Worm Breeder's Gazette 12(4): 28 (October 1, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The glp-l protein (GLP1) is required for at least two inductive events in C. elegans development. Zygotically, glp-1 activity is required for induction of germline proliferation by the somatic distal tip cell, and maternally it is needed for induction of an anterior pharynx. The glp-1 gene appears to encode a receptor based on evidence from mosaic analysis, tissue localization, and sequence data.
The amino acid sequence of GLP1 indicates that it is a transmembrane protein with a predicted molecular weight of 144 kD. It also has 12 consensus sites for N-linked glycosylation. In order to biochemically characterize GLP1 ,we have begun work on identifying GLP1 on Western blots with the antibodies used for tissue localization (see Crittenden and Kimble, WBG 12 #3, p.85, 1992). Since GLP1 is predicted to be a glycoprotein, we used lectin binding to enrich for GLP1 prior to Western blots.
Using wheat germ lectin binding as an enrichment technique, we detect a 160 kD protein on Western blots with antibodies to three different regions in the N-terminal region of the protein, and antibodies to one region in the C-terminal region of the protein. The protein is believed to be the full length product due to its size and because antibodies to regions throughout the protein recognize it. In order to confirm the identification of this protein as GLP1 ,Western blots were done of protein isolated by lectin binding from glp-1 mutants. Two mutants, q35 and q172 ,which each have a glp-1 sequence that predicts a GLP1 product smaller than the wild type protein (Mango et al., Nature 352, p.811, 1991; Kodoyianni et al., Mol Biol Cell, in press)were used for confirmation experiments. In both mutants, the full length protein was recognized at a smaller molecular weight on Western blots, confirming the 160 kD protein as GLP1 .
We also detect two fragments of GLP1 on Western blots, an N-terminal fragment and a C-terminal fragment. The N-terminal fragment is approximately 100kD and is recognized by antibodies to three regions in the N-terminus of GLP1 .This fragment was recognized at a smaller molecular weight on Western blots of protein from q172 worms, which are mutants with an inframe deletion in the N-terminus of GLP1 .The C-terminal fragment is approximately 95 kD and is recognized by antibodies to one region in the C-terminus of GLP1 .This fragment was recognized at a smaller molecular weight on Western blots of protein from q35 worms, which are mutants with a premature stop codon that results in a truncated C-terminus. Therefore Western blots with these two mutants confirm that the 100 kD and 95 kD proteins are also GLP1 .
The fragments of GLP1 appear to be at least twice as abundant as the full-length GLP1 on Western blots. It is unclear whether these fragments are degradation products of GLP1 ,or are active proteins resulting from post-translational proteolysis. In gld-1 mutants, which have a tumorous germline (T. Schedl, see 1991 worm meeting abstract) and express GLP1 of the same molecular weight as wildtype, the abundance of total GLP1 is approximately ten times that of wildtype and the proportion of GLP1 fragments to full length GLP1 remains the same. However in gld-1 ; glp-1 ( q172 )mutants, the proportion of N-terminal fragment to full-length GLP1 is much smaller than in wildtype and gld-1 worms. It is possible that the fragments are unstable degradation products in these mutants and are therefore detected at lower quantities. Alternatively, it is possible that these fragments are required for glp-1 function, and so are detected in smaller amounts in mutants that have GLP1 which is less active.
Mango et al., Nature 352, p.811, 1991.
Kodoyianni et al., Mol Biol Cell, in press.
T. Schedl, see 1991 worm meeting abstract.