Glycosylation is one of the most frequent and most diverse posttranscriptional modifications of proteins, but still relatively little is known about its role in a living organism. It plays an important role in development and normal physiology and has also been shown to be altered in several human diseases. Protein O-fucosylation is found in small cysteine-knotted protein domains, like certain EGF- and thrombospondin type 1 repeats (TSRs). In TSRs it has been found in a conserved consensus sequence in Thrombospondin, Properdin and F-spondin from platelets, blood serum and different cell culture models respectively, as shown by our previous studies. We are currently characterizing the mammalian enzyme POFUT-2, which selectively adds O-fucose to the serine or threonine residues of this sequence, and its homolog, PAD-2, in C. elegans. SPON-1, the C. elegans homolog of F-spondin, has five TSR domains, four of which contain the conserved consensus sequence for O-fucosylation. That makes it a good candidate for investigating whether TSR domains are O-fucosylated in vivo in the nematode. To address this question, we cloned the C-terminal part of SPON-1 that contains the TSR domains and are expressing it in wt, and
pad-2(
tm1756) genetic backgrounds. Subsequently, we will purify the expressed polypeptide and analyze its glycosylation by mass spectrometry. A further aim of our research is to investigate whether O-fucosylation is important for SPON-1 activity in vivo. For that purpose we adopted the model from the literature (Chisholm group, personal communication) in which a genetic interaction was shown between
spon-1(
ju430) and the gene encoding the integrin <font face=symbol>a</font> subunit,
ina-1(
gm144), during development: PVQ neurons (left and right) are placed ventrally in the tail region and during embryonic development send axons parallel to each other along the ventral cord to the nerve ring. HSN neuroblasts migrate during embryonic development, from the tail to the vulva.
ina-1(
gm144) mutants show severe defects in both of those processes.
spon-1(
ju430) rescues the midline crossing defect of PVQ neurons and the migration defects of the HSN neuroblast of
ina-1(
gm144). We hypothesized that if SPON-1 is O-fucosylated and if this modification is important for the genetic interaction with
ina-1, then by knocking out PAD-2, we should observe the same rescue of the
ina-1 phenotype. Our first results did not show an obvious rescue of either the PVQ nor HSN phenotype in the
ina-1(
gm144);
pad-2(
tm1756) double mutant. We are now investigating the effect of the over-expression of the SPON-1 C-terminus in both wt and
pad-2 (
tm1756) backgrounds.