disulfide bond between regulatory and catalytic domains suggesting that the N-terminal region of Nudel may be required for proper function of the Nudel protein. In addition, I have investigated the potential role of the LDL-A modules in Nudel.
Using 2D-gel electrophoresis and site-directed mutagenesis, I have obtained evidence that the N-terminal region of Nudel contains a site for glycosaminoglycan attachment that is required for dorsoventral patterning. Disruption of this site blocks a disulfide-based association between N- and C-terminal Nudel polypeptides and activation of Nudel's protease domain. I discuss how a GAG chain on Nudel is required for Nudel protease activation.